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Anthocyanins in maize (Zea mays L.) kernels determine the plant's color and can enhance its resistance. Selenium (Se) significantly impacts plant growth, development, and secondary metabolic regulation. However, the molecular mechanisms by which Se regulates anthocyanin synthesis in waxy corn remain unclear. This study employed integrated transcriptomic and metabolomic analyses to investigate the mechanisms through which selenium influences anthocyanin synthesis in yellow and purple waxy corn. The results showed that maize varieties with higher anthocyanin content had higher selenium enrichment capacity in their kernels. Under selenium stress, HN2025 exhibited 1,904 more differentially expressed genes (DEGs) and 140 more differential metabolites compared to HN5. The expression levels of anthocyanin synthesis-related genes and transcription factors such as phenylalanine ammonia-lyase, flavonoid 3-hydroxylase (F3H), dihydroflavonol reductase (DFR), chalcone synthase (CHS), cinnamate-4-hydroxylase (C4H), anthocyanin 5,3-Oglucosyltransferases, and anthocyanidin reductase, MYB, and bHLH were strongly induced in HN2025. Metabolomic analysis revealed significant enrichment in anthocyanin biosynthesis, flavonoid and flavonol biosynthesis, glutathione metabolism, phenylalanine biosynthesis, and phenylalanine metabolism under selenium treatment. Three up-regulated PAL genes and one C4H gene were significantly enriched with DAMs in phenylalanine metabolism, phenylpropanoid biosynthesis, flavonoid biosynthesis, and anthocyanin biosynthesis, resulting in significant differences between HN5 and HN2025 in selenium-induced anthocyanin metabolism-related pathways. These findings provide a theoretical basis for understanding the effects of selenium on the molecular regulatory mechanisms of anthocyanin biosynthesis in maize kernels. [ABSTRACT FROM AUTHOR]

Camellia hainanica is one of the camellia plants distributed in tropical regions, and its regeneration system and genetic transformation are affected by callus browning. However, the underlying mechanism of Camellia hainanica callus browning formation remains largely unknown. To investigate the metabolic basis and molecular mechanism of the callus browning of Camellia hainanica, histological staining, high-throughput metabolomics, and transcriptomic assays were performed on calli with different browning degrees (T1, T2, and T3). The results of histological staining revealed that the brown callus cells had obvious lignification and accumulation of polyphenols. Widely targeted metabolomics revealed 1190 differentially accumulated metabolites (DAMs), with 53 DAMs annotated as phenylpropanoids and flavonoids. Comparative transcriptomics revealed differentially expressed genes (DEGs) of the T2 vs. T1 associated with the biosynthesis and regulation of flavonoids and transcription factors in Camellia hainanica. Among them, forty-four enzyme genes associated with flavonoid biosynthesis were identified, including phenylalaninase (PAL), 4-coumaroyl CoA ligase (4CL), naringenin via flavanone 3-hydroxylase (F3H), flavonol synthase (FLS), Chalcone synthase (CHS), Chalcone isomerase (CHI), hydroxycinnamoyl-CoA shikimate transferase (HCT), Dihydroflavonol reductase (DFR), anthocyanin reductase (LAR), anthocyanin synthetase (ANS), and anthocyanin reductase (ANR). Related transcription factors R2R3-MYB, basic helix-loop-helix (bHLH), and WRKY genes also presented different expression patterns in T2 vs. T1. These results indicate that the browning of calli in Camellia hainanica is regulated at both the transcriptional and metabolic levels. The oxidation of flavonoids and the regulation of related structural genes and transcription factors are crucial decisive factors. This study preliminarily revealed the molecular mechanism of the browning of the callus of Camellia hainanensis, and the results can provide a reference for the anti-browning culture of Camellia hainanica callus. [ABSTRACT FROM AUTHOR]

Jasmonates, including jasmonic acid (JA) and its derivatives such as methyl jasmonate (MeJA) or jasmonly isoleucine (JA-Ile), regulate plant responses to various biotic and abiotic stresses. In this study, we applied exogenous MeJA onto Senna tora leaves subjected to wounding and conducted a transcriptome deep sequencing analysis at 1 (T1), 3 (T3), 6 (T6), and 24 (T24) h after MeJA induction, along with the pretreatment control at 0 h (T0). Out of 18,883 mapped genes, we identified 10,048 differentially expressed genes (DEGs) between the T0 time point and at least one of the four treatment times. We detected the most DEGs at T3, followed by T6, T1, and T24. We observed the upregulation of genes related to JA biosynthesis upon exogenous MeJA application. Similarly, transcript levels of genes related to flavonoid biosynthesis increased after MeJA application and tended to reach their maximum at T6. In agreement, the flavonols kaempferol and quercetin reached their highest accumulation at T24, whereas the levels of the anthraquinones aloe-emodin, emodin, and citreorosein remained constant until T24. This study highlights an increase in flavonoid biosynthesis following both MeJA application and mechanical wounding, whereas no significant influence is observed on anthraquinone biosynthesis. These results provide insights into the distinct regulatory pathways of flavonoid and anthraquinone biosynthesis in response to MeJA and mechanical wounding. [ABSTRACT FROM AUTHOR]

Gene innovation plays an essential role in trait evolution. Rhizobial symbioses, the most important N2‐fixing agent in agricultural systems that exists mainly in Leguminosae, is one of the most attractive evolution events. However, the gene innovations underlying Leguminosae root nodule symbiosis (RNS) remain largely unknown. Here, we investigated the gene gain event in Leguminosae RNS evolution through comprehensive phylogenomic analyses. We revealed that Leguminosae‐gain genes were acquired by gene duplication and underwent a strong purifying selection. Kyoto Encyclopedia of Genes and Genomes analyses showed that the innovated genes were enriched in flavonoid biosynthesis pathways, particular downstream of chalcone synthase (CHS). Among them, Leguminosae‐gain type Ⅱ chalcone isomerase (CHI) could be further divided into CHI1A and CHI1B clades, which resulted from the products of tandem duplication. Furthermore, the duplicated CHI genes exhibited exon–intron structural divergences evolved through exon/intron gain/loss and insertion/deletion. Knocking down CHI1B significantly reduced nodulation in Glycine max (soybean) and Medicago truncatula; whereas, knocking down its duplication gene CHI1A had no effect on nodulation. Therefore, Leguminosae‐gain type Ⅱ CHI participated in RNS and the duplicated CHI1A and CHI1B genes exhibited RNS functional divergence. This study provides functional insights into Leguminosae‐gain genetic innovation and sub‐functionalization after gene duplication that contribute to the evolution and adaptation of RNS in Leguminosae. [ABSTRACT FROM AUTHOR]

To reveal the effect of sucrose concentration on the production of secondary metabolites, a metabolome and transcriptome joint analysis was carried out using callus induced from grape variety Mio Red cambial meristematic cells. We identified 559 metabolites—mainly flavonoids, phenolic acids, and stilbenoids—as differential content metabolites (fold change ≥2 or ≤0.5) in at least one pairwise comparison of treatments with 7.5, 15, or 30 g/L sucrose in the growing media for 15 or 30 days (d). Resveratrol, viniferin, and amurensin contents were highest at 15 d of subculture; piceid, ampelopsin, and pterostilbene had higher contents at 30 d. A transcriptome analysis identified 1310 and 498 (at 15 d) and 1696 and 2211 (at 30 d) differentially expressed genes (DEGs; log2(fold change) ≥ 1, p < 0.05) in 7.5 vs. 15 g/L and 15 vs. 30 g/L sucrose treatments, respectively. In phenylpropane and isoflavone pathways, DEGs encoding cinnamic acid 4-hydroxylase, chalcone synthase, chalcone isomerase, and flavanone 3-hydroxylase were more highly expressed at 15 d than at 30 d, while other DEGs showed different regulation patterns corresponding to sucrose concentrations and cultivation times. For all three sucrose concentrations, the stilbene synthase (STS) gene exhibited significantly higher expression at 15 vs. 30 d, while two resveratrol O-methyltransferase (ROMT) genes related to pterostilbene synthesis showed significantly higher expression at 30 vs. 15 d. In addition, a total of 481 DEGs were annotated as transcription factors in pairwise comparisons; an integrative analysis suggested MYB59, WRKY20, and MADS8 as potential regulators responding to sucrose levels in flavonoid and stilbene biosynthesis in grape callus. Our results provide valuable information for high-efficiency production of flavonoids and stilbenes using grape callus. [ABSTRACT FROM AUTHOR]

Cotton aphids (Aphis gossypii Glover) cause harm by feeding on phloem sap and spreading plant viruses to lily. Understanding the mechanisms by which aphids infest lily plants is crucial for effective aphid management and control. In this study, we investigated the activity of antioxidants, integrated nontargeted metabolomes and transcriptomes of lilies infested by cotton aphids to explore the changes in lily leaves. Overall, the results indicated that the catalase (CAT) activity in the leaves of the lily plants was greater than that in the leaves of the control plants. A comprehensive identification of 604 substances was conducted in the leaves. Furthermore, the differentially abundant metabolite analysis revealed the enrichment of phenylalanine metabolism and α-linolenic acid metabolism. Moreover, 3574 differentially expressed genes (DEGs), whose expression tended to increase, were linked to glutathione metabolism and phenylpropanoid biosynthesis. In addition, the integrated analysis revealed that the defensive response of lily leaves to aphids is manifested through antioxidant reactions, phenylpropane and flavonoid biosynthesis, and α-linolenic acid metabolism. Finally, the key metabolites were CAT, glutathione, coumaric acid, and jasmonic acid, along with the key genes chalcone synthase (CHS), phenylalanine ammonia-lyase (PAL), and 12-oxo-phytodienoic acid reductase (OPR). Accordingly, the findings of this research elucidate the molecular and metabolic reactions of A. gossypii in lily plants, offering valuable insights for developing aphid resistance strategies in lily farming. [ABSTRACT FROM AUTHOR]

Sunburn stress is one of the main environmental stress factors that seriously affects the fruit development and quality of Chinese olive, a tropical and subtropical fruit in south China. Therefore, the understanding of the changes in physiological, biochemical, metabolic, and gene expression in response to sunburn stress is of great significance for the industry and breeding of Chinese olive. In this study, the different stress degrees of Chinese olive fruits, including serious sunburn injury (SSI), mild sunburn injury (MSI), and ordinary (control check, CK) samples, were used to identify the physiological and biochemical changes and explore the differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) by using transcriptomics and metabolomics. Compared with CK, the phenotypes, antioxidant capacity, and antioxidant-related enzyme activities of sunburn stress samples changed significantly. Based on DEG-based KEGG metabolic pathway analysis of transcriptomics, the polyphenol and flavonoid-related pathways, including phenylpropanoid biosynthesis, sesquiterpenoid, and triterpenoid biosynthesis, monoterpene biosynthesis, carotenoid biosynthesis, isoflavonoid biosynthesis, flavonoid biosynthesis, were enriched under sunburn stress of Chinese olive. Meanwhile, 33 differentially accumulated polyphenols and 99 differentially accumulated flavonoids were identified using metabolomics. According to the integration of transcriptome and metabolome, 15 and 8 DEGs were predicted to regulate polyphenol and flavonoid biosynthesis in Chinese olive, including 4-coumarate-CoA ligase (4CL), cinnamoyl-CoA reductase (CCR), cinnamoyl-alcohol dehydrogenase (CAD), chalcone synthase (CHS), flavanone-3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS). Additionally, the content of total polyphenols and flavonoids was found to be significantly increased in MSI and SSI samples compared with CK. Our research suggested that the sunburn stress probably activates the transcription of the structural genes involved in polyphenol and flavonoid biosynthesis in Chinese olive fruits to affect the antioxidant capacity and increase the accumulation of polyphenols and flavonoids, thereby responding to this abiotic stress. [ABSTRACT FROM AUTHOR]

Abstract Background Polyketide synthases (PKSs) are classified into three types based on their enzyme structures. Among them, type III PKSs, catalyzing the iterative condensation of malonyl-coenzyme A (CoA) with a CoA-linked starter molecule, are important synthases of valuable natural products. However, low efficiency and byproducts formation often limit their applications in recombinant overproduction. Results Herein, a rapid growth selection system is designed based on the accumulation and derepression of toxic acyl-CoA starter molecule intermediate products, which could be potentially applicable to most type III polyketides biosynthesis. This approach is validated by engineering both chalcone synthases (CHS) and host cell genome, to improve naringenin productions in Escherichia coli. From directed evolution of key enzyme CHS, beneficial mutant with ~ threefold improvement in capability of naringenin biosynthesis was selected and characterized. From directed genome evolution, effect of thioesterases on CHS catalysis is first discovered, expanding our understanding of byproduct formation mechanism in type III PKSs. Taken together, a whole-cell catalyst producing 1082 mg L−1 naringenin in flask with E value (evaluating product specificity) improved from 50.1% to 96.7% is obtained. Conclusions The growth selection system has greatly contributed to both enhanced activity and discovery of byproduct formation mechanism in CHS. This research provides new insights in the catalytic mechanisms of CHS and sheds light on engineering highly efficient heterologous bio-factories to produce naringenin, and potentially more high-value type III polyketides, with minimized byproducts formation.

Melatonin (Mt) functions as a growth regulator and multifunctional signaling molecule in plants, thereby playing a crucial role in promoting growth and orchestrating protective responses to various abiotic stresses. However, the mechanism whereby exogenous Mt protects Lonicera japonica Thunb. (L. japonica) against salt stress has not been fully elucidated. Therefore, this study aimed to elucidate how exogenous Mt alleviates sodium chloride (NaCl) stress in L. japonica seedlings. Salt-sensitive L. japonica seedlings were treated with an aqueous solution containing 150 mM of NaCl and aqueous solutions containing various concentrations of Mt. The results revealed that treatment of NaCl-stressed L. japonica seedlings with a 60 µM aqueous solution of Mt significantly enhanced vegetative plant growth by scavenging reactive oxygen species and thus reducing oxidative stress. The latter was evidenced by decreases in electrical conductivity and malondialdehyde (MDA) concentrations. Moreover, Mt treatment led to increases in the NaCl-stressed L. japonica seedlings' total chlorophyll content, soluble sugar content, and flavonoid content, demonstrating that Mt treatment improved the seedlings' tolerance of NaCl stress. This was also indicated by the NaCl-stressed L. japonica seedlings exhibiting marked increases in the activities of antioxidant enzymes (superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase) and in photosynthetic functions. Furthermore, Mt treatment of NaCl-stressed L. japonica seedlings increased their expression of phenylalanine ammonia-lyase 1 (PAL1), phenylalanine ammonia-lyase 2 (PAL2), calcium-dependent protein kinase (CPK), cinnamyl alcohol dehydrogenase (CAD), flavanol synthase (FLS), and chalcone synthase (CHS). In conclusion, our results demonstrate that treatment of L. japonica seedlings with a 60 µM aqueous solution of Mt significantly ameliorated the detrimental effects of NaCl stress in the seedlings. Therefore, such treatment has substantial potential for use in safeguarding medicinal plant crops against severe salinity. [ABSTRACT FROM AUTHOR]

SUMMARY: Due to the chelation of phosphorus in the soil, it becomes unavailable for plant growth and development. The mechanisms by which phosphorus‐solubilizing bacteria activate immobilized phosphorus to promote the growth and development of woody plants, as well as the intrinsic molecular mechanisms, are not clear. Through the analysis of microbial communities in the rhizosphere 16S V3–V4 and a homologous gene encoding microbial alkaline phosphomonoesterase (phoD) in phosphate‐efficient (PE) and phosphate‐inefficient apple rootstocks, it was found that PE significantly enriched beneficial rhizobacteria. The best phosphorus‐solubilizing bacteria, Bacillus sp. strain 7DB1 (B2), was isolated, purified, and identified from the rhizosphere soil of PE rootstocks. Incubating with Bacillus B2 into the rhizosphere of apple rootstocks significantly increased the soluble phosphorus and flavonoid content in the rhizosphere soil. Simultaneously, this process stimulates the root development of the rootstocks and enhances plant phosphorus uptake. After root transcriptome sequencing, candidate transcription factor MhMYB15, responsive to Bacillus B2, was identified through heatmap and co‐expression network analysis. Yeast one‐hybrid, electrophoretic mobility shift assay, and LUC assay confirmed that MhMYB15 can directly bind to the promoter regions of downstream functional genes, including chalcone synthase MhCHS2 and phosphate transporter MhPHT1;15. Transgenic experiments with MhMYB15 revealed that RNAi‐MhMYB15 silenced lines failed to induce an increase in flavonoid content and phosphorus levels in the roots under the treatment of Bacillus B2, and plant growth was slower than the control. In conclusion, MhMYB15 actively responds to Bacillus B2, regulating the accumulation of flavonoids and the uptake of phosphorus, thereby influencing plant growth and development. Significance Statement: In the rhizosphere soil of apple rootstocks on different phosphorus‐efficient/inefficient rootstocks, there exist distinct microbial communities. MhMYB15 can respond to the rhizobacterium Bacillus B2, and interact with flavonoids (MhCHS2) and phosphate transport proteins (MhPHT1;15) to promote plant root development and phosphate absorption. [ABSTRACT FROM AUTHOR]

The application of double-stranded RNAs (dsRNAs) to plant surfaces has emerged as a promising tool for manipulating gene expression in plants and pathogens, offering new opportunities for crop improvement. While research has shown the capability of exogenous dsRNAs to silence genes, the full spectrum of their impact, particularly on the intricate network of microRNAs (miRNAs), remains largely unexplored. Here, we show that the exogenous application of chalcone synthase (CHS)-encoding dsRNA to the rosette leaves of Arabidopsis thaliana induced extensive alterations in the miRNA profile, while non-specific bacterial neomycin phosphotransferase II (NPTII) dsRNA had a minimal effect. Two days after treatment, we detected 60 differentially expressed miRNAs among the 428 miRNAs found in the A. thaliana genome. A total of 59 miRNAs were significantly changed after AtCHS-dsRNA treatment compared with water and NPTII-dsRNA, and 1 miRNA was significantly changed after AtCHS-dsRNA and NPTII-dsRNA compared with the water control. A comprehensive functional enrichment analysis revealed 17 major GO categories enriched among the genes potentially targeted by the up- and downregulated miRNAs. These categories included processes such as aromatic compound biosynthesis (a pathway directly related to CHS activity), heterocycle biosynthesis, RNA metabolism and biosynthesis, DNA transcription, and plant development. Several predicted targets of upregulated and downregulated miRNAs, including APETALA2, SCL27, SOD1, GRF1, AGO2, PHB, and PHV, were verified by qRT-PCR. The analysis showed a negative correlation between the expression of miRNAs and the expression of their predicted targets. Thus, exogenous plant gene-specific dsRNAs induce substantial changes in the plant miRNA composition, ultimately affecting the expression of a wide range of genes. These findings have profound implications for our understanding of the effects of exogenously induced RNA interference, which can have broader effects beyond targeted mRNA degradation, affecting the expression of other genes through miRNA regulation. [ABSTRACT FROM AUTHOR]

Naringenin is a plant polyphenol, widely explored due to its interesting biological activities, namely anticancer, antioxidant, and anti-inflammatory. Due to its potential applications and attempt to overcome the industrial demand, there has been an increased interest in its heterologous production. The microbial biosynthetic pathway to produce naringenin is composed of tyrosine ammonia-lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI). Herein, we targeted the efficient de novo production of naringenin in Escherichia coli by performing a step-by-step validation and optimization of the pathway. For that purpose, we first started by expressing two TAL genes from different sources in three different E. coli strains. The highest p-coumaric acid production (2.54 g/L) was obtained in the tyrosine-overproducing M-PAR-121 strain carrying TAL from Flavobacterium johnsoniae (FjTAL). Afterwards, this platform strain was used to express different combinations of 4CL and CHS genes from different sources. The highest naringenin chalcone production (560.2 mg/L) was achieved by expressing FjTAL combined with 4CL from Arabidopsis thaliana (At4CL) and CHS from Cucurbita maxima (CmCHS). Finally, different CHIs were tested and validated, and 765.9 mg/L of naringenin was produced by expressing CHI from Medicago sativa (MsCHI) combined with the other previously chosen genes. To our knowledge, this titer corresponds to the highest de novo production of naringenin reported so far in E. coli. Key points: • Best enzyme and strain combination were selected for de novo naringenin production. • After genetic and operational optimizations, 765.9 mg/L of naringenin was produced. • This de novo production is the highest reported so far in E. coli. [ABSTRACT FROM AUTHOR]

Phenylpropanoid metabolism plays an important role in cantaloupe ripening and senescence, but the mechanism of ozone regulation on phenylpropanoid metabolism remains unclear. This study investigated how ozone treatment modulates the levels of secondary metabolites associated with phenylpropanoid metabolism, the related enzyme activities, and gene expression in cantaloupe. Treating cantaloupes with 15 mg/m3 of ozone after precooling can help maintain postharvest hardness. This treatment also enhances the production and accumulation of secondary metabolites, such as total phenols, flavonoids, and lignin. These metabolites are essential components of the phenylpropanoid metabolic pathway, activating enzymes like phenylalanine ammonia‐lyase, cinnamate 4‐hydroxylase, 4CL, chalcone synthase, and chalcone isomerase. The results of the transcriptional expression patterns showed that differential gene expression related to phenylpropanoid metabolism in the peel of ozone‐treated cantaloupes was primarily observed during the middle and late storage stages. In contrast, the pulp exhibited significant differential gene expression mainly during the early storage stage. Furthermore, it was observed that the level of gene expression in the peel was generally higher than that in the pulp. The correlation between the relative amount of gene changes in cantaloupe, activity of selected enzymes, and concentration of secondary metabolites could be accompanied by positive regulation of the phenylpropanoid metabolic pathway. Therefore, ozone stress induction positively enhances the biosynthesis of flavonoids in cantaloupes, leading to an increased accumulation of secondary metabolites. Additionally, it also improves the postharvest storage quality of cantaloupes. [ABSTRACT FROM AUTHOR]

Rhododendron simsii Planchon is an important ornamental species in the northern hemisphere. Flower color is an important objective of Rhododendron breeding programs. However, information on anthocyanin synthesis in R. simsii is limited. In this research, the regulatory mechanism of anthocyanin biosynthesis in R. simsii was performed through the integrated analysis of metabolome and RNA-seq. A total of 805 and 513 metabolites were screened by positive and negative ionization modes, respectively, In total, 79 flavonoids contained seven anthocyanidins, 42 flavanones, 10 flavans, 13 flavones, and seven flavonols. Methylated and glycosylated derivatives took up the most. Differentially accumulated metabolites were mainly involved in "flavone and flavonol biosynthesis", "cyanoamino acid metabolism", "pyrimidine metabolism", and "phenylalanine metabolism" pathways. For flavonoid biosynthesis, different expression of shikimate O-hydroxycinnamoyltransferase, caffeoyl-CoA O-methyltransferase, flavonoid 3′-monooxygenase, flavonol synthase, dihydroflavonol 4-reductase/flavanone 4-reductase, F3′5′H, chalcone synthase, leucoanthocyanidin reductase, and 5-O-(4-coumaroyl)-D-quinate 3′-monooxygenase genes ultimately led to different accumulations of quercetin, myricetin, cyanidin, and eriodictyol. In flavone and flavonol biosynthesis pathway, differential expression of F3′5′H, flavonoid 3′-monooxygenase and flavonol-3-O-glucoside/galactoside glucosyltransferase genes led to the differential accumulation of quercetin, isovitexin, and laricitrin. This research will provide a biochemical basis for further modification of flower color and genetic breeding in R. simsii and related Rhododendron species. [ABSTRACT FROM AUTHOR]

To explore the regulatory mechanism of endogenous hormones in the synthesis of anthocyanins in Anoectochilus roxburghii (Wall.) Lindl (A. roxburghii) under different light intensities, this study used metabolomics and transcriptomics techniques to identify the key genes and transcription factors involved in anthocyanin biosynthesis. We also analyzed the changes in and correlations between plant endogenous hormones and anthocyanin metabolites under different light intensities. The results indicate that light intensity significantly affects the levels of anthocyanin glycosides and endogenous hormones in leaves. A total of 38 anthocyanin-related differential metabolites were identified. Under 75% light transmittance (T3 treatment), the leaves exhibited the highest anthocyanin content and differentially expressed genes such as chalcone synthase (CHS), flavonol synthase (FLS), and flavonoid 3′-monooxygenase (F3′H) exhibited the highest expression levels. Additionally, 13 transcription factors were found to have regulatory relationships with 7 enzyme genes, with 11 possessing cis-elements responsive to plant hormones. The expression of six genes and two transcription factors was validated using qRT-PCR, with the results agreeing with those obtained using RNA sequencing. This study revealed that by modulating endogenous hormones and transcription factors, light intensity plays a pivotal role in regulating anthocyanin glycoside synthesis in A. roxburghii leaves. These findings provide insights into the molecular mechanisms underlying light-induced changes in leaf coloration and contribute to our knowledge of plant secondary metabolite regulation caused by environmental factors. [ABSTRACT FROM AUTHOR]

Although nitrogen deficiency and sucrose are linked to anthocyanin synthesis, the potential role of sucrose in regulating anthocyanin biosynthesis under low nitrogen conditions (LN) in purple lettuce (Lactuca sativa L.) remains unclear. We found that adding exogenous sucrose enhanced anthocyanin biosynthesis but significantly inhibited lettuce growth at high concentrations. Optimal results were obtained using 1 mmol/L sucrose in a low-nitrogen nutrient solution (LN + T1). Chlorophyll fluorescence imaging indicated that the addition of exogenous sucrose induced mild stress. Meanwhile, the activities of antioxidant enzymes (SOD, CAT, and POD) and antioxidant capacity were both enhanced. The mild stress activated the antioxidant system, thereby promoting the accumulation of anthocyanins induced by exogenous sucrose. LN + T1 (low nitrogen nutrient solution supplemented with 1 mmol/L sucrose) up-regulated enzyme genes in the biosynthetic pathway of anthocyanins, including phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), dihydroflavonol reductase (DFR), flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), flavone synthase II (FNSII), and anthocyanidin synthase (ANS). Additionally, various transcription factors such as AP2/ERF, MYB, bHLH, C2H2, NAC, C2C2, HB, MADS, bZIP, and WRKY were found to be up-regulated. This study elucidates the regulatory mechanism of anthocyanin metabolism in response to the addition of exogenous sucrose under low nitrogen conditions and provides a nutrient solution formula to enhance anthocyanin content in modern, high-quality agricultural cultivation. [ABSTRACT FROM AUTHOR]

4-methyl-5-vinylthiazole (MVT) is a significant volatile of caladium (Caladium bicolor) which produces a very high level of thiamin thiazole synthase (THI4) in male flowers. We explored transcriptomes upregulating MVT using RNA-seq during the six developmental stages of the male flower (Day−10 to Day0) in C. bicolor 'Tapestry'. THI4 was the highest transcript throughout the male flower development. Additionally, the genes showing the high expression associated with floral volatiles of caladium on Day0 were trans-resveratrol di-O-methyltransferase (ROMT), chalcone synthase (CHS), 3-ketoacyl-CoA thiolase 2 (KAT2), and linalool synthase (TPS). These four genes correspond to the following elevated volatiles of caladium: 1,3,5-trimethoxybenzene, MVT, indole, methyl salicylate, and linalool on Day0 compared to Day−10. The upstream THI4 gene was cloned to drive a fluorescent gene (ZsGreen1) in transient and stable transgenic petunia and tobacco plants, showing the gene expression only in the male tissue. The tissue-specific expression of the caladium THI4 promoter could benefit crop production with minimal modification of plants. Investigating transcriptomes associated with caladium fragrance can help provide insight into understanding the regulatory mechanisms of floral volatiles of caladium. [ABSTRACT FROM AUTHOR]

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Naturally synthesized secondarymetabolites in plants are considered an important source of drugs, food additives, etc. Among them, research on natural plant medicinal components and their synthesis mechanisms has always been of high concern. We identified a novel medicinal floral crop, Plumbago auriculata L., that can be treated with methyl jasmonate (MeJA) for the rapid or sustainable production of natural bioactives from hairy roots. In the study, we globally analyzed the changes in the accumulation of plumbagin and others in the hairy roots of Plumbago auriculata L. hairy roots (PAHR) 15834 in P. auriculata L. based on 100 mmol/L of MeJA treatment by RNA-seq profiling, and we found that there was a significant increase in the accumulation of plumbagin and saponin before 24 h. To explain the principle of co-accumulation, it showed thatMeJA induced JA signaling and the shikimic acid pathway, and the methylvaleric acid (MVA) pathway was activated downstream subsequently by the Mfuzz and weighted gene coexpression analysis. Under the shared metabolic pathway, the high expression of PAL3 and HMGR promoted the activity of the "gateway enzymes" phenylalanine ammonia lyase (PAL) and 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), which respectively induced the high expression of key reaction enzyme genes, including chalcone synthase (CHS), isopentenyl diphosphate (IPP), and farnesyl pyrophosphate synthase (FPS), that led to the synthesis of plumbagin and saponin. We speculated that large amounts of ketones and/or aldehydes were formed under the action of these characteristic enzymes, ultimately achieving their coaccumulation through polyketone and high-level sugar and amino acid metabolism. The study results provided a theoretical basis for carrying out the factory refinement and biosynthesis of plumbagin and saponins and also provided new ideas for fully exploiting multifunctional agricultural crops and plants and developing new agricultural by-products. [ABSTRACT FROM AUTHOR]

The application of phosphorus-solubilizing bacteria is an effective method for increasing the available phosphorus content and inhibiting wheat uptake of heavy metals. However, further research is needed on the mechanism by which phosphorus-solubilizing bacteria inhibit cadmium (Cd) uptake in wheat roots and its impact on the expression of root-related genes. Here, the effects of strain Klebsiella aerogenes M2 on Cd absorption in wheat and the expression of root-related Cd detoxification and immobilization genes were determined. Compared with the control, strain M2 reduced (64.1–64.6%) Cd uptake by wheat roots. Cd fluorescence staining revealed that strain M2 blocked the entry of exogenous Cd into the root interior and enhanced the immobilization of Cd by cell walls. Forty-seven genes related to Cd detoxification, including genes encoding peroxidase, chalcone synthase, and naringenin 3-dioxygenase, were upregulated in the Cd+M2 treatment. Strain M2 enhanced the Cd resistance and detoxification activity of wheat roots through the regulation of flavonoid biosynthesis and antioxidant enzyme activity. Moreover, strain M2 regulated the expression of genes related to phenylalanine metabolism and the MAPK signaling pathway to enhance Cd immobilization in roots. These results provide a theoretical basis for the use of phosphorus-solubilizing bacteria to remediate Cd-contaminated fields and reduce Cd uptake in wheat. [ABSTRACT FROM AUTHOR]

Background: Polyketide synthases (PKSs) are classified into three types based on their enzyme structures. Among them, type III PKSs, catalyzing the iterative condensation of malonyl-coenzyme A (CoA) with a CoA-linked starter molecule, are important synthases of valuable natural products. However, low efficiency and byproducts formation often limit their applications in recombinant overproduction. Results: Herein, a rapid growth selection system is designed based on the accumulation and derepression of toxic acyl-CoA starter molecule intermediate products, which could be potentially applicable to most type III polyketides biosynthesis. This approach is validated by engineering both chalcone synthases (CHS) and host cell genome, to improve naringenin productions in Escherichia coli. From directed evolution of key enzyme CHS, beneficial mutant with ~ threefold improvement in capability of naringenin biosynthesis was selected and characterized. From directed genome evolution, effect of thioesterases on CHS catalysis is first discovered, expanding our understanding of byproduct formation mechanism in type III PKSs. Taken together, a whole-cell catalyst producing 1082 mg L−1 naringenin in flask with E value (evaluating product specificity) improved from 50.1% to 96.7% is obtained. Conclusions: The growth selection system has greatly contributed to both enhanced activity and discovery of byproduct formation mechanism in CHS. This research provides new insights in the catalytic mechanisms of CHS and sheds light on engineering highly efficient heterologous bio-factories to produce naringenin, and potentially more high-value type III polyketides, with minimized byproducts formation. [ABSTRACT FROM AUTHOR]

Simple Summary: The presence of secondary metabolites like flavonoids, alkaloids, and phenolic compounds in white mulberry (Morus alba) make it an ornamental tree mainly in the Asian subcontinent. These secondary metabolites are synthesized through various biosynthetic pathways. Flavonoids like rutin, quercetin, and kaempferol are produced via the phenylpropanoid and flavonoid biosynthetic pathways. The key enzymes responsible for this pathway include phenylalanine ammonia-lyase, chalcone synthase, and flavonoid hydroxylases. Phenolic compounds like resveratrol and oxyresveratrol are produced via the phenylpropanoid pathway using phenylalanine as a precursor. Presences of these secondary metabolites, like in the case of flavonoids (rutin and quercetin), in the mulberry tree have potential biological activities like antioxidant, anti-inflammatory and neuroprotective effects. Also, they have been found to have great medicinal importance, for example in treating diabetes, obesity and neurodegenerative diseases. Also, phenolic compounds like reservertrol exhibit cardio-protective, anticancer, and anti-aging properties. Traditional natural products have been the focus of research to explore their medicinal properties. One such medicinally important plant is the white mulberry, Morus alba, widely distributed in the Asian subcontinent. It is one of the most cultivated species of mulberry tree and has attracted more focus from researchers because of its abundance in phytochemicals as well as multipurpose uses. The leaves, fruits and other parts of the white mulberry plant act as a source of valuable bioactive compounds like flavonoids, phenolic acids, terpenoids and alkaloids. These secondary metabolites have manifold healthy uses as they possess antioxidant, anti-inflammatory, antidiabetic, neutrotrophic, and anticancer properties. Despite the increasing scientific interest in this plant, there are very few reviews that highlight the phytochemistry and biological potential of white mulberry for biomedical research. To this end, this review elaborates the phytochemistry, biosynthetic pathways and pharmacological activities of the glycoside flavonoids of Morus alba. A comprehensive analysis of the available literature indicates that Morus alba could emerge as a promising natural agent to combat diverse conditions including diabetes, cancer, inflammation and infectious diseases. To achieve such important objectives, it is crucial to elucidate the biosynthesis and regulation mechanisms of the bioactive compounds in white mulberry as well as the multifaceted pharmacological effects attributed to this plant resource. The present review paper is intended to present a summary of existing scientific data and a guide for further research in the phytochemistry and pharmacology of white mulberry. Further, a biosynthetic pathway analysis of the glycoside flavonoid in mulberry is also given. Lastly, we discuss the pros and cons of the current research to ensure the prudent and effective therapeutic value of mulberry for promoting human and animal health. [ABSTRACT FROM AUTHOR]

The purpose of this study was to explore the effects of different LED light quality on the germination of alfalfa seeds and the content of photosynthetic pigments and phenolic compounds in seedlings, so as to provide a basis for further light quality control technology for the sprout vegetable production and industrial production of alfalfa plants. [Methods] The effects of 10 different LED light quality(red light R660 and R630, blue light B450 and B465, green light G520, yellow light Y590, red and blue combination R5B2, R1B1 and R2B5, white light W)on seed germination, seedling growth, anatomy, and phenolic compound synthesis of alfalfa‘WL656'were studied using dark treatment (D) as the control. [Results] Different treatments had no effect on the germination rate, germination potential and germination index of alfalfa seeds. Among monochromatic light treatments, both pure red lights promoted radicle elongation of alfalfa seedlings; B465 treatment significantly increased the stem diameter, epidermal cell thickness, cortical thickness, and individual vessel area of the hypocotyl of seedlings, and B450 significantly increased the diameter of vascular bundles. Different monochromatic light had no significant effect on chlorophyll a content, while the chlorophyll b content was lowest under two different blue light treatments, and the carotenoid content was highest under R660 treatment. Both pure blue lights promoted the accumulation of anthocyanins, total phenols and flavonoids in seedlings, but seedlings contained almost no anthocyanins under G520, Y590 and D treatments. R630 increased the chalcone synthase (CHS) activity in the hypocotyl, and G520 significantly increased the CHS activity in cotyledons. W increased the anthocyanin synthase (ANS) activity in the hypocotyl of seedlings, and Y590 increased the ANS activity in seedlings. Among the three combinations of red and blue light, with the decrease of red light ratio, stem diameter, cortical thickness and CHS activity in hypocotyl decreased, the content of root length, epidermal cell thickness, vascular bundle diameter, and anthocyanins of hypocotyls decreased first and then increased, and the content of photosynthetic pigments and CHS activity in seedling cotyledons increased. The content of total phenols and flavonoids in hypocotyl was highest under R2B5 treatment, and there was no difference in the content of three phenolic compounds in cotyledons. ANS activity in hypocotyl was the highest under R1B1 treatment, and ANS activity in cotyledon was the highest under R5B2 treatment. [Conclusions] Pure red light promoted seedling elongation after seed germination, pure blue light promoted the formation of anthocyanins, flavonoids, and total phenolsin seedlings, and the influence of red and blue combination light on the physiological characteristics of seedlings was more complicated. Taking into account the growth and physiological indicators, this study preliminarily believed that R2B5 treatment was a suitable LED light quality for the growth of alfalfa seedlings as it had moderate growth, high chlorophyll and phenolic content. [ABSTRACT FROM AUTHOR]

The flavonoids in citrus fruits are crucial physiological regulators and natural bioactive products of high pharmaceutical value. Melatonin is a pleiotropic hormone that can regulate plant morphogenesis and stress resistance and alter the accumulation of flavonoids in these processes. However, the direct effect of melatonin on citrus flavonoids remains unclear. In this study, nontargeted metabolomics and transcriptomics were utilized to reveal how exogenous melatonin affects flavonoid biosynthesis in "Bingtangcheng" citrus fruits. The melatonin treatment at 0.1 mmol L−1 significantly increased the contents of seven polymethoxylated flavones (PMFs) and up-regulated a series of flavonoid pathway genes, including 4CL (4-coumaroyl CoA ligase), FNS (flavone synthase), and FHs (flavonoid hydroxylases). Meanwhile, CHS (chalcone synthase) was down-regulated, causing a decrease in the content of most flavonoid glycosides. Pearson correlation analysis obtained 21 transcription factors co-expressed with differentially accumulated flavonoids, among which the AP2/EREBP members were the most numerous. Additionally, circadian rhythm and photosynthesis pathways were enriched in the DEG (differentially expressed gene) analysis, suggesting that melatonin might also mediate changes in the flavonoid biosynthesis pathway by affecting the fruit's circadian rhythm. These results provide valuable information for further exploration of the molecular mechanisms through which melatonin regulates citrus fruit metabolism. [ABSTRACT FROM AUTHOR]

Cold response in plants is mediated by metabolic adjustments of hormones and defense systems which support survival, growth, and crop productivity. Hence, a comparative analysis of metaboliteschangewas conducted in conjunction with oxidative damages in two chickpea (Cicer arietinum L.) genotypes differing in cold-tolerance (Sel96th11439 and ILC533) during coldstress (4°C). In sensitive genotype, cold stress increased H2O2 and MDA contents by 47 and 57%, respectively, without any significant changes in tolerant genotype. During stress, unlike the tolerant genotype, the growth of sensitive genotype was markedly inhibited (by 11%) compared to control conditions. During the initial stages of cold responses, ABA content in tolerant genotype reached its peak, showing 77% increase 3 days post stress (dps), whereas the sensitive genotype showed 20% raise 6 dps. Gibberellin (GA) content in the tolerant genotype was 16% higher than the sensitive genotype 1 dps. Compared to control conditions, indole acetic acid (IAA) content attained its maximum level in tolerant and sensitive genotypes at 1 and 6 dps, respectively. 15% increase in phenol compoundsin tolerant genotype was concomitant with heightened antioxidant capacity, as well as increased in flavonoid and anthocyanin contents by 46, 75 and 200% respectively. At 6 dps, a significant increase in transcript levels of chalcone synthase (15.3-fold), phenylalanine ammonia-lyase (3.5-fold), and DELLA (4.2-fold) genes were observed in tolerant genotype at 6 dps. It can be concluded that ability to develop defense responses towards cold stress was related to integrating time-dependent co-regulation patterns of hormone-metabolites with effective stability of plant pigments and growth. [ABSTRACT FROM AUTHOR]

The current study sought to determine the effects of Colpomenia sinuosa and Sargassum linifolium aqueous extracts on non-enzymatic and enzymatic antioxidants in Spinacia oleracea L. leaf extract. Glutathione synthase (GSS, EC: 6.3.2.3) and -glutamyl cysteine synthetase (-GCS, EC: 6.3.2.2) are involved in the glutathione biosynthesis process. Treatment of S. oleracea with seaweed extracts increased the level of reduced glutathione (GSH), oxidized glutathione (GSSG), and total glutathione at lower concentrations. Total phenols and total flavonoids in S. oleracea leaves accumulated more rapidly after treatment with seaweed extracts. The activity of the enzyme’s phenylalanine ammonia lyase (PAL, EC: 4.3.1.25), chalcone synthase (CHS, EC: 2.3. 1.74), and chalcone isomerase (CHI, EC: 5.5.1.6) involved in the production of phenylpropanoid and flavonoid in S. oleracea leaves rose in a dose-dependent manner. Glutathione reductase (GR, EC 1.6.4.2), glutathione peroxidase (GPX, EC: 1.11.1.9), and glutathione-S-transferase (GST, EC: 2.5.1.18) are examples of antioxidant enzymes whose activity were also elevated by the treatment. In comparison to untreated plants, the treated plants' S. oleracea leaf extract significantly reduced superoxide anion, 1, 1-diphenyl-2-picrylhydrazyl (DPPH), and hydroxyl radicals. In light of this, the current findings imply that the use of seaweed extracts was found to boost the activity of the antioxidant system in Spinacea oleracea [ABSTRACT FROM AUTHOR]

Key message: Through a map-based cloning approach, a gene coding for an R2R3-MYB transcription factor was identified as a causal gene for theI locus controlling the dominant white bulb color in onion. White bulb colors in onion (Allium cepa L.) are determined by either the C or I loci. The causal gene for the C locus was previously isolated, but the gene responsible for the I locus has not been identified yet. To identify candidate genes for the I locus, an approximately 7-Mb genomic DNA region harboring the I locus was obtained from onion and bunching onion (A. fistulosum) whole genome sequences using two tightly linked molecular markers. Within this interval, the AcMYB1 gene, known as a positive regulator of anthocyanin production, was identified. No polymorphic sequences were found between white and red AcMYB1 alleles in the 4,860-bp full-length genomic DNA sequences. However, a 4,838-bp LTR-retrotransposon was identified in the white allele, in the 79-bp upstream coding region from the stop codon. The insertion of this LTR-retrotransposon created a premature stop codon, resulting in the replacement of 26 amino acids with seven different residues. A molecular marker was developed based on the insertion of this LTR-retrotransposon to genotype the I locus. A perfect linkage between bulb color phenotypes and marker genotypes was observed among 5,303 individuals of segregating populations. The transcription of AcMYB1 appeared to be normal in both red and white onions, but the transcription of CHS-A, which encodes chalcone synthase and is involved in the first step of the anthocyanin biosynthesis pathway, was inactivated in the white onions. Taken together, an aberrant AcMYB1 protein produced from the mutant allele might be responsible for the dominant white bulb color in onions. [ABSTRACT FROM AUTHOR]

Chalcone synthase (CHS) and chalcone isomerase (CHI) catalyze the first two committed steps of the flavonoid pathway that plays a pivotal role in the growth and reproduction of land plants, including UV protection, pigmentation, symbiotic nitrogen fixation, and pathogen resistance. Based on the obtained X-ray crystal structures of CHS, CHI, and chalcone isomerase-like protein (CHIL) from the same monocotyledon, Panicum virgatum, along with the results of the steady-state kinetics, spectroscopic/thermodynamic analyses, intermolecular interactions, and their effect on each catalytic step are proposed. In addition, PvCHI's unique activity for both naringenin chalcone and isoliquiritigenin was analyzed, and the observed hierarchical activity for those type-I and -II substrates was explained with the intrinsic characteristics of the enzyme and two substrates. The structure of PvCHS complexed with naringenin supports uncompetitive inhibition. PvCHS displays intrinsic catalytic promiscuity, evident from the formation of p-coumaroyltriacetic acid lactone (CTAL) in addition to naringenin chalcone. In the presence of PvCHIL, conversion of p-coumaroyl-CoA to naringenin through PvCHS and PvCHI displayed ~400-fold increased Vmax with reduced formation of CTAL by 70%. Supporting this model, molecular docking, ITC (Isothermal Titration Calorimetry), and FRET (Fluorescence Resonance Energy Transfer) indicated that both PvCHI and PvCHIL interact with PvCHS in a non-competitive manner, indicating the plausible allosteric effect of naringenin on CHS. Significantly, the presence of naringenin increased the affinity between PvCHS and PvCHIL, whereas naringenin chalcone decreased the affinity, indicating a plausible feedback mechanism to minimize spontaneous incorrect stereoisomers. These are the first findings from a three-body system from the same species, indicating the importance of the macromolecular assembly of CHS-CHI-CHIL in determining the amount and type of flavonoids produced in plant cells. [ABSTRACT FROM AUTHOR]

Ginkgo is an economic tree species with high medicinal value, and flavonols are its main medicinal components. This research was conducted to investigate the molecular mechanism underlying the influence of Ultraviolet A (UVA) treatment on the synthesis of ginkgo flavonols with the aim of increasing their content. Ginkgo full-sib hybrid offspring were used as test materials. The phenylalanine ammonialyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate: CoA ligase (4CL) enzyme activities, as well as flavonol contents, were measured under the same intensity of white light (300 μmol·m−2·s−1) with the addition of 20, 40, and 60 μmol·m−2·s−1 UVA separately after 20 days of treatment. The control check (CK) and treatment with the highest flavonol content were chosen for transcriptome sequencing analysis. The results showed that the PAL, C4H, and 4CL enzyme activities, as well as the flavonol and totalflavonol glycoside contents, of ginkgo hybrid progeny differed significantly under different UVA treatments. They showed a tendency to increase and then decrease, reaching a maximum value under UVA-4 (40 μmol·m−2·s−1 ultraviolet UVA light intensity) treatment. Ribonucleic acid (RNA) sequencing revealed the presence of 4165 genes with differential expression, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the metabolic pathways commonly enriched across all four comparison groups included 'phenylpropanoid biosynthesis', while the pathways commonly enriched in green-leaf ginkgo UVA-4 treatment (TL), yellow-leaf ginkgo mutant CK treatment (CKY), and green-leaf ginkgo CK treatment (CKL) were related to 'flavonoid biosynthesis'. Treatment with UVA light led to the increased expression of PAL and 4CL enzymes in the phenylpropanoid biosynthesis pathway, as well as increased expression of chalcone synthase (CHS), Flavanone 3-hydroxylase (F3H), and flavonol synthase (FLS) enzymes in the flavonoid biosynthesis pathway, thereby promoting the synthesis of ginkgo flavonols. In summary, the use of 40 μmol·m−2·s−1 UVA treatment for 20 days significantly increased the flavonol content and the expression of related enzyme genes in ginkgo hybrid offspring, enhancing ginkgo flavonoids and increasing the medicinal value of ginkgo. [ABSTRACT FROM AUTHOR]

Abstract Background This study aimed to investigate the alterations in biochemical and physiological responses of oat plants exposed to antimony (Sb) contamination in soil. Specifically, we evaluated the effectiveness of an arbuscular mycorrhizal fungus (AMF) and olive mill waste (OMW) in mitigating the effects of Sb contamination. The soil was treated with a commercial strain of AMF (Rhizophagus irregularis) and OMW (4% w/w) under two different levels of Sb (0 and 1500 mg kg−1 soil). Results The combined treatment (OMW + AMF) enhanced the photosynthetic rate (+ 40%) and chlorophyll a (+ 91%) and chlorophyll b (+ 50%) content under Sb condition, which in turn induced more biomass production (+ 67–78%) compared to the contaminated control plants. More photosynthesis in OMW + AMF-treated plants gives a route for phenylalanine amino acid synthesis (+ 69%), which is used as a precursor for the biosynthesis of secondary metabolites, including flavonoids (+ 110%), polyphenols (+ 26%), and anthocyanins (+ 63%) compared to control plants. More activation of phenylalanine ammonia-lyase (+ 38%) and chalcone synthase (+ 26%) enzymes in OMW + AMF-treated plants under Sb stress indicated the activation of phenylpropanoid pathways in antioxidant metabolites biosynthesis. There was also improved shifting of antioxidant enzyme activities in the ASC/GSH and catalytic pathways in plants in response to OMW + AMF and Sb contamination, remarkably reducing oxidative damage markers. Conclusions While individual applications of OMW and AMF also demonstrated some degree of plant tolerance induction, the combined presence of AMF with OMW supplementation significantly enhanced plant biomass production and adaptability to oxidative stress induced by soil Sb contamination.

Background: The mechanisms by which the apple MdPYL9 gene mediates the response to drought stress remain unclear. Here, transcriptome and metabolome analyses of apple plants under drought were used to investigate the mechanisms by which MdPYL9 regulates the response to drought stress in apple. MdPYL9-overexpressed transgenic and non-transgenic apple histoculture seedlings were rooted, transplanted, and subjected to drought treatments to clarify the mechanisms underlying the responses of apples to drought stress through phenotypic observations, physiological and biochemical index measurements, and transcriptomic and metabolomic analyses. Results: Under drought stress treatment, transgenic plants were less affected by drought stress than non-transgenic plants. Decreases in the net photosynthetic rate, stomatal conductance, and transpiration rate of transgenic apple plants were less pronounced in transgenic plants than in non-transgenic plants, and increases in the intercellular CO2 concentration were less pronounced in transgenic plants than in non-transgenic plants. The relative electrical conductivity and content of malondialdehyde, superoxide anion, and hydrogen peroxide were significantly lower in transgenic plants than in non-transgenic plants, and the chlorophyll content and activities of antioxidant enzymes (superoxide dismutase, peroxidase, and catalase) were significantly higher in transgenic plants than in non-transgenic plants. The number of differentially expressed genes (DEGs) involved in the response to drought stress was lower in transgenic plants than in non-transgenic plants, and the most significant and highly annotated DEGs in the transgenic plants were involved in the flavonoid biosynthesis pathway, and the most significant and highly annotated DEGs in control plants were involved in the phytohormone signal transduction pathway. The number of differentially accumulated metabolites involved in the response to drought stress was lower in transgenic plants than in non-transgenic plants, and up-regulated metabolites were significantly enriched in apigenin-7-O-glucoside in transgenic plants and in abscisic acid in non-transgenic plants. In the flavonoid biosynthetic pathway, the expression of genes encoding chalcone synthase (CHS) and chalcone isomerase (CHI) was more significantly down-regulated in non-transgenic plants than in transgenic plants, and the expression of the gene encoding 4-coumarate-CoA ligase (4CL) was more significantly up-regulated in transgenic plants than in non-transgenic plants, which resulted in the significant up-regulation of apigenin-7-O-glucoside in transgenic plants. Conclusions: The above results indicated that the over-expression of MdPYL9 increased the drought resistance of plants under drought stress by attenuating the down-regulation of the expression of genes encoding CHS and CHI and enhancing the up-regulated expression of the gene encoding 4CL, which enhanced the content of apigenin-7-O-glucoside. [ABSTRACT FROM AUTHOR]

Pinus sibirica is primarily distributed in Siberia. Owing to its excellent cold resistance and development potential, it has become an important introduced tree species in the Greater Xing'an area of China. Pine wilt disease, triggered by the pine wood nematode (PWN, Bursaphelenchus xylophilus), constitutes a profoundly critical affliction within forest ecosystems. Its incidence has extended to the northeastern region of China in recent years. To explore the potential host status of P. sibirica in the Greater Xing'an area for PWN and to elucidate the responses following inoculation, artificial inoculation, transcriptomics, and metabolomics methods were used. In the artificial inoculation experiments, quantitative analysis of nematode populations within the trees demonstrated that PWN exhibited normal growth and reproductive capabilities within P. sibirica. Subsequently, transcriptome and metabolome sequencing were conducted at four time points before disease onset (3-, 5-, 7-, and 9-days post inoculation). Gene trend analysis and differentially expressed gene screening were employed and the results indicated that genes associated with the flavonoid biosynthesis pathway exhibited predominant enrichment among the upregulated genes. Metabolome analysis showed that the abundance of flavonoidrelated metabolites in P. sibirica increased after inoculation with PWN. Integrated analysis of transcriptome and metabolome revealed that after PWN inoculation in P. sibirica, two chalcone synthase (chs) genes and a chalcone isomerase (chi) gene were significantly upregulated, and the upregulation should accumulate naringenin, pinocembrin, and apigenin to help P. sibirica resist infection of PWN. The results suggested that flavonoid biosynthesis pathway continued to respond after P. sibirica was infected with PWN and played an important role in the interaction between P. sibirica and PWN. [ABSTRACT FROM AUTHOR]

Nitrogen is one of the important factors restricting the development of sesame planting and industry in China. Cultivating sesame varieties tolerant to low nitrogen is an effective way to solve the problem of crop nitrogen deficiency. To date, the mechanism of low nitrogen tolerance in sesame has not been elucidated at the transcriptional level. In this study, two sesame varieties Zhengzhi HL05 (ZZ, nitrogen efficient) and Burmese prolific (MD, nitrogen inefficient) in low nitrogen were used for RNA-sequencing. A total of 3964 DEGs (differentially expressed genes) and 221 DELs (differentially expressed lncRNAs) were identified in two sesame varieties at 3d and 9d after low nitrogen stress. Among them, 1227 genes related to low nitrogen tolerance are mainly located in amino acid metabolism, starch and sucrose metabolism and secondary metabolism, and participate in the process of transporter activity and antioxidant activity. In addition, a total of 209 pairs of lncRNA-mRNA were detected, including 21 pairs of trans and 188 cis. WGCNA (weighted gene co-expression network analysis) analysis divided the obtained genes into 29 modules; phenotypic association analysis identified three low-nitrogen response modules; through lncRNA-mRNA co-expression network, a number of hub genes and cis/trans-regulatory factors were identified in response to low-nitrogen stress including GS1-2 (glutamine synthetase 1–2), PAL (phenylalanine ammonia-lyase), CHS (chalcone synthase, CHS), CAB21 (chlorophyll a-b binding protein 21) and transcription factors MYB54, MYB88 and NAC75 and so on. As a trans regulator, lncRNA MSTRG.13854.1 affects the expression of some genes related to low nitrogen response by regulating the expression of MYB54, thus responding to low nitrogen stress. Our research is the first to provide a more comprehensive understanding of DEGs involved in the low nitrogen stress of sesame at the transcriptome level. These results may reveal insights into the molecular mechanisms of low nitrogen tolerance in sesame and provide diverse genetic resources involved in low nitrogen tolerance research. [ABSTRACT FROM AUTHOR]

Chalcone synthase (CHS) is a key enzyme that catalyzes the first committed step of flavonoid biosynthetic pathway. It plays a vital role not only in maintaining plant growth and development, but also in regulating plant response to environmental hazards. However, the systematic phylogenomic analysis of CHS gene family in a wide range of plant species has not been reported yet. To fill this knowledge gap, a large-scale investigation of CHS genes was performed in 178 plant species covering green algae to dicotyledons. A total of 2,011 CHS and 293 CHS-like genes were identified and phylogenetically divided into four groups, respectively. Gene distribution patterns across the plant kingdom revealed the origin of CHS can be traced back to before the rise of algae. The gene length varied largely in different species, while the exon structure was relatively conserved. Selection pressure analysis also indicated the conserved features of CHS genes on evolutionary time scales. Moreover, our synteny analysis pinpointed that, besides genome-wide duplication and tandem duplication, lineage specific transposition events also occurred in the evolutionary trajectory of CHS gene family. This work provides novel insights into the evolution of CHS gene family and may facilitate further research to better understand the regulatory mechanism of traits relating to flavonoid biosynthesis in diverse plants. [ABSTRACT FROM AUTHOR]

Background: This study aimed to investigate the alterations in biochemical and physiological responses of oat plants exposed to antimony (Sb) contamination in soil. Specifically, we evaluated the effectiveness of an arbuscular mycorrhizal fungus (AMF) and olive mill waste (OMW) in mitigating the effects of Sb contamination. The soil was treated with a commercial strain of AMF (Rhizophagus irregularis) and OMW (4% w/w) under two different levels of Sb (0 and 1500 mg kg−1 soil). Results: The combined treatment (OMW + AMF) enhanced the photosynthetic rate (+ 40%) and chlorophyll a (+ 91%) and chlorophyll b (+ 50%) content under Sb condition, which in turn induced more biomass production (+ 67–78%) compared to the contaminated control plants. More photosynthesis in OMW + AMF-treated plants gives a route for phenylalanine amino acid synthesis (+ 69%), which is used as a precursor for the biosynthesis of secondary metabolites, including flavonoids (+ 110%), polyphenols (+ 26%), and anthocyanins (+ 63%) compared to control plants. More activation of phenylalanine ammonia-lyase (+ 38%) and chalcone synthase (+ 26%) enzymes in OMW + AMF-treated plants under Sb stress indicated the activation of phenylpropanoid pathways in antioxidant metabolites biosynthesis. There was also improved shifting of antioxidant enzyme activities in the ASC/GSH and catalytic pathways in plants in response to OMW + AMF and Sb contamination, remarkably reducing oxidative damage markers. Conclusions: While individual applications of OMW and AMF also demonstrated some degree of plant tolerance induction, the combined presence of AMF with OMW supplementation significantly enhanced plant biomass production and adaptability to oxidative stress induced by soil Sb contamination. [ABSTRACT FROM AUTHOR]

Ginkgo biloba L. is a valuable medicinal plant known for its high content of flavonoids and terpenoids in the leaves of young trees. Pruning can increase leaf yield in ginkgo plantations; however, it is unclear how the intensity of pruning affects leaf yield and quality. In addition, G. biloba exhibits low cutting rooting rates, which limits its efficiency in asexual propagation. In our study, we compared consecutive pruning with varying levels of intensity, including top pruning, light pruning, and heavy pruning, to evaluate the effects of pruning on leaf yield and cutting rooting. The results showed that these three pruning methods all contributed to an increase in the number of new branches, the leaf weight, and the flavonoid content in five-year-old trees. Among them, the effect of light pruning was the best, with a 150% increase in branch number, a 130% increase in leaf weight, and a 40.6% increase in flavonoid content. The secondary pruning further increased leaf area by 22.3%, indicating that secondary pruning further enhanced the rejuvenation of plants and increased leaf yield. At the transcriptional level, pruning can significantly change the expression of genes related to bud sprouting, resulting in a particularly significant increase in SHR expression in the buds. Pruning also promoted the expression of important genes related to flavonoid synthesis, including chalcone synthase (CHS), flavonoid 3′-hydroxylase (F3′H), flavonol synthase (FLS), and dihydroflavonol reductase (DFR). Furthermore, we demonstrated a significant increase in the rooting rate of these second-pruned branch cuttings and screened the optimal hormone ratio for rooting, which is 1.5 μM MeJA + 400 mg/L NAA + 100 mg/L Uniconazole-P. These results suggest that secondary pruning can effectively rejuvenate plants to promote cutting rooting in G. biloba. This method can not only be used to improve the yield and quality of ginkgo leaves, but also for cutting propagation. [ABSTRACT FROM AUTHOR]

Flower color is an important ornamental feature that is often modulated by the contents of flavonoids. Chalcone synthase is the first key enzyme in the biosynthesis of flavonoids, but little is known about the role of R. delavayi CHS in flavonoid biosynthesis. In this paper, three CHS genes (RdCHS1-3) were successfully cloned from R. delavayi flowers. According to multiple sequence alignment and a phylogenetic analysis, only RdCHS1 contained all the highly conserved and important residues, which was classified into the cluster of bona fide CHSs. RdCHS1 was then subjected to further functional analysis. Real-time PCR analysis revealed that the transcripts of RdCHS1 were the highest in the leaves and lowest in the roots; this did not match the anthocyanin accumulation patterns during flower development. Biochemical characterization displayed that RdCHS1 could catalyze p-coumaroyl-CoA and malonyl-CoA molecules to produce naringenin chalcone. The physiological function of RdCHS1 was checked in Arabidopsis mutants and tobacco, and the results showed that RdCHS1 transgenes could recover the color phenotypes of the tt4 mutant and caused the tobacco flower color to change from pink to dark pink through modulating the expressions of endogenous structural and regulatory genes in the tobacco. All these results demonstrate that RdCHS1 fulfills the function of a bona fide CHS and contributes to flavonoid biosynthesis in R. delavayi. [ABSTRACT FROM AUTHOR]

Introduction: The mulberry industry has thrived in China for millennia, offering significant ecological and economic benefits. However, the prevalence of mulberry ring rot disease poses a serious threat to the quality and yield of mulberry leaves. Methods: In this study, we employed a combination of transcriptomic and metabolomic analyses to elucidate the changes occurring at the transcriptional and metabolic levels in Morus notabilis in response to this disease infestation. Key metabolites identified were further validated through in vitro inhibition experiments. Results: The findings revealed significant enrichment in Kyoto Encyclopedia of Genes and Genomes pathways, particularly those related to flavonoid biosynthesis. Notably, naringenin, kaempferol, and quercetin emerged as pivotal players in M. notabilis' defense mechanism against this disease pathogen. The upregulation of synthase genes, including chalcone synthase, flavanone-3- hydroxylase, and flavonol synthase, suggested their crucial roles as structural genes in this process. In vitro inhibition experiments demonstrated that kaempferol and quercetin exhibited broad inhibitory properties, while salicylic acid and methyl jasmonate demonstrated efficient inhibitory effects. Discussion: This study underscores the significance of the flavonoid biosynthesis pathway in M. notabilis' defense response against mulberry ring rot disease, offering a theoretical foundation for disease control measures. [ABSTRACT FROM AUTHOR]

Background: Quinoa leaves demonstrate a diverse array of colors, offering a potential enhancement to landscape aesthetics and the development of leisure-oriented sightseeing agriculture in semi-arid regions. This study utilized integrated transcriptomic and metabolomic analyses to investigate the mechanisms underlying anthocyanin synthesis in both emerald green and pink quinoa leaves. Results: Integrated transcriptomic and metabolomic analyses indicated that both flavonoid biosynthesis pathway (ko00941) and anthocyanin biosynthesis pathway (ko00942) were significantly associated with anthocyanin biosynthesis. Differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) were analyzed between the two germplasms during different developmental periods. Ten DEGs were verified using qRT-PCR, and the results were consistent with those of the transcriptomic sequencing. The elevated expression of phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), 4-coumarate CoA ligase (4CL) and Hydroxycinnamoyltransferase (HCT), as well as the reduced expression of flavanone 3-hydroxylase (F3H) and Flavonol synthase (FLS), likely cause pink leaf formation. In addition, bHLH14, WRKY46, and TGA indirectly affected the activities of CHS and 4CL, collectively regulating the levels of cyanidin 3-O-(3", 6"-O-dimalonyl) glucoside and naringenin. The diminished expression of PAL, 4CL, and HCT decreased the formation of cyanidin-3-O-(6"-O-malonyl-2"-O-glucuronyl) glucoside, leading to the emergence of emerald green leaves. Moreover, the lowered expression of TGA and WRKY46 indirectly regulated 4CL activity, serving as another important factor in maintaining the emerald green hue in leaves N1, N2, and N3. Conclusion: These findings establish a foundation for elucidating the molecular regulatory mechanisms governing anthocyanin biosynthesis in quinoa leaves, and also provide some theoretical basis for the development of leisure and sightseeing agriculture. [ABSTRACT FROM AUTHOR]

"The Genetics of Dominant and Recessive Blossom Traits" by Graham Goode is an article that explores the genetic concepts of dominant and recessive inheritance in African violets. The author discusses Gregor Mendel's experiments with pea plants in the 1860s to understand how traits are passed down from one generation to the next. The article explains that dominant traits are stronger than recessive traits, and provides examples of different blossom colors and their dominant or recessive nature. It also mentions the role of bees in pollinating African violets and how certain flower colors attract them. The article concludes by listing dominant and recessive traits for African violet blossoms. Additionally, the article delves into the chemical pathways and genetic factors that determine the colors and traits of African violets. It explains how certain genes and enzymes influence the production of pigments, such as anthocyanins, which give the flowers their colors. The article also explores the inheritance patterns of these traits and how they can be manipulated through hybridization. It acknowledges that there is still much to learn about the complex genetics and biochemistry of African violets. [Extracted from the article]

Variegated flowers, characterized by the presence of different colors in flowers, have high economic and aesthetic values. The main pigment in the orchid's purple flowers is anthocyanin, while the chalcone synthase (CHS) gene is the key to anthocyanin biosynthesis. Analysis of the CHS gene can reveal some changes, including mutations, in the process of color patterning in flowers. This study aims to determine the structure of the CHS gene related to color patterning in Dendrobium 'Enobi' and Phalaenopsis hybrid with variegated flowers. The methods applied in this study are floral morphology observation, DNA isolation, CHS gene amplification, anthocyanin measurement, and bioinformatic analysis. Morphologically, the variegated pattern has appeared since the flowers were still in the bud on both orchids. Based on the anthocyanin content analysis, the difference in the genus is not directly related to the differences in the flower's anthocyanin content. In addition, the purple zone in the D. 'Enobi' and Phalaenopsis hybrid has a longer fragment of CHS than the white zone. Our analysis suggested several mutations in the white zone and differences in the type and location of several conserved domain proteins. Mutations at the CHS gene fragment might cause decreased anthocyanin pigment formation in the white region. [ABSTRACT FROM AUTHOR]

Background and aims: A greenhouse experiment was conducted to analyze the physiological behavior of Acacia arabica in relation to concentration of phenolic compounds during drought and subsequent recovery. Methods: Seedlings were exposed to water shortage during 32 days and then rehydrated and allowed to recover during 15 days. Parameters related to plant water status, photosynthesis and oxidative stress were quantified after 7, 15 and 32 days of stress, and after 7 and 15 days of recovery. Phenolic acids, flavonoids and enzyme activities involved in their synthesis were quantified. Results: 88% of plants remained alive during the stress period and 10% died during recovery. Drought reduced water and osmotic potentials, stomatal conductance, net photosynthesis, instantaneous transpiration, non-photochemical quenching and flavanols concentration and increased proline, malondialdehyde, phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) activities, DPPH scavenging, hydroxycinnamic acids, hydroxybenzoic acids and flavonols concentration. Genes coding for PAL and chalcone synthase (CHS) were slightly downregulated at the end of drought while gene coding for C4H was upregulated at the beginning of drought. Parameters related to oxidative stress as well as hydroxybenzoic acids and flavonols remained at high values throughout the recovery period. Genes coding for C4H and CHS were up-regulated during recovery. Conclusions: Acacia arabica is resistant to water stress at the seedling stage and able to recover after stress relief. Recovery induces a specific physiological status in terms of transpiration rates and management of oxidative stress in relation to flavonols and hydroxybenzoic acids concentrations. [ABSTRACT FROM AUTHOR]

Cassava is susceptible to mites, especially Tetranychus cinnabarinus. Secondary metabolism products such as flavonoids play an important role as antimicrobial metabolites protecting plants against biotic stressors including fungal, pathogen, bacterial, and pest defense. The chalcone synthase (CHS) is the initial step of the phenylpropanoid pathway for producing flavonoids and is the gatekeeper of the pathway. Until recently, the CHS genes family has not been systematically studied in cassava. Thirty-nine CHS genes were identified from the cassava genome database. Based on phylogenetic and sequence composition analysis, these CHSs were divided into 3 subfamilies. Within the same subfamily, the gene structure and motif compositions of these CHS genes were found to be quite conserved. Duplication events, particularly segmental duplication of the cassava CHS genes, were identified as one of the main driving force of its expansion. Various cis-elements contained in the promoter might regulate the gene expression patterns of MeCHS. Protein-protein interaction (PPI) network analysis showed that MeCHS1 and MeCHS10 protein are more closely related to other family members. The expression of MeCHS genes in young leaves was higher than that in other tissues, and their expression varies even within the same tissue. Coincidentally, these CHS genes of most LAP subclasses were highly expressed in young leaves. The verified MeCHS genes showed consistent with the real-time reverse transcription quantitative PCR (RT-qPCR) and proteomic expression in protected and affected leaves respectively, indicating that these MeCHS genes play crucial roles in the response to T. cinnabarinus. This study is the first to comprehensively expatiate the information on MeCHS family members. These data will further enhance our understanding both the molecular mechanisms and the effects of CHS genes. In addition, the results will help to further clarify the effects on T. cinnabarinus and provide a theoretical basis for the potential functions of the specific CHS gene in resistance to mites and other biotic stress. [ABSTRACT FROM AUTHOR]

Purpose: The utilization of pyrogenic organic matter and organic fertilizers is widespread in agroforestry ecosystems for the purpose of augmenting soil nutrient levels and crop yields. Nonetheless, the impacts of these fertilizers on the yield and quality of Tetrastigma hemsleyanum Diels et Gilg, as well as the underlying mechanisms mediating these processes, remain inadequately understood. Methods: In a potting experiment, we studied the effects of pyrogenic organic matter (20 and 40 mg C g−1 soil) and organic fertilizer (5% and 10%) on the yield and quality of T. hemsleyanum. Soil chemical properties (available K, AK; available P, AP; available N, AN; organic matter, OM), soil enzymes (urease, protease, and cellobiohydrolase), plant growth (plant height, stem thick, and fresh root weight), flavonoid-related enzymes (phenylalanine ammonia lyase, PAL; chalcone synthase, CHS; and chalcone isomerase, CHI), and total flavonoids were measured to elucidate the effects of the two fertilizers. Results: Compared with the control, plant height, fresh root weight, and total flavonoid concentration (increased by 1.60 and 2.70 times, respectively) were enhanced by low and high biochar applications, whereas organic fertilizer addition only had a positive impact on fresh root weight. Furthermore, biochar supplementation enhanced growth, quality, and total flavonoid content by elevating AK, AP, AN, and OM levels, as well as increasing urease, protease, and CHI activities. Fresh root weight following the addition of organic fertilizer addition was significantly correlated with AK, AP, and protease activity. Conclusions: The addition of pyrogenic organic matter addition is found to be a more suitable strategy than organic fertilizer addition for effectively enhancing the yield and quality of T. hemsleyanum. [ABSTRACT FROM AUTHOR]

PAP2 (Production of anthocyanin segment 2) encodes MYB transcription factor, and regulates anthocyanin synthesis by forming MYB-bHLH-WD40 complex with bHLH and WD40. In this study we cloned PAP2 gene in Brassica, and preliminarily verified the function of PAP2, providing a reference for further understanding the regulatory mechanism of anthocyanin synthesis in Brassica. Two and one copies of PAP2 gene were cloned from Brassica carinata and Brassica nigra, respectively, and named as BcaB.PAP2, BcaC.PAP2, BniB.PAP2. These three genes are composed of 3 exons and 2 introns, encoding 247 amino acids. Based on the PAP2 gene sequences cloned in this study and other reported PAP2 gene sequences of Brassica, three pairs of primers were designed to detect the PAP2 gene of A, B and C genomes respectively, and the genomic origin of PAP2 of six species in the U's Triangle of Brassica could be distinguished by allele specific PCR. The BjuB.PAP2 gene from the B genome of Brassica juncea was selected to construct an overexpression vector and transformed into Arabidopsis thaliana. The transgenic Arabidopsis leaves turned purple, indicating that PAP2 gene is regulating anthocyanin synthesis. After shading treatment for 10 days, the purple color of Brassica carinata lightened, and the anthocyanin content decreased by 41.22%. Quantitative PCR and transcriptomic studies showed that the expression of BcaB.PAP2 and BcaC.PAP2 in the leaves of shading plants decreased. The expression of structural genes for anthocyanin synthesis, such as chalcone synthase gene (CHS), chalcone isomerase gene (CHI), flavanone 3-hydroxylase gene (F3H), dihydroflavonol reductase gene (DFR), anthocyanin synthase gene (ANS), flavonoid 3-glucosyltransferase gene (UFGT) and other genes were also decreased. This study completed the cloning of PAP2 gene of Brassica carinata and Brassica nigra, and carried out their evolutionary analysis. Allele specific PCR primers were designed according to the sequences of PAP2 gene cloned in this experiment and other reported genes to provide molecular markers for the genomic transmission identification of PAP2 gene in Brassica interspecific hybrids. Through the transformation of BjuB.PAP2 gene into Arabidopsis plants, it was found that PAP2 gene was involved in the accumulation of anthocyanins. It was found that the expression of BcaPAP2 gene and anthocyanin synthesis structure gene were induced by light after shading. This study cloned the PAP2 gene in Brassica and preliminarily verified its function, providing a reference for further understanding the regulatory mechanism of anthocyanin synthesis in Brassica plants. [ABSTRACT FROM AUTHOR]

Jasmonates, including jasmonic acid (JA) and its derivatives such as methyl jasmonate (MeJA) or jasmonly isoleucine (JA-Ile), regulate plant responses to various biotic and abiotic stresses. In this study, we applied exogenous MeJA onto Senna tora leaves subjected to wounding and conducted a transcriptome deep sequencing analysis at 1 (T1), 3 (T3), 6 (T6), and 24 (T24) h after MeJA induction, along with the pretreatment control at 0 h (T0). Out of 18,883 mapped genes, we identified 10,048 differentially expressed genes (DEGs) between the T0 time point and at least one of the four treatment times. We detected the most DEGs at T3, followed by T6, T1, and T24. We observed the upregulation of genes related to JA biosynthesis upon exogenous MeJA application. Similarly, transcript levels of genes related to flavonoid biosynthesis increased after MeJA application and tended to reach their maximum at T6. In agreement, the flavonols kaempferol and quercetin reached their highest accumulation at T24, whereas the levels of the anthraquinones aloe-emodin, emodin, and citreorosein remained constant until T24. This study highlights an increase in flavonoid biosynthesis following both MeJA application and mechanical wounding, whereas no significant influence is observed on anthraquinone biosynthesis. These results provide insights into the distinct regulatory pathways of flavonoid and anthraquinone biosynthesis in response to MeJA and mechanical wounding.

Seed propagation is the main method of mulberry expansion in China, an important economic forest species. However, seed germination is the most sensitive stage to various abiotic stresses, especially salinity stress. To reveal the molecular regulatory mechanism of mulberry seed germination under salt stress, flavonoid metabolomics and transcriptomics analyses were performed on mulberry seeds germinated under 50 and 100 mmol/L NaCl stress. Analysis of the flavonoid metabolome revealed that a total of 145 differential flavonoid metabolites (DFMs) were classified into 9 groups, 40 flavonols, 32 flavones, 16 chalcones and 14 flavanones. Among them, 61.4% (89) of the DFMs accumulated continuously with increasing salt concentration, reaching the highest level at a 100 mmol/L salt concentration; these DFMs included quercetin-3-O-glucoside (isoquercitrin), kaempferol (3,5,7,4'-tetrahydroxyflavone), quercetin-7-O-glucoside, taxifolin (dihydroquercetin) and apigenin (4',5,7-trihydroxyflavone), indicating that these flavonoids may be key metabolites involved in the response to salt stress. Transcriptional analysis identified a total of 3055 differentially expressed genes (DEGs), most of which were enriched in flavonoid biosynthesis (ko00941), phenylpropanoid biosynthesis (ko00940) and biosynthesis of secondary metabolites (ko01110). Combined analysis of flavonoid metabolomic and transcriptomic data indicated that phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), flavonol synthase (FLS), bifunctional dihydroflavonol 4-reductase/flavanone 4-reductase (DFR) and anthocyanidin reductase (ANR) were the key genes involved in flavonoid accumulation during mulberry seed germination under 50 and 100 mmol/L NaCl stress. In addition, three transcription factors, MYB, bHLH and NAC, were involved in the regulation of flavonoid accumulation under salt stress. The results of quantitative real-time PCR (qRT‒PCR) validation showed that the expression levels of 11 DEGs, including 7 genes involved in flavonoid biosynthesis, under different salt concentrations were consistent with the transcriptomic data, and parallel reaction monitoring (PRM) results showed that the expression levels of 6 key enzymes (proteins) involved in flavonoid synthesis were consistent with the accumulation of flavonoids. This study provides a new perspective for investigating the regulatory role of flavonoid biosynthesis in the regulation of mulberry seed germination under salt stress at different concentrations. [ABSTRACT FROM AUTHOR]

• In silico methods are used to examine the characteristics of GbPAL, GbCHS, GbDFR, and GbANS structural genes in Gynura bicolor. • Detailed prediction of the physico-chemical properties, protein classifications and models, gene ontologies associated with GbPAL, GbCHS, GbDFR, and GbANS have been provided and verified in silico. • GbPAL showed a secondary structure dominated by helix (mainly alpha), GbCHS and GbDFR (Alpha-beta), and GbANS (mainly beta). • Phylogenetic tree was constructed to evaluate the relationship of GbPAL, GbCHS, GbDFR, and GbANS with other Asteraceae plants. • GbPAL, GbCHS, GbDFR, and GbANS enzymes have been modelled and accurately docked with their appropriate substrates. Gynura bicolor is a member of the Asteraceae family, possessing important pharmaceutical compounds such as flavonoids, anthocyanins, and terpenoids. Genetic and molecular studies focusing on the biosynthesis of flavonoids are indispensable to determining their regulation. GbPAL, GbCHS, GbDFR , and GbANS are genes encoding Phenylalanine ammonia lyase, Chalcone synthase, Dihydroflavonol reductase, and Anthocyanidin synthase enzymes, resepectively and are involved in the biosynthesis of flavonoids in G. bicolor, resepectively. There are still a few studies concerning the characterization of these genes. In this study, the structural enzyme sequences were selected to be characterized in silico using bioinformatics software. The phylogenetic analysis demonstrated that the genes in question were closely related to several species of the Asteraceae family. Our data revealed that the G. bicolor PAL, CHS, DFR and ANS genes are closely related to the sequence of genes found in Chrysanthemum morifolium. Furthermore, the CHS, DFR and ANS of G. bicolor also tightly correlated with sequence from Callistephus chinensis. Further analysis also demonstrated that all conserved motifs contain active functional and catalytic domain supporting the flavonoid-related enzyme functions. All physicochemical parameters used in this study also meet the prediction criteria. The investigation of secondary structure prediction using two instruments (PSIPRED and SOPMA) revealed that all enzymes tested possess similar α-helix (H), β-sheet (E), and coil (C) composition. GbPAL showed a secondary structure dominated by helix (mainly alpha), GbCHS and GbDFR (Alpha-beta), and GbANS (mainly beta). Each enzyme was modeled, with RMSD values superimposed on the template: 0.125 Å (GbPAL), 0.061 Å (GbCHS), 0.065 Å (GbDFR), and 0.089 Å (GbANS). The 3D model evaluation was checked using four parameters, including PROCHECK, Verify3D, ERRAT, and QMEAN-Z score. All the parameters verified that the model showed a high quality (more than 90 % of the favored region), high ressemblence of the 3D and 1D amino acid sequence, the 3D modeles were acceptable since they possesed high resolution of the protein structure (2.5 – 3 Å), and the enzyme models have Z-Score values in the permissible range. The accuracy and reliability of the generated models met Ramachandran value in comparison to experimentally resolved protein templates. The molecular docking analysis showed that each substrate compound docked at the real binding site in every tested enzyme. Furthermore, they also showed similar results compared to other experimentally observed proteins. The overall findings provide an insight into the molecular characteristics of flavonoid-related genes, which might enrich our understanding for further genomic application of the Gynura bicolor. [ABSTRACT FROM AUTHOR]