MeJA Treatment Research Articles

SlMYC2 interacted with the SlTOR promoter and mediated JA signaling to regulate growth and fruit quality in tomato.

Tomato (Solanum lycopersicum) is a major vegetable crop cultivated worldwide. The regulation of tomato growth and fruit quality has long been a popular research topic. MYC2 is a key regulator of the interaction between jasmonic acid (JA) signaling and other signaling pathways, and MYC2 can integrate the interaction between JA signaling and other hormone signals to regulate plant growth and development. TOR signaling is also an essential regulator of plant growth and development. However, it is unclear whether MYC2 can integrate JA signaling and TOR signaling during growth and development in tomato. Here, MeJA treatment and SlMYC2 overexpression inhibited the growth and development of tomato seedlings and photosynthesis, but increased the sugar-acid ratio and the contents of lycopene, carotenoid, soluble sugar, total phenol and flavonoids, indicating that JA signaling inhibited the growth of tomato seedlings and altered fruit quality. When TOR signaling was inhibited by RAP, the JA content increased, and the growth and photosynthesis of tomato seedlings decreased, indicating that TOR signaling positively regulated the growth and development of tomato seedlings. Further yeast one-hybrid assays showed that SlMYC2 could bind directly to the SlTOR promoter. Based on GUS staining analysis, SlMYC2 regulated the transcription of SlTOR, indicating that SlMYC2 mediated the interaction between JA and TOR signaling by acting on the promoter of SlTOR. This study provides a new strategy and some theoretical basis for tomato breeding.

Genome-Wide Identification of Auxin-Responsive GH3 Gene Family in Saccharum and the Expression of ScGH3-1 in Stress Response.

Gretchen Hagen3 (GH3), one of the three major auxin-responsive gene families, is involved in hormone homeostasis in vivo by amino acid splicing with the free forms of salicylic acid (SA), jasmonic acid (JA) or indole-3-acetic acid (IAA). Until now, the functions of sugarcane GH3 (SsGH3) family genes in response to biotic stresses have been largely unknown. In this study, we performed a systematic identification of the SsGH3 gene family at the genome level and identified 41 members on 19 chromosomes in the wild sugarcane species, Saccharum spontaneum. Many of these genes were segmentally duplicated and polyploidization was the main contributor to the increased number of SsGH3 members. SsGH3 proteins can be divided into three major categories (SsGH3-I, SsGH3-II, and SsGH3-III) and most SsGH3 genes have relatively conserved exon-intron arrangements and motif compositions. Diverse cis-elements in the promoters of SsGH3 genes were predicted to be essential players in regulating SsGH3 expression patterns. Multiple transcriptome datasets demonstrated that many SsGH3 genes were responsive to biotic and abiotic stresses and possibly had important functions in the stress response. RNA sequencing and RT-qPCR analysis revealed that SsGH3 genes were differentially expressed in sugarcane tissues and under Sporisorium scitamineum stress. In addition, the SsGH3 homolog ScGH3-1 gene (GenBank accession number: OP429459) was cloned from the sugarcane cultivar (Saccharum hybrid) ROC22 and verified to encode a nuclear- and membrane-localization protein. ScGH3-1 was constitutively expressed in all tissues of sugarcane and the highest amount was observed in the stem pith. Interestingly, it was down-regulated after smut pathogen infection but up-regulated after MeJA and SA treatments. Furthermore, transiently overexpressed Nicotiana benthamiana, transduced with the ScGH3-1 gene, showed negative regulation in response to the infection of Ralstonia solanacearum and Fusarium solani var. coeruleum. Finally, a potential model for ScGH3-1-mediated regulation of resistance to pathogen infection in transgenic N. benthamiana plants was proposed. This study lays the foundation for a comprehensive understanding of the sequence characteristics, structural properties, evolutionary relationships, and expression of the GH3 gene family and thus provides a potential genetic resource for sugarcane disease-resistance breeding.

Promoter of Vegetable Soybean GmTIP1;6 Responds to Diverse Abiotic Stresses and Hormone Signals in Transgenic Arabidopsis.

Tonoplast intrinsic proteins (TIPs), a sub-family of aquaporins (AQPs), are known to play important roles in plant abiotic stress responses. However, evidence for the promoters of TIPs involvement in abiotic stress processes remains scarce. In this study, the promoter of the vegetable soybean GmTIP1;6 gene, which had the highest similarity to TIP1-type AQPs from other plants, was cloned. Expression pattern analyses indicated that the GmTIP1;6 gene was dramatically induced by drought, salt, abscisic acid (ABA), and methyl jasmonate (MeJA) stimuli. Promoter analyses revealed that the GmTIP1;6 promoter contained drought, ABA, and MeJA cis-acting elements. Histochemical staining of the GmTIP1;6 promoter in transgenic Arabidopsis corroborated that it was strongly expressed in the vascular bundles of leaves, stems, and roots. Beta-glucuronidase (GUS) activity assays showed that the activities of the GmTIP1;6 promoter were enhanced by different concentrations of polyethylene glycol 6000 (PEG 6000), NaCl, ABA, and MEJA treatments. Integrating these results revealed that the GmTIP1;6 promoter could be applied for improving the tolerance to abiotic stresses of the transgenic plants by promoting the expression of vegetable soybean AQPs.

Cloning, Subcellular Location and Expression Analysis of Grape Myb Gene

MYB gene plays an important role in plant growth, development and response to abiotic stress. In the present study, ‘Yatomi Rosa’ grape (Vitis vinifera L.) was used as the test material. Two MYB, VvMYBB1 and VvMYBA3 gene were obtained by homologous cloning, and their subcellular location, different organs, and expression patterns under stress treatment and hormone induction were obtained. VvMYBB1 and VvMYBA3 proteins were located in the nucleus and belonged to nuclear proteins. They were highly expressed in roots and flowers. These genes might have a negative effect on the formation of peel color. VvMYB1 gene and VvMYBA3 gene might play an important role in drought resistance and salt stress resistance. Under the induction of exogenous hormones, relative expression of VvMYBB1 gene was higher than that of VvMYBA3 gene under IAA, ETH, SA and MeJA treatment. Relative expression of VvMYBB1 gene was lower than that of VvMYBA3 gene under 6-BA, GA3 and ABA treatment. It showed that MYB gene played an important role in the development of different organs of grapes, fruit color, and response to abiotic stress and exogenous hormones induction in grape. Bangladesh J. Bot. 51(3): 499-506, 2022 (September)

Identification and expression analysis of the grape pentatricopeptide repeat (PPR) gene family in abiotic stress.

The pentatricopeptide repeat (PPR) is one of the largest gene family in plants, and play important role in regulating plant growth, development and abiotic stress response. However, PPR genes have been poorly studied in grapes. In this study, based on the grape genome database, bioinformatics methods and quantitative real-time PCR (qRT-PCR) were used to identify the VvPPR family and the response to abiotic stress. A total of 181 PPR genes were identified in grape and divided into two subfamilies. Subcellular localization predicted that this gene family mainly functions in chloroplasts, nucleus, and mitochondria. Protein-protein interaction prediction indicated that there may be interaction between VvPPR44,53 and VvPPR44. The promoter region of VvPPR gene family contained various cis-acting elements, which were related to light and hormone. Expression pattern analysis showed that the VvPPR gene family was highly expressed in grape leaves, buds and carpel tissues. qRT-PCR results showed that the expression of VvPPR genes in roots was higher than stems and leaves under NAA, SA, ABA, MeJA and GA3 treatments. VvPPR8 was significantly up-regulated after GA3 and MeJA treatment for 24h, VvPPR53 was significantly up-regulated after SA, NAA, ABA and MeJA treatment. In addition, In grape leaves, VvPPR53 was up-regulated under PEG, Nacl and 4℃ treatments. These data indicate that VvPPR gene family members are responsive to hormones and abiotic stresses, and that there are some differences in the degree of response and expression in different grape tissues. This study provides a certain theoretical basis for grape resistance breeding.

The UDP-glycosyltransferase MtUGT84A1 regulates anthocyanin accumulation and plant growth via JA signaling in Medicago truncatula

Anthocyanins confer plants beneficial qualities and play protective roles under extreme light conditions. Here, we characterized a novel UDP flavonoid 3- O -glucosyltransferase ( UFGT ) gene MtUGT84A1 and demonstrated its involvement in anthocyanin accumulation and feedback inhibition of JA signaling in M. truncatula. The loss-of-function mutants mtugt84a1–1 and mtugt84a1–2 exhibited emerald hypocotyls and dwarfism. In vitro and in vivo assays revealed that MtUGT84A1 glycosylated peonidin and delphinidin at the 3-OH position. Constitutive expression of MtUGT84A1 rescued the phenotypic abnormalities of mtugt84a1 and significantly increased the anthocyanin content in the MtUGT84A1 -overexpression plants. Moreover, in mtugt84a1 , the contents of JA and JA isoleucine (JA-Ile) were doubled. The transcription level of MtMYC2 was significantly increased in mtugt84a1 , while decreased in the MtUGT84A1- overexpression plants, indicating MtUGT84A1 inhibits JA pathway. Under high-intensity light or MeJA treatment, MtUGT84A1 was elevated causing drastic accumulation of anthocyanins in the wildtype, but the pigments in mtugt84a1 did not alter clearly. Therefore, MtUGT84A1 not only functions as a structural gene for the final step of anthocyanin biosynthesis, but also regulates JA signaling by feedback inhibition. The findings unveil the critical roles of MtUGT84A1 -modulated glycosylation of anthocyanidins and shed light on the feedback regulatory loop of MtUGT84A1 in JA-mediated biosynthesis of natural products. • MtUGT84A1 encodes UDP flavonoid 3- O -glucosyltransferase. • MtUGT84A1 glucosylates peonidin and delphinidin at the 3-OH position. • MtUGT84A1 regulates JA signaling by feedback inhibition. • MtUGT84A1 modulates both anthocyanin accumulation and shoot growth of M. truncatula.

Molecular Cloning and Functional Analysis of IrUGT86A1-like Gene in Medicinal Plant Isodon rubescens (Hemsl.) Hara.

The synthesis of secondary metabolites in plants often includes glycosylation modifications. Often, the final step of constructing plant secondary metabolites is completed by glycosylation transferases, which are also involved in many cell processes. In this study, a UDP-glycosyltransferase gene (UGT) was amplified from Isodon rubescens (Hemsl.) Hara with RT-PCR and named IrUGT86A1-like (GenBank: MZ913258). Here, we found that IrUGT86A1-like gene is 1450 bp in length and encodes for 479 amino acids. Bioinformatics analysis revealed that IrUGT86A1-like is a stable and hydrophilic protein, located in the cytoplasm with a transmembrane domain. Phylogenetic analysis showed that IrUGT86A1-like protein has the closest genetic relationship with the UDP-glycosyltransferase 86A1-like protein (XP_042054241.1) of Salvia splendens. RT-qPCR analysis demonstrated that the expression of IrUGT86A1-like gene varied in different tissues; leaves had the highest expression followed by flowers, stems, and roots had the lowest expression. This expression trend is similar to the distribution of oridonin content in different tissues of I. rubescens. Additionally, IrUGT86A1-like gene was found to be positively enhanced by NaCl and MeJA treatment, and in contrast was down-regulated by ABA treatment. Finally, the prokaryotic expression vector pEASY®-Blunt E1-IrUGT86A1 was successfully used to express about 53 KD of IrUGT86A1-like protein. This research builds a foundation for further investigation on the function of this gene in the synthesis and modification of secondary metabolites.

Genome-Wide Identification and Characterization of G2-Like Transcription Factor Genes in Moso Bamboo (Phyllostachys edulis).

G2-like (GLK) transcription factors contribute significantly and extensively in regulating chloroplast growth and development in plants. This study investigated the genome-wide identification, phylogenetic relationships, conserved motifs, promoter cis-elements, MCScanX, divergence times, and expression profile analysis of PeGLK genes in moso bamboo (Phyllostachys edulis). Overall, 78 putative PeGLKs (PeGLK1–PeGLK78) were identified and divided into 13 distinct subfamilies. Each subfamily contains members displaying similar gene structure and motif composition. By synteny analysis, 42 orthologous pairs and highly conserved microsynteny between regions of GLK genes across moso bamboo and maize were found. Furthermore, an analysis of the divergence times indicated that PeGLK genes had a duplication event around 15 million years ago (MYA) and a divergence happened around 38 MYA between PeGLK and ZmGLK. Tissue-specific expression analysis showed that PeGLK genes presented distinct expression profiles in various tissues, and many members were highly expressed in leaves. Additionally, several PeGLKs were significantly up-regulated under cold stress, osmotic stress, and MeJA and GA treatment, implying that they have a likelihood of affecting abiotic stress and phytohormone responses in plants. The results of this study provide a comprehensive understanding of the moso bamboo GLK gene family, as well as elucidating the potential functional characterization of PeGLK genes.

Identification of the Citrus Superoxide Dismutase Family and Their Roles in Response to Phytohormones and Citrus Bacterial Canker

Superoxide dismutases (SODs) play critical roles in plants, especially in the maintenance of redox homeostasis. The response of SODs in Citrus (Citrus sinensis (L.) Osbeck) to citrus bacterial canker (CBC) infection were investigated. The CsSODs were identified, and their gene structures, phylogeny, conserved domains and motifs, predicted interactions, and chromosomal distribution were analyzed. CsSOD expression in response to stress-related plant hormones (salicylic acid, SA; methyl jasmonate, MeJA; and abscisic acid, ABA) and Xanthomonas citri subsp. citri (Xcc) infection were also investigated. Thirteen CsSODs were identified in C. sinensis, including four Fe/MnSODs and nine Cu/ZnSODs with typical functional domains. The CsSODs were distributed on chromosomes 3, 5, 7, and 8. Specific hormone-response motifs were identified in the gene promoter regions. Ten genes were induced by MeJA treatment, as shown by qRT-PCR, and were upregulated in the CBC-susceptible Wanjincheng citrus variety, while CsSOD06 and CsSOD08 were upregulated by ABA in both the Wanjincheng and the CBC-resistant Kumquat varieties. Xcc infection significantly altered the levels of most CsSODs. The overexpression of CsSOD06 and CsSOD08 resulted in increased hydrogen peroxide levels and SOD activity. Our findings highlight the significance of SOD enzymes in the plant response to pathogen infection and have a potential application for breeding CBC-tolerant citrus varieties.

Genome-wide analysis of basic helix-loop-helix genes in Dendrobium catenatum and functional characterization of DcMYC2 in jasmonate-mediated immunity to Sclerotium delphinii.

Dendrobium catenatum, belonging to the Orchidaceae, is a precious Chinese herbal medicine. Sclerotium delphinii (P1) is a broad-spectrum fungal disease, which causes widespread loss in the near-wild cultivation of D. catenatum. Thus, resistance breeding of D. catenatum has become the key to solve this problem. The basic helix–loop–helix (bHLH) gene family is closely related to plant resistance to external stresses, but the related research in D. catenatum is not deep enough yet. Phylogenetic analysis showed that 108 DcbHLH genes could be divided into 23 subgroups. Promoter cis-acting elements revealed that DcbHLHs contain a large number of stress-related cis-acting elements. Transcriptome analysis of MeJA and P1 treatment manifested that exogenous MeJA can change the expression pattern of most bHLH genes, especially the IIIe subgroup, including inhibiting the expression of DcbHLH026 (MYC2a) and promoting the expression of DcbHLH027 (MYC2b). Subcellular localization indicated that they were located in the nucleus. Furthermore, exogenous MeJA treatment significantly delayed disease time and reduced lesion size after infection with P1. DcMYC2b-overexpression Arabidopsis lines showed significantly smaller lesions after being infected with P1 than the wild type, indicating that DcMYC2b functions as an important positive regulator in D. catenatum defense against P1. Our findings shed more insights into the critical role of the DcbHLH family in plants and the resistance breeding of D. catenatum.

Elicitor Induced JA-Signaling Genes Are Associated with Partial Tolerance to Hemibiotrophic Pathogen Phytophthora capsici in Capsicum chinense

Phytophthora capsici causes root and stem rot disease in Capsicum. However, molecular mechanisms underlying this pathosystem are little known. The use of elicitors as tools that trigger defense responses to biotic stresses to study molecular plant defense has increased. In this study, early defense induced in the susceptible cultivar C. chinense using three elicitors to assess its role during interaction with hemibiotrophic P. capsici. The response to infection by phenotypic analyses across the time during disease development in seedlings treated with elicitors was compared. Likewise; defense-gene expression were investigated by qRT-PCR. A total of five resistance genes were used as markers of signaling pathways mediated by jasmonate/ethylene (JA/ET) and salicylic acid (SA). Further, six R genes analogs (CcRGAS) related to oomycete-defense were employed. The results showed that elicitors MeJA and b-aminobutyric acid (BABA) slightly reduced disease symptoms. Moreover, MeJA or BABA treatments followed by challenge with P. capsici up-regulated the expression level of genes related to the JA/ET signaling pathway (CcLOX2, CcPDF1 and CcETR1). Furthermore, MeJA treatment followed by challenge triggered a significant induction of de CcRGAS and CcRPP13 expression within 24 h of inoculation. This suggests that in the early defense mechanisms against P. capsici JA signaling plays an important role.

A Tomato Putative Metalloprotease SlEGY2 Plays a Positive Role in Thermotolerance

Intramembrane proteases play very important roles in plants, such as chloroplast development, flower morphology, and response to abiotic stress. In this study, a putative metalloprotease gene, homologous to Ethylene-dependent Gravitropism deficient and Yellow-green2 (EGY2) of Arabidopsis, was isolated from tomato (Solanum lycopersicum) plants and named SlEGY2. We found that SlEGY2 was a member of the metalloprotease family M50 which contained conserved motifs HEXXH and NPDG and was localized in the chloroplast. SlEGY2 antisense transgenic tomato plants (AS) have similar hypocotyls phenotype to the Arabidopsis egy2 mutant. Heat (42 °C), PEG, ABA and MeJA treatments can upregulate the expression of SlEGY2. Under heat stress, SlEGY2 AS lines are more sensitive, with more water loss (lower fresh weight), seriously damaged membrane, and ROS accumulation, but lower activities of APX and CAT. In addition, suppression of SlEGY2 decreases the content of chlorophyll and photosynthetic activities, especially photosystem II. These results suggest that SlEGY2 can regulate the thermotolerance of tomatoes by affecting ROS accumulation and photosynthetic activities.

Transcriptome analysis reveals regulation mechanism of methyl jasmonate-induced terpenes biosynthesis in Curcuma wenyujin.

Curcuma wenyujin is the source plant of three traditional Chinese medicines, which have been widely used in clinical treatment over 1000 years. The content of terpenes, the major medicinal active ingredients, is relatively low in this plant. Studies have shown that MeJA can promote terpenes biosynthesis in plants. However, the mechanism underlying the effect of MeJA in C. wenyujin remains unclear. In this work, the transcriptome of C. wenyujin leaves with MeJA treatment was analyzed to elucidate the regulation mechanism of MeJA-mediated terpene biosynthesis. Based on the RNA-seq data, 7,246 unigenes were differentially expressed with MeJA treatment. Expression pattern clustering of DEGs revealed that unigenes, related to JA biosynthesis and signal transduction, responded to exogenous MeJA stimulation on the early stage and maintained throughout the process. Subsequently, unigenes related to terpene biosynthesis pathway showed a significant up-regulation with 6 h treatment. The analysis results suggested that MeJA induced the expression of JA biosynthesis genes (such as LOXs, AOSs, AOCs, OPRs, and MFPs) and JA signal transduction core genes (JAZs and MYCs) to activate JA signaling pathway. Meanwhile, downstream JA-responsive genes presented up-regulated expression levels such as AACT, HMGSs, HMGRs, DXSs, DXRs, MCTs, HDSs, and HDRs, thus promoting terpenes biosynthesis. The transcriptional expressions of these genes were validated by qRT-PCR. In addition, six CwTPS genes in response to MeJA were identified. With MeJA treatment, the expression levels of CwTPSs were increased as well as those of the transcription factors MYB, NAC, bZIP, WRKY, AP2/ERF, and HLH. These TFs might potentially regulate terpenes biosynthesis. These results provide insights for regulation mechanism of terpenes biosynthesis.

Integrated metabolomics and transcriptome analysis on flavonoid biosynthesis in flowers of safflower (Carthamus tinctorius L.) during colour-transition.

BackgroundSafflower (Carthamus tinctorius L.), well known for its flower, is widely used as a dye and traditional Chinese medicine. Flavonoids, especially flavonoid glycosides, are the main pigments and active components. However, their biosynthesis is largely unknown. Interestingly, the colour of flowers in safflower changed from yellow to red during flower development, while much of the gene and chemical bases during colour transition are unclear.MethodsIn this research, widely targeted metabolomics and transcriptomics were used to elucidate the changes in flavonoid biosynthesis from the gene and chemical points of view in flowers of safflower during colour transition. The screening of differential metabolites depended on fold change and variable importance in project (VIP) value. Differential expressed genes (DEGs) were screened by DESeq2 method. RT-PCR was used to analyse relative expressions of DEGs.ResultsA total of 212 flavonoid metabolites, including hydroxysafflor yellow A, carthamin and anthocyanins, were detected and showed a large difference. The candidate genes of glycosyltransferases and flavonoid hydroxylase that might participate in flavonoid glycoside biosynthesis were screened. Ten candidate genes were screened. Through integrated metabolomics and transcriptome analysis, a uridine diphosphate glucose glycosyltransferase gene, CtUGT9 showed a significant correlation with flavonoid glycosides in safflower. In addition, expression analysis showed that CtUGT9 was mainly expressed in the middle development of flowers and was significantly upregulated under MeJA treatment. Our results indicated that CtUGT9 might play an important role in flavonoid glycoside biosynthesis during colour-transition in safflower.

Investigation of Brazilian grape juice metabolomic profile changes caused by methyl jasmonate pre‐harvest treatment

SummaryGrape juice is a major source of potential health‐promoting bioactive polyphenols, especially for children and those who do not consume wine. Since the subtropical climate may negatively affect the concentrations of grape polyphenols, especially anthocyanins, elicitors such as methyl jasmonate (MeJa) could be used to promote polyphenol biosynthesis. This work aimed at investigating the impact of MeJa treatment on grape juice produced via a traditional low‐cost process from two Vitis labrusca cultivars and in two Brazilian regions. The untargeted LC–MS analytical protocol demonstrated that Isabel Precoce juices strongly benefited from MeJa treatment, especially regarding their anthocyanic profile, regardless of the cultivation region. Known MeJa markers in wine and V. vinifera grapes (flavanols, flavonols and stilbenes) in this experiment had mixed behaviours depending on the region/variety/cultivation. Moreover, it was found that all the detected hydroxycinnamates were influenced by the treatment, especially the concentration of their glucosides, which was increased. Glutathione, 2‐S‐glutathionyl caftaric acid and indole lactic acid glucoside were identified for the first time as MeJa treatment biomarkers in grape products, indicating a possible positive effect on juice antioxidant properties.

Characterization and Functional Analysis of GhNAC82, A NAM Domain Gene, Coordinates the Leaf Senescence in Upland Cotton (Gossypium hirsutum L.).

In the process of growth and development, cotton exhibits premature senescence under various abiotic stresses, impairing yield and fiber quality. NAC (NAM, ATAF1,2, and CUC2) protein widely distributed in land plants, play the critical role in responding to abiotic stress and regulating leaf senescence. We have identified and functional analyzed a NAM domain gene GhNAC82 in upland cotton, it was located on the A11 chromosome 4,921,702 to 4,922,748 bp, only containing one exon. The spatio-temporal expression pattern analysis revealed that it was highly expressed in root, torus, ovule and fiber development stage. The results of qRT-PCR validated that GhNAC82 negatively regulated by salt stress, drought stress, H2O2 stress, IAA treatment, and ethylene treatment, positively regulated by the ABA and MeJA treatment. Moreover, heterologous overexpression of GhNAC82 results in leaf premature senescence and delays root system development in Arabidopsis thaliana. The phenotype of delayed-senescence was performed after silencing GhNAC82 by VIGS in premature cotton. Taken together, GhNAC82 was involved in different abiotic stress pathways and play important roles in negatively regulating leaf premature senescence.

Genome-Wide Characterization of the Methyl CpG Binding Domain-Containing Proteins in Watermelon and Functional Analysis of Their Roles in Disease Resistance Through Ectopic Overexpression in Arabidopsis thaliana.

Methyl-CPG-Binding Domain (MBD) proteins play important roles in plant growth, development, and stress responses. The present study characterized the MBD families in watermelon and other cucurbit plants regarding the gene numbers and structures, phylogenetic and syntenic relationships, evolution events, and conserved domain organization of the MBD proteins. The watermelon ClMBD proteins were found to be localized in nucleus, and ClMBD2 and ClMBD3 interacted with ClIDM2 and ClIDM3. ClMBD2 bound to DNA harboring methylated CG sites but not to DNA with methylated CHG and CHH sites in vitro. The ClMBD genes exhibited distinct expression patterns in watermelon plants after SA and MeJA treatment and after infection by fungal pathogens Fusarium oxysporum f.sp. niveum and Didymella bryoniae. Overexpression of ClMBD2, ClMBD3, or ClMBD5 in Arabidopsis resulted in attenuated resistance against Botrytis cinerea, accompanied by down-regulated expression of AtPDF1.2 and increased accumulation of H2O2 upon B. cinerea infection. Overexpression of ClMBD1 and ClMBD2 led to down-regulated expression of AtPR1 and decreased resistance while overexpression of ClMBD5 resulted in up-regulated expression of AtPR1 and increased resistance against Pseudomonas syringae pv. tomato DC3000. Transcriptome analysis revealed that overexpression of ClMBD2 in Arabidopsis up-regulated the expression of a small set of genes that negatively regulate Arabidopsis immunity. These data suggest the importance of some ClMBD genes in plant immunity and provide the possibility to improve plant immunity through modification of specific ClMBD genes.

Integrative Analysis of Elicitor-Induced Camptothecin Biosynthesis in Camptotheca acuminata Plantlets Through a Combined Omics Approach.

Treatments with abiotic elicitors can efficiently induce the accumulation of specialized metabolites in plants. We used a combined omics approach to analyze the elicitation effects of MeJa, AgNO3, and PEG on camptothecin (CPT) biosynthesis in Camptotheca acuminata plantlets. Untargeted analyses revealed that treatments with MeJa, AgNO3, and PEG significantly inhibited the photosynthetic pathway and promoted carbon metabolism and secondary metabolic pathways. The CPT levels increased by 78.6, 73.3, and 50.0% in the MeJa, AgNO3, and PEG treatment groups, respectively. Using C. acuminata plantlets after elicitation treatment, we mined and characterized 15 new alkaloids, 25 known CPT analogs and precursors, 9 iridoid biosynthetic precursors, and 15 tryptamine biosynthetic precursors based on their MS/MS fragmentation spectra. Using 32 characterized genes involved in CPT biosynthesis as bait, we mined 12 prioritized CYP450 genes from the 416 CYP450 candidates that had been identified based on co-expression analysis, conserved domain analysis, and their elicitation-associated upregulation patterns. This study provides a comprehensive perspective on CPT biosynthesis in C. acuminata plantlets after abiotic elicitation. The findings enable us to elucidate the previously unexplored CYP450-mediated oxidation steps for CPT biosynthesis.

Methyl Jasmonate Activates the 2C Methyl-D-erithrytol 2,4-cyclodiphosphate Synthase Gene and Stimulates Tanshinone Accumulation in Salvia miltiorrhiza Solid Callus Cultures.

The present study characterizes the 5′ regulatory region of the SmMEC gene. The isolated fragment is 1559 bp long and consists of a promoter, 5′UTR and 31 nucleotide 5′ fragments of the CDS region. In silico bioinformatic analysis found that the promoter region contains repetitions of many potential cis-active elements. Cis-active elements associated with the response to methyl jasmonate (MeJa) were identified in the SmMEC gene promoter. Co-expression studies combined with earlier transcriptomic research suggest the significant role of MeJa in SmMEC gene regulation. These findings were in line with the results of the RT-PCR test showing SmMEC gene expression induction after 72 h of MeJa treatment. Biphasic total tanshinone accumulation was observed following treatment of S. miltiorrhiza solid callus cultures with 50–500 μM methyl jasmonate, with peaks observed after 10–20 and 50–60 days. An early peak of total tanshinone concentration (0.08%) occurred after 20 days of 100 μM MeJa induction, and a second, much lower one, was observed after 50 days of 50 μM MeJa stimulation (0.04%). The dominant tanshinones were cryptotanshinone (CT) and dihydrotanshinone (DHT). To better understand the inducing effect of MeJa treatment on tanshinone biosynthesis, a search was performed for methyl jasmonate-responsive cis-active motifs in the available sequences of gene proximal promoters associated with terpenoid precursor biosynthesis. The results indicate that MeJa has the potential to induce a significant proportion of the presented genes, which is in line with available transcriptomic and RT-PCR data.

Identification and Functional Analysis of Two Alcohol Dehydrogenase Genes Involved in Catalyzing the Reduction of (Z)-3-Hexenal into (Z)-3-Hexenol in Tea Plants (Camellia sinensis).

Alcohol dehydrogenase (ADH) is a vital enzyme in the biosynthesis pathway of six-carbon volatiles in plants. However, little is known about its functions in tea plants. Here, we identified two ADH genes (CsADH1 and CsADH2). An in vitro protein expression assay showed that both CsADH1 and CsADH2 proteins can catalyze the reduction of (Z)-3-hexenal into (Z)-3-hexenol. Subcellular localization revealed that both CsADH1 and CsADH2 proteins were predominantly localized in the nucleus and cytosol. CsADH1 had high transcripts in young stems in autumn, while CsADH2 showed extremely high expression levels in stems and roots. The expression of CsADH2 was mainly downregulated under ABA treatment, while CsADH1 and CsADH2 transcripts were significantly lower under MeJA treatment at 12 and 24 h. Under cold treatment, CsADH1 transcripts first decreased and then increased, while CsADH2 demonstrated an almost opposite expression pattern. Notably, CsADH2 was significantly upregulated under simulated Ectropis obliqua invasion. Gene suppression by antisense oligonucleotides (AsODNs) demonstrated that AsODN_ADH2 treatment significantly reduced CsADH2 transcripts and the abundance of (Z)-3-hexenol products. The results indicate that the two CsADH genes may play an important role in response to (a)biotic stresses and in the process of (Z)-3-hexenol biosynthesis.