Deep learning-based quantification and transcriptomic profiling reveal a methyl jasmonate-mediated glandular trichome formation pathway in Cannabis sativa.

Cannabis glandular trichomes (GTs) are economically and biotechnologically important structures that have a remarkable morphology and capacity to produce, store, and secrete diverse classes of secondary metabolites. However, our understanding of the developmental changes and the underlying molecular processes involved in cannabis GT development is limited. In this study, we developed Cannabis Glandular Trichome Detection Model (CGTDM), a deep learning-based model capable of differentiating and quantifying three types of cannabis GTs with a high degree of efficiency and accuracy. By profiling at eight different time points, we captured dynamic changes in gene expression, phenotypes, and metabolic processes associated with GT development. By integrating weighted gene co-expression network analysis with CGTDM measurements, we established correlations between phenotypic variations in GT traits and the global transcriptome profiles across the developmental gradient. Notably, we identified a module containing methyl jasmonate (MeJA)-responsive genes that significantly correlated with stalked GT density and cannabinoid content during development, suggesting the existence of a MeJA-mediated GT formation pathway. Our findings were further supported by the successful promotion of GT development in cannabis through exogenous MeJA treatment. Importantly, we have identified CsMYC4 as a key transcription factor that positively regulates GT formation via MeJA signaling in cannabis. These findings provide novel tools for GT detection and counting, as well as valuable information for understanding the molecular regulatory mechanism of GT formation, which has the potential to facilitate the molecular breeding, targeted engineering, informed harvest timing, and manipulation of cannabinoid production.

Genome-wide identification and integrated analysis of the FAR1/FHY3 gene family and genes expression analysis under methyl jasmonate treatment in Panax ginseng C. A. Mey.

Ginseng (Panax ginseng C. A. Mey.) is an important and valuable medicinal plant species used in traditional Chinese medicine, and its metabolite ginsenoside is the primary active ingredient. The FAR1/FHY3 gene family members play critical roles in plant growth and development as well as participate in a variety of physiological processes, including plant development and signaling of hormones. Studies have indicated that methyl jasmonate treatment of ginseng adventitious roots resulted in a significant increase in the content of protopanaxadiol ginsenosides. Therefore, it is highly significant to screen the FAR1/FHY3 gene family members in ginseng and preliminarily investigate their expression patterns in response to methyl jasmonic acid signaling. In this study, we screened and identified the FAR1/FHY3 family genes in the ginseng transcriptome databases. And then, we analyzed their gene structure and phylogeny, chromosomal localization and expression patterns, and promoter cis-acting elements, and made GO functional annotations on the members of this family. After that, we treated the ginseng adventitious roots with 200 mM methyl jasmonate and investigated the trend of the expression of four genes containing the largest number of methyl jasmonate cis-acting elements at different treatment times. All four genes were able to respond to methyl jasmonate, the most significant change was in the PgFAR40 gene. This study provides data support for subsequent studies of this family member in ginseng and provides experimental reference for subsequent validation of the function of this family member under methyl jasmonic acid signaling.

MeJA-mediated enhancement of salt-tolerance of Populus wutunensis by 5-aminolevulinic acid.

BACKGROUND: 5-Aminolevulinic acid (ALA) is a natural and environmentally benign multifunctional plant growth regulator involved in the regulation of plant tolerance to various environmental stresses. This research aimed to explore the molecular mechanisms of salt tolerance in Populus wutunensis induced by exogenous ALA using physiological and transcriptomic analyses. RESULTS: Physiological results showed that 50 mg·L- 1 ALA-treatment significantly reduced the malondialdehyde (MDA) content and the relative electrical conductivity (REC) and enhanced antioxidant activities of enzymes such as SOD, POD and CAT in salt-stressed P. wutunensis seedlings. Transcriptome analysis identified ALA-induced differentially expressed genes (DEGs) associating with increased salt-tolerance in P. wutunensis. GO and KEGG enrichment analyses showed that ALA activated the jasmonic acid signaling and significantly enhanced the protein processing in endoplasmic reticulum and the flavonoid biosynthesis pathways. Results of the hormone-quantification by LC-MS/MS-based assays showed that ALA could increase the accumulation of methyl jasmonate (MeJA) in salt-stressed P. wutunensis. Induced contents of soluble proteins and flavonoids by exogenous ALA in salt-treated seedlings were also correlated with the MeJA content. CONCLUSION: 5-aminolevulinic acid improved the protein-folding efficiency in the endoplasmic reticulum and the flavonoid-accumulation through the MeJA-activated jasmonic acid signaling, thereby increased salt-tolerance in P. wutunensis.

Impacts of methyl jasmonate on Selaginella martensii: volatiles, transcriptomics, phytohormones, and gas exchange.

Methyl jasmonate (MeJA) induces various defence responses in seed plants, but for early plant lineages, information on the potential of jasmonates to elicit stress signalling and trigger physiological modifications is limited. The spikemoss Selaginella martensii was exposed to a range of MeJA concentrations (0, 10, 25, and 50 mM), and biogenic volatile organic compound (BVOC) emissions, photosynthetic rate (A), and stomatal conductance (gs) were continuously measured. In addition, changes in phytohormone concentrations and gene expression were studied. Enhancement of methanol, lipoxygenase pathway volatiles and linalool emissions, and reductions in A and gs, were MeJA dose-dependent. Before MeJA treatment, the concentration of 12-oxo-phytodienoic acid (OPDA) was 7-fold higher than jasmonic acid (JA). MeJA treatment rapidly increased OPDA and JA concentrations (within 30 min), with the latter more responsive. Some genes involved in BVOC biosynthesis and OPDA-specific response were up-regulated at 30 min after MeJA spraying, whereas those in the JA signalling pathway were not affected. Although JA was synthesized in S. martensii, OPDA was prioritized as a signalling molecule upon MeJA application. MeJA inhibited primary and enhanced secondary metabolism; we propose that fast-emitted linalool could serve as a marker of elicitation of stress-induced metabolism in lycophytes.

Identification and promoter analysis of a GA-stimulated transcript 1 gene from Jatropha curcas.

KEY MESSAGE: Overexpression of JcGAST1 promotes plant growth but inhibits pistil development. The pyrimidine box and CGTCA motif of the JcGAST1 promoter were responsible for the GA and MeJA responses. Members of the gibberellic acid-stimulated Arabidopsis (GASA) gene family play roles in plant growth and development, particularly in flower induction and seed development. However, there is still relatively limited knowledge of GASA genes in Jatropha curcas. Herein, we identified a GASA family gene from Jatropha curcas, namely, JcGAST1, which encodes a protein containing a conserved GASA domain. Sequence alignment showed that the JcGAST1 protein shares 76% sequence identity and 80% sequence similarity with SlGAST1. JcGAST1 had higher expression and protein levels in the female flowers than in the male flowers. Overexpression of JcGAST1 in tobacco promotes plant growth but inhibits pistil development. JcGAST1 expression was upregulated by GA and downregulated by MeJA. Promoter analysis indicated that the pyrimidine box and CGTCA motif were the GA- and MeJA-responsive elements of the JcGAST1 promoter. Using a Y1H screen, six transcription factors were found to interact with the pyrimidine box, and three transcription factors were found to interact with the CGTCA motif. Overall, the results of this study improve our understanding of the JcGAST1 gene and provide useful information for further studies.

Metabolic Responses of the Microalga Neochloris oleoabundans to Extracellular Self- and Nonself-DNA.

Stressed organisms identify intracellular molecules released from damaged cells due to trauma or pathogen infection as components of the innate immune response. These molecules called DAMPs (Damage-Associated Molecular Patterns) are extracellular ATP, sugars, and extracellular DNA, among others. Animals and plants can recognize their own DNA applied externally (self-exDNA) as a DAMP with a high degree of specificity. However, little is known about the microalgae responses to damage when exposed to DAMPs and specifically to self-exDNAs. Here we compared the response of the oilseed microalgae Neochloris oleoabundans to self-exDNA, with the stress responses elicited by nonself-exDNA, methyl jasmonate (MeJA) and sodium bicarbonate (NaHCO3). We analyzed the peroxidase enzyme activity related to the production of reactive oxygen species (ROS), as well as the production of polyphenols, lipids, triacylglycerols, and phytohormones. After 5 min of addition, self-exDNA induced peroxidase enzyme activity higher than the other elicitors. Polyphenols and lipids were increased by self-exDNA at 48 and 24 h, respectively. Triacylglycerols were increased with all elicitors from addition and up to 48 h, except with nonself-exDNA. Regarding phytohormones, self-exDNA and MeJA increased gibberellic acid, isopentenyladenine, and benzylaminopurine at 24 h. Results show that Neochloris oleoabundans have self-exDNA specific responses.

Seed Priming with MeJa Prevents Salt-Induced Growth Inhibition and Oxidative Damage in Sorghum bicolor by Inducing the Expression of Jasmonic Acid Biosynthesis Genes.

Salinity is one of the major detrimental abiotic stresses at the forefront of deterring crop productivity globally. Although the exogenous application of phytohormones has formerly proven efficacious to plants, their effect on the moderately stress-tolerant crop "Sorghum bicolor" remains elusive. To investigate this, S. bicolor seeds primed with methyl jasmonate (0; 10 and 15 µM MeJa) were exposed to salt (200 mM NaCl) stress, and their morpho-physiological, biochemical, and molecular attributes were measured. Salt stress significantly decreased shoot length and fresh weight by 50%, whereas dry weight and chlorophyll content were decreased by more than 40%. Furthermore, salt-stress-induced oxidative damage was evident by the formation of brown formazan spots (indicative of H2O2 production) on sorghum leaves and a more than 30% increase in MDA content. However, priming with MeJa improved growth, increased chlorophyll content, and prevented oxidative damage under salt stress. While 15 µM MeJa maintained proline content to the same level as the salt-stressed samples, total soluble sugars were maintained under 10 µM MeJa, indicating a high degree of osmotic adjustment. Shriveling and thinning of the epidermis and xylem tissues due to salt stress was prevented by MeJa, followed by a more than 70% decrease in the Na+/K+ ratio. MeJa also reversed the FTIR spectral shifts observed for salt-stressed plants. Furthermore, salt stress induced the expression of the jasmonic acid biosynthesis genes; linoleate 92-lipoxygenase 3, allene oxide synthase 1, allene oxide cyclase, and 12-oxophytodienoate reductase 1. In MeJa-primed plants, their expression was reduced, except for the 12-oxophytodienoate reductase 1 transcript, which further increased by 67%. These findings suggest that MeJa conferred salt-stress tolerance to S. bicolor through osmoregulation and synthesis of JA-related metabolites.

Comprehensive Analysis of the Catalase (CAT) Gene Family and Expression Patterns in Rubber Tree (Hevea brasiliensis) under Various Abiotic Stresses and Multiple Hormone Treatments.

Catalase (CAT) is one of the key enzymes involved in antioxidant defense systems and mainly scavenges H2O2 and plays a vital role in plant growth, development, and various adverse stresses. To date, a systematic study of the CAT gene family in rubber tree has not been reported. In this study, five HbCAT gene family members were identified from the rubber tree genome, and these were mainly clustered into two subfamilies. Gene structure and motif analysis showed that exon-intron and motif patterns were conserved across different plant species. Sequence analysis revealed that HbCAT proteins contain one active catalytic site, one heme-ligand signature sequence, three conserved amino acid residues (His, Tyr, and Asn), and one peroxisome-targeting signal 1 (PTS1) sequence. Fragment duplication is a selection pressure for the evolution of the HbCAT family based on Ka/Ks values. Analysis of cis-acting elements in the promoters indicated that HbCAT gene expression might be regulated by abscisic acid (ABA), salicylic acid (SA), and MYB transcription factors; furthermore, these genes might be involved in plant growth, development, and abiotic stress responses. A tissue-specific expression analysis showed that HbCATs gradually increased with leaf development and were highly expressed in mature leaves. Gene expression profiling exhibited the differential expression of the HbCATs under cold, heat, drought, and NaCl stresses. Our results provide comprehensive information about the HbCAT gene family, laying the foundation for further research on its function in rubber tree.

Transcriptome characterization of candidate genes for heat tolerance in perennial ryegrass after exogenous methyl Jasmonate application.

Methyl jasmonate (MeJA) plays a role in improving plant stress tolerance. The molecular mechanisms associated with heat tolerance mediated by MeJA are not fully understood in perennial grass species. The study was designed to explore transcriptomic mechanisms underlying heat tolerance by exogenous MeJA in perennial ryegrass (Lolium perenne L.) using RNA-seq. Transcriptomic profiling was performed on plants under normal temperature (CK), high temperature for 12 h (H), MeJA pretreatment (T), MeJA pretreatment + H (T-H), respectively. The analysis of differentially expressed genes (DEGs) showed that H resulted in the most DEGs and T had the least, compared with CK. Among them, the DEGs related to the response to oxygen-containing compound was higher in CKvsH, while many genes related to photosynthetic system were down-regulated. The DEGs related to plastid components was higher in CKvsT. GO and KEGG analysis showed that exogenous application of MeJA enriched photosynthesis related pathways under heat stress. Exogenous MeJA significantly increased the expression of genes involved in chlorophyll (Chl) biosynthesis and antioxidant metabolism, and decreased the expression of Chl degradation genes, as well as the expression of heat shock transcription factor - heat shock protein (HSF-HSP) network under heat stress. The results indicated that exogenous application of MeJA improved the heat tolerance of perennial ryegrass by mediating expression of genes in different pathways, such as Chl biosynthesis and degradation, antioxidant enzyme system, HSF-HSP network and JAs biosynthesis.

Methyl jasmonate induces selaginellin accumulation in Selaginella convoluta.

INTRODUCTION: Selaginellins are specialized metabolites and chemotaxonomic markers for Selaginella species. Despite the growing interest in these compounds as a result of their bioactivities, they are accumulated at low levels in the plant. Hence, their isolation and chemical characterization are often difficult, time consuming, and limiting for biological tests. Elicitation with the phytohormone methyl jasmonate (MeJA) could be a strategy to increase the content of selaginellins addressing their low availability problem, that also impairs pharmacological investigations. MATHERIALS AND METHODS: In this study, we examined MeJA elicitation in Selaginella convoluta plants, a medicinal plant found in northeastern Brazil, by treating them with two different concentrations (MeJA: 50 and 100 µM), followed by chemical profiling after 12, 24 and 48 h after application. Samples were harvested and analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). RESULTS AND DISCUSSCION: MeJA treatment significantly impacted the chemical phenotype. Regarding shoots differences in the time-dependent increased accumulation of all metabolites when plants were subjected to 100 µM MeJA were observed while in roots, most metabolites had their concentrations decreased in a time-dependent fashion at the same conditions. Results support organ, MeJA concentration and time post-treatment dependence of specialized metabolite accumulation, mainly the flavonoids and selaginellins. The amount of Selaginellin G in shoots of MeJA-treated specimens increased in 5.63-fold relative to control. The molecular networking approach allowed for the putative annotation of 64 metabolites, among them, the MeJA treatment followed by targeted metabolome analysis also allowed to annotate seven unprecedented selaginellins. Additionally, the in silico bioactive potential of the annotated selaginellins highlighted targets related to neurodegenerative disorders, antiproliferative, and antiparasitic issues. Taken together, data point out MeJA exposure as a strategy to induce potentially bioactive selaginellins accumulation in S. convoluta, this approach could enable a deep investigation about the metabolic function of these metabolites in the genus as well as regarding pharmacological exploration of the undervalued potential.

Exploring the Effect of Methyl Jasmonate on the Expression of microRNAs Involved in Biosynthesis of Active Compounds of Rosemary Cell Suspension Cultures through RNA-Sequencing.

Our aim in the experiment was to study the effects of methyl jasmonates (MeJA) on the active compounds of rosemary suspension cells, the metabolites' change of contents under different concentrations of MeJA, including 0 (CK), 10 (M10), 50 (M50) and 100 µM MeJA (M100). The results demonstrated that MeJA treatments promoted the accumulation of rosmarinic acid (RA), carnosic acid (CA), flavonoids, jasmonate (JA), gibberellin (GA), and auxin (IAA); but reduced the accumulations of abscisic acid (ABA), salicylic acid (SA), and aspartate (Asp). In addition, 50 and 100 µM MeJA promoted the accumulation of alanine (Ala) and glutamate (Glu), and 50 µM MeJA promoted the accumulation of linoleic acid and alpha-linolenic acid in rosemary suspension cells. Comparative RNA-sequencing analysis of different concentrations of MeJA showed that a total of 30, 61, and 39 miRNAs were differentially expressed in the comparisons of CKvsM10, CKvsM50, CKvsM100, respectively. The analysis of the target genes of the differentially expressed miRNAs showed that plant hormone signal transduction, linoleic acid, and alpha-linolenic acid metabolism-related genes were significantly enriched. In addition, we found that miR160a-5p target ARF, miR171d_1 and miR171f_3 target DELLA, miR171b-3p target ETR, and miR156a target BRI1, which played a key role in rosemary suspension cells under MeJA treatments. qRT-PCR of 12 differentially expressed miRNAs and their target genes showed a high correlation between the RNA-seq and the qRT-PCR result. Amplification culture of rosemary suspension cells in a 5 L stirred bioreactor showed that cell biomass accumulation in the bioreactor was less than that in the shake flask under the same conditions, and the whole cultivation period was extended to 14 d. Taken together, MeJA promoted the synthesis of the active compounds in rosemary suspension cells in a wide concentration range via concentration-dependent differential expression patterns. This study provided an overall view of the miRNAs responding to MeJA in rosemary.

Characterization of Virus-Inducible Orchid Argonaute 5b Promoter and Its Functional Characterization in Nicotiana benthamiana during Virus Infection.

Plant ARGONAUTES (AGOs) play a significant role in the defense against viral infection. Previously, we have demonstrated that AGO5s encoded in Phalaenopsis aphrodite subsp. formosana (PaAGO5s) took an indispensable part in defense against major viruses. To understand the underlying defense mechanism, we cloned PaAGO5s promoters (pPaAGO5s) and analyzed their activity in transgenic Nicotiana benthamiana using ß-glucuronidase (GUS) as a reporter gene. GUS activity analyses revealed that during Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) infections, pPaAGO5b activity was significantly increased compared to pPaAGO5a and pPaAGO5c. Analysis of pPaAGO5b 5'-deletion revealed that pPaAGO5b_941 has higher activity during virus infection. Further, yeast one-hybrid analysis showed that the transcription factor NbMYB30 physically interacted with pPaAGO5b_941 to enhance its activity. Overexpression and silencing of NbMYB30 resulted in up- and downregulation of GUS expression, respectively. Exogenous application and endogenous measurement of phytohormones have shown that methyl jasmonate and salicylic acid respond to viral infections. NbMYB30 overexpression and its closest related protein, PaMYB30, in P. aphrodite subsp. formosana reduced CymMV accumulation in P. aphrodite subsp. formosana. Based on these discoveries, this study uncovers the interaction between virus-responsive promoter and the corresponding transcription factor in plants.

Genome-wide dynamic network analysis reveals the potential genes for MeJA-induced growth-to-defense transition.

BACKGROUND: Methyl jasmonate (MeJA), which has been identified as a lipid-derived stress hormone, mediates plant resistance to biotic/abiotic stress. Understanding MeJA-induced plant defense provides insight into how they responding to environmental stimuli. RESULT: In this work, the dynamic network analysis method was used to quantitatively identify the tipping point of growth-to-defense transition and detect the associated genes. As a result, 146 genes were detected as dynamic network biomarker (DNB) members and the critical defense transition was identified based on dense time-series RNA-seq data of MeJA-treated Arabidopsis thaliana. The GO functional analysis showed that these DNB genes were significantly enriched in defense terms. The network analysis between DNB genes and differentially expressed genes showed that the hub genes including SYP121, SYP122, WRKY33 and MPK11 play a vital role in plant growth-to-defense transition. CONCLUSIONS: Based on the dynamic network analysis of MeJA-induced plant resistance, we provide an important guideline for understanding the growth-to-defense transition of plants' response to environment stimuli. This study also provides a database with the key genes of plant defense induced by MeJA.

CRISPR/Cas9-mediated mutagenesis of WRKY3 and WRKY4 function decreases salt and Me-JA stress tolerance in Arabidopsis thaliana.

BACKGROUND: WRKY transcription factor is involved in regulation of plant growth and development, response to biotic and abiotic stresses, including homologous WRKY3 and WRKY4 genes which play a vital role in regulating plants defense response to pathogen and drought stress. METHODS AND RESULTS: To investigate the function of AtWRKY3 and AtWRKY4 genes in regulating salt and Me-JA stresses, the loss-of-function mutations were generated by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) system in Arabidopsis thaliana. Several independent transgenic lines with single or double mutations were obtained via Agrobacterium-mediated transformation. The knockout lines of AtWRKY3 and AtWRKY4 genes were successfully achieved and confirmed by qRT-PCR technology. Expression analysis showed that AtWRKY3 and AtWRKY4 genes had significantly up-regulated under salt and Me-JA stresses. The growth of double mutant plants under salt or Me-JA stresses were significantly inhibited compared with corresponding wild type (WT) plants, especially their root lengths. Moreover, the double mutant plants displayed salt and Me-JA sensitivity phenotypic characteristics, such as the increased relative electrolyte leakage (REL) and a substantial reduce in the activities of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities. CONCLUSION: Taken together, these data suggested that the simultaneous modification of homologous gene copies of WRKY are established using CRISPR/Cas9 system in A. thaliana and the loss of AtWRKY3 and AtWRKY4 has an effect on ROS scavenging pathways to reduce stress tolerance.

Synergistic Effect of Methyl Jasmonate and Abscisic Acid Co-Treatment on Avenanthramide Production in Germinating Oats.

The oat (Avena sativa L.) is a grain of the Poaceae grass family and contains many powerful anti-oxidants, including avenanthramides as phenolic alkaloids with anti-inflammatory, anti-oxidant, anti-itch, anti-irritant, and anti-atherogenic activities. Here, the treatment of germinating oats with methyl jasmonate (MeJA) or abscisic acid (ABA) resulted in 2.5-fold (582.9 mg/kg FW) and 2.8-fold (642.9 mg/kg FW) increase in avenanthramide content, respectively, relative to untreated controls (232.6 mg/kg FW). Moreover, MeJA and ABA co-treatment synergistically increased avenanthramide production in germinating oats to 1505 mg/kg FW. Individual or combined MeJA and ABA treatment increased the expression of genes encoding key catalytic enzymes in the avenanthramide-biosynthesis pathway, including hydroxycinnamoyl-CoA:hydrocyanthranilate N-hydroxycinnamoyl transferase (HHT). Further analyses showed that six AsHHT genes were effectively upregulated by MeJA or ABA treatment, especially AsHHT4 for MeJA and AsHHT5 for ABA, thereby enhancing the production of all three avenanthramides in germinating oats. Specifically, AsHHT5 exhibited the highest expression following MeJA and ABA co-treatment, indicating that AsHHT5 played a more crucial role in avenanthramide biosynthesis in response to MeJA and ABA co-treatment of germinating oats. These findings suggest that elicitor-mediated metabolite farming using MeJA and ABA could be a valuable method for avenanthramide production in germinating oats.

Jasmonic Acid Plays a Pivotal Role in Pollen Development and Fertility Regulation in Different Types of P(T)GMS Rice Lines.

Two-line hybrid rice systems represent a new technical approach to utilizing the advantages of rice hybrids. However, the mechanism underlying the male sterile-line fertility transition in rice remains unclear. Peiai 64S (PA64S) is a photoperiod- and thermo-sensitive genic male sterile (PTGMS) line in which male sterility manifests at an average temperature above 23.5 °C under long-day (LD) conditions. Nongken 58S (NK58S) is a LD-sensitive genic male sterile (PGMS) rice that is sterile under LD conditions (above 13.75 h-day). In contrast, D52S is a short-day (SD)-PGMS line that manifests male sterility under SD conditions (below 13.5 h-day). In this study, we obtained fertile and sterile plants from all three lines and performed transcriptome analyses on the anthers of the plants. Gene ontology (GO) analysis suggested that the differentially expressed genes identified were significantly enriched in common terms involved in the response to jasmonic acid (JA) and in JA biosynthesis. On the basis of the biochemical and molecular validation of dynamic, tissue-specific changes in JA, indole-3-acetic acid (IAA) levels, gibberellin (GA) levels, and JA biosynthetic enzyme activities and expression, we proposed that JA could play a pivotal role in viable pollen production through its initial upregulation, constant fluctuation and leaf-spikelet signaling under certain fertility-inducing conditions. Furthermore, we also sprayed methyl jasmonate (MEJA) and salicylhydroxamic acid (SHAM) on the plants, thereby achieving fertility reversal in the PGMS lines NK58S and D52S, with 12.91-63.53% pollen fertility changes. Through qPCR and enzyme activity analyses, we identified two key enzymes-allene oxide synthase (AOS) and allene oxide cyclase (AOC)-that were produced and upregulated by 20-500-fold in PGMS in response to spraying; the activities of these enzymes reversed pollen fertility by influencing the JA biosynthetic pathway. These results provide a new understanding of hormone interactions and networks in male-sterile rice based on the role of JA that will help us to better understand the potential regulatory mechanisms of fertility development in rice in the future.

Cerium-Promoted Ginsenosides Accumulation by Regulating Endogenous Methyl Jasmonate Biosynthesis in Hairy Roots of Panax ginseng.

Among rare earth elements, cerium has the unique ability of regulating the growth of plant cells and the biosynthesis of metabolites at different stages of plant development. The signal pathways of Ce3+-mediated ginsenosides biosynthesis in ginseng hairy roots were investigated. At a low concentration, Ce3+ improved the elongation and biomass of hairy roots. The Ce3+-induced accumulation of ginsenosides showed a high correlation with the reactive oxygen species (ROS), as well as the biosynthesis of endogenous methyl jasmonate (MeJA) and ginsenoside key enzyme genes (PgSS, PgSE and PgDDS). At a Ce3+ concentration of 20 mg L-1, the total ginsenoside content was 1.7-fold, and the total ginsenosides yield was 2.7-fold that of the control. Malondialdehyde (MDA) content and the ROS production rate were significantly higher than those of the control. The activity of superoxide dismutase (SOD) was significantly activated within the Ce3+ concentration range of 10 to 30 mg L-1. The activity of catalase (CAT) and peroxidase (POD) strengthened with the increasing concentration of Ce3+ in the range of 20-40 mg L-1. The Ce3+ exposure induced transient production of superoxide anion (O2•-) and hydrogen peroxide (H2O2). Together with the increase in the intracellular MeJA level and enzyme activity for lipoxygenase (LOX), there was an increase in the gene expression level of MeJA biosynthesis including PgLOX, PgAOS and PgJMT. Our results also revealed that Ce3+ did not directly influence PgSS, PgSE and PgDDS activity. We speculated that Ce3+-induced ROS production could enhance the accumulation of ginsenosides in ginseng hairy roots via the direct stimulation of enzyme genes for MeJA biosynthesis. This study demonstrates a potential approach for understanding and improving ginsenoside biosynthesis that is regulated by Ce3+-mediated signal transduction.

Genome-wide identification and functional analysis of the TIFY gene family in the response to multiple stresses in Brassica napus L.

BACKGROUND: TIFY is a plant-specific protein family with a diversity of functions in plant development and responses to stress and hormones, which contains JASMONATE ZIM-domain (JAZ), TIFY, PPD and ZML subfamilies. Despite extensive studies of TIFY family in many other species, TIFY has not yet been characterized in Brassica napus. RESULTS: In this study, we identified 77, 36 and 39 TIFY family genes in the genome of B. napus, B. rapa and B. oleracea, respectively. Results of the phylogenetic analysis indicated the 170 TIFY proteins from Arabidopsis, B. napus, B. rapa and B. oleracea could be divided into 11 groups: seven JAZ groups, one PPD group, one TIFY group, and two ZIM/ZML groups. The molecular evolutionary analysis showed that TIFY genes were conserved in Brassicaceae species. Gene expression profiling and qRT-PCR revealed that different groups of BnaTIFY members have distinct spatiotemporal expression patterns in normal conditions or following treatment with different abiotic/biotic stresses and hormones. The BnaJAZ subfamily genes were predominantly expressed in roots and up-regulated by NaCl, PEG, freezing, methyl jasmonate (MeJA), salicylic acid (SA) and Sclerotinia sclerotiorum in leaves, suggesting that they have a vital role in hormone signaling to regulate multiple stress tolerance in B. napus. CONCLUSIONS: The extensive annotation and expression analysis of the BnaTIFY genes contributes to our understanding of the functions of these genes in multiple stress responses and phytohormone crosstalk in B. napus.

Bioactive compounds induced in Physalis angulata L. by methyl-jasmonate: an investigation of compound accumulation patterns and biosynthesis-related candidate genes.

KEY MESSAGE: We employed both metabolomic and transcriptomic approaches to explore the accumulation patterns of physalins, flavonoids and chlorogenic acid in Physalis angulata and revealed the genes associated with the biosynthesis of bioactive compounds under methyl-jasmonate (MeJA) treatment. Physalis angulata L. is an annual Solanaceae plant with a number of medicinally active compounds. Despite the potential pharmacological benefits of P. angulata, the scarce genomic information regarding this plant has limited the studies on the mechanisms of bioactive compound biosynthesis. To facilitate the basic understanding of the main chemical constituent biosynthesis pathways, we performed both metabolomic and transcriptomic approaches to reveal the genes associated with the biosynthesis of bioactive compounds under methyl-jasmonate (MeJA) treatment. Untargeted metabolome analysis showed that most physalins, flavonoids and chlorogenic acid were significantly upregulated. Targeted HPLC-MS/MS analysis confirmed variations in the contents of two important representative steroid derivatives (physalins B and G), total flavonoids, neochlorogenic acid, and chlorogenic acid between MeJA-treated plants and controls. Transcript levels of a few steroid biosynthesis-, flavonoid biosynthesis-, and chlorogenic acid biosynthesis-related genes were upregulated, providing a potential explanation for MeJA-induced active ingredient synthesis in P. angulata. Systematic correlation analysis identified a number of novel candidate genes associated with bioactive compound biosynthesis. These results may help to elucidate the regulatory mechanism underlying MeJA-induced active compound accumulation and provide several valuable candidate genes for further functional study.

Integrated metabolomics and transcriptome analysis on flavonoid biosynthesis in safflower (Carthamus tinctorius L.) under MeJA treatment.

BACKGROUND: Safflower (Carthamus tinctorius L.) is an important cash crop, of which the dried tube flower is not only an important raw material for dyes and cosmetics but also an important herb widely used in traditional Chinese medicine. The pigment and bioactive compounds are composed of flavonoids (mainly quinone chalcones), and studies have reported that MeJA can promote the biosynthesis of quinone chalcones, but the mechanism underlying the effect of MeJA in safflower remains unclear. Here, we attempt to use metabolomics and transcriptome technologies to analyse the molecular mechanism of flavonoid biosynthesis under MeJA treatment in safflower. RESULTS: Based on a UHPLC-ESI-MS/MS detection platform and a self-built database (including hydroxysafflor yellow A, HSYA), a total of 209 flavonoid metabolites were detected, and 35 metabolites were significantly different after treatment with MeJA. Among them, 24 metabolites were upregulated upon MeJA treatment, especially HSYA. Eleven metabolites were downregulated after MeJA treatment. Integrated metabolomics and transcriptome analysis showed that MeJA might upregulate the expression of upstream genes in the flavonoid biosynthesis pathway (such as CHSs, CHIs and HCTs) and downregulate the expression of downstream genes (such as F3Ms, ANRs and ANSs), thus promoting the biosynthesis of quinone chalcones, such as HSYA. The transcription expressions of these genes were validated by real-time PCR. In addition, the promoters of two genes (CtCHI and CtHCT) that were significantly upregulated under MeJA treatment were cloned and analysed. 7 and 3 MeJA response elements were found in the promoters, respectively. CONCLUSIONS: MeJA might upregulate the expression of the upstream genes in the flavonoid biosynthesis pathway and downregulate the expression of the downstream genes, thus promoting the biosynthesis of quinone chalcones. Our results provide insights and basic data for the molecular mechanism analysis of flavonoid synthesis in safflower under MeJA treatment.