Key Enzyme Genes Research Articles

Citrus pomace co-fermentation improved its protein and amino acids by Bacillus amyloliquefaciens and Candida utilis

High-value utilization of citrus pomace (CP) is of great significance to the sustainable development of the citrus industry. Co-fermentation of CP was investigated using the autochthonous probiotic bacteria, Bacillus amyloliquefaciens BF2 and Candida utilis GIM 2.9. Crude protein, soluble protein, and small peptide content of CP increased by 40.01%, 923.53%, and 626.67% after fermentation, respectively. Essential amino acids increased from 28.51% to 47.04% of total free amino acids. Leucine level reached 4.04 mg/g DW, increasing by 1293.10%, and produced histidine, valine, cysteine, and tyrosine. Scanning electron microscopy results showed that BF2 and GIM 2.9 could effectively degrade and utilize the CP fibers. Enzymolysis co-fermentation (ECF) system secreted higher levels of non-starch polysaccharide enzymes and proteases than co-fermentation (CF) system. Acid protease activity was positively correlated with soluble protein and free amino acid contents. BF2 possessed 63 essential amino acid-biosynthesis genes, nearly twice as many as that of non-essential amino acids. Eight key enzyme genes were identified for leucine biosynthesis in the BF2 genome, suggesting that BF2 played a key role in leucine biosynthesis. Our results provide a potential method for the high-value utilization of CP as a protein feed additive or dietary supplement.

Systematic identification of chemical forms of key terpene synthase in Cinnamomum camphora

Cinnamomum camphora is an important economic tree species in China. According to the type and content of main components in the volatile oil of leaf, C. camphora were divided into five chemotypes, including borneol-type, camphor-type, linalool-type, cineole-type, and nerolidol-type. Terpene synthase(TPS) is the key enzyme for the formation of these compounds. Although several key enzyme genes have been identified, the biosynthetic pathway of(+)-borneol, which has the most economic value, has not been reported. In this study, nine terpenoid synthase genes CcTPS1-CcTPS9 were cloned through transcriptome analysis of four chemical-type leaves. After the recombinant protein was induced by Escherichia coli, geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) were used as substrates for enzymatic reaction, respectively. Both CcTPS1 and CcTPS9 could catalyze GPP to produce bornyl pyrophosphate, which could be hydrolyzed by phosphohydrolase to obtain(+)-borneol, and the product of(+)-borneol accounted for 0.4% and 89.3%, respectively. Both CcTPS3 and CcTPS6 could catalyze GPP to generate a single product linalool, and CcTPS6 could also react with FPP to generate nerolidol. CcTPS8 reacted with GPP to produce 1,8-cineol(30.71%). Nine terpene synthases produced 9 monoterpene and 6 sesquiterpenes. The study has identified the key enzyme genes responsible for borneol biosynthesis in C. camphora for the first time, laying a foundation for further elucidating the molecular mechanism of chemical type formation and cultivating new varieties of borneol with high yield by using bioengineering technology.

Molecular Cloning and Characterization of WRKY12, A Pathogen Induced WRKY Transcription Factor from Akebia trifoliata.

WRKY transcription factors (TFs), which are plant-specific TFs, play significant roles in plant defense. Here, a pathogen-induced WRKY gene, named AktWRKY12, which was the homologous gene of AtWRKY12, was isolated from Akebia trifoliata. The AktWRKY12 gene has a total length of 645 nucleotides and an open reading frame (ORF) encoding 214 amino acid polypeptides. The characterizations of AktWRKY12 were subsequently performed with the ExPASy online tool Compute pI/Mw, PSIPRED and SWISS-MODEL softwares. The AktWRKY12 could be classified as a member of WRKY group II-c TFs based on sequence alignment and phylogenetic analysis. The results of tissue-specific expression analysis revealed that the AktWRKY12 gene was expressed in all the tested tissues, and the highest expression level was detected in A. trifoliata leaves. Subcellular localization analysis showed that AktWRKY12 was a nuclear protein. Results showed that the expression level of AktWRKY12 significantly increased in A. trifoliata leaves with pathogen infection. Furthermore, heterologous over-expression of AktWRKY12 in tobacco resulted in suppressed expression of lignin synthesis key enzyme genes. Based on our results, we speculate that AktWRKY12 might play a negative role in A. trifoliata responding to biotic stress by regulating the expression of lignin synthesis key enzyme genes during pathogen infection.

An endophytic fungus Schizophyllum commune isolated from Panax ginseng enhances hairy roots growth and ginsenoside biosynthesis.

Using endophytic fungal elicitors to increase the accumulation of valuable secondary metabolites in plant tissue culture is an effective biotechnology strategy. In this study, a collection of 56 strains of endophytic fungi were isolated from different organs of cultivated Panax ginseng, of which seven strains can be symbiotically co-cultured with the hairy roots of P. ginseng. Further experiments observed that strain 3R-2, identified as endophytic fungus Schizophyllum commune, can not only infect hairy roots but also promote the accumulation of specific ginsenosides. This was further verified because S. commune colonization significantly affected the overall metabolic profile of ginseng hairy roots. By comparing the effects of S. commune mycelia and its mycelia extract (EM) on ginsenoside production in P. ginseng hairy roots, the EM was confirmed to be a relatively better stimulus elicitor. Additionally, the introduction of EM elicitor can significantly enhance the expressions of key enzyme genes of pgHMGR, pgSS, pgSE, and pgSD involved in the biosynthetic pathway of ginsenosides, which was deemed the most relevant factor for promoting ginsenosides production during the elicitation period. In conclusion, this study is the first to show that the EM of endophytic fungus S. commune can be considered as an effective endophytic fungal elicitor for increasing the biosynthesis of ginsenosides in hairy root cultures of P. ginseng.

WSSV exploits AMPK to activate mTORC2 signaling for proliferation by enhancing aerobic glycolysis

AMPK plays significant roles in the modulation of metabolic reprogramming and viral infection. However, the detailed mechanism by which AMPK affects viral infection is unclear. The present study aims to determine how AMPK influences white spot syndrome virus (WSSV) infection in shrimp (Marsupenaeus japonicus). Here, we find that AMPK expression and phosphorylation are significantly upregulated in WSSV-infected shrimp. WSSV replication decreases remarkably after knockdown of Ampkα and the shrimp survival rate of AMPK-inhibitor injection shrimp increases significantly, suggesting that AMPK is beneficial for WSSV proliferation. Mechanistically, WSSV infection increases intracellular Ca2+ level, and activates CaMKK, which result in AMPK phosphorylation and partial nuclear translocation. AMPK directly activates mTORC2-AKT signaling pathway to phosphorylate key enzymes of glycolysis in the cytosol and promotes expression of Hif1α to mediate transcription of key glycolytic enzyme genes, both of which lead to increased glycolysis to provide energy for WSSV proliferation. Our findings reveal a novel mechanism by which WSSV exploits the host CaMKK-AMPK-mTORC2 pathway for its proliferation, and suggest that AMPK might be a target for WSSV control in shrimp aquaculture.

Effect of nicosulfuron on dynamic changes in the starch-sugar interconversion in sweet maize (Zea mays L.)

Starch is an important reserve of sugar, and starch-sugar conversion in plants plays an important role in the response of plants to various abiotic stresses. Nicosulfuron is a post-emergence herbicide commonly applied to maize fields. However, it is unclear how sucrose and starch in sweet corn are converted to accommodate nicosulfuron stress. Field and pot experiments were conducted to study the effects of nicosulfuron on the sugar metabolism enzymes, starch metabolism enzymes, non-enzyme substances, and expression of key enzyme genes in leaves and roots of sweet maize seedlings. Accordingly, this research compared the responses of the sister lines HK301 and HK320, which are nicosulfuron tolerant and sensitive, respectively. Under nicosulfuron stress, compared with HK301 seedlings, the accumulation of stem and root dry matter of HK320 seedlings was significantly reduced, resulting in a lower root-to-shoot ratio. Compared with HK320 seedlings, nicosulfuron stress significantly increased the sucrose, soluble sugar, and starch contents in HK301 leaves and roots. This may be related to the enhanced carbohydrate metabolism under nicosulfuron stress, including significant changes in sugar metabolism enzyme activity and the levels of SPS and SuSys expression. Further, under nicosulfuron stress, sucrose transporter genes (SUC 1, SUC 2, SWEET 13a, and SWEET 13b) in the leaves and roots of HK301 seedlings were significantly upregulated. Our results emphasize that changes in sugar distribution, metabolism, and transport can improve the adaptability of sweet maize to nicosulfuron stress.

L-Glutamate maintains the quality of apple fruit by mediating carotenoid, sorbitol and sucrose metabolisms.

l-Glutamate is involved in many important chemical reactions in horticultural products and improves postharvest disease resistance. Quality decline of apple fruit caused by senescence and fungus invasion often leads to tremendous losses during logistics. This study was performed to evaluate the variations of quality attributes, carotenoid, sorbitol and sucrose metabolisms in apples (cv. Qiujin) after l-glutamate dipping treatment. l-Glutamate immersion maintained high values of L*, a* and b*, flesh firmness, titratable acidity, as well as the total soluble solids, soluble sugar, reducing sugar and ascorbic acid contents in apples. l-Glutamate also decreased mass loss, respiratory rate and ethylene release, enhanced sucrose synthase-cleavage, acid invertase and neutral invertase activities, whereas reduced sorbitol dehydrogenase, sucrose phosphate synthase, sucrose synthase synthesis and sorbitol oxidase activities in apples. Moreover, l-glutamate inhibited lutein, β-carotene and lycopene accumulation, and down-regulated phytoene synthase, lycopene β-cyclase, ζ-carotene desaturase, phytoene desaturase, carotenoid isomerase, ζ-carotene isomerase and carotenoids cleavage dioxygenase gene expressions, but up-regulated 9-cis-epoxycarotenoid dioxygenase gene expression in apples. Postharvest l-glutamate dipping treatment can keep apple quality by modulating key enzyme activity and gene expression in sorbitol, sucrose and carotenoid metabolisms. © 2023 Society of Chemical Industry.

The Heat Shock Transcription Factor PtHSF1 Mediates Triacylglycerol and Fucoxanthin Synthesis by Regulating the Expression of GPAT3 and DXS in Phaeodactylum tricornutum.

In addition to being important primary productive forces in marine ecosystems, diatoms are also rich in bioactive substances such as triacylglycerol and fucoxanthin. However, little is known about the transcriptional mechanisms underlying the biosynthesis of these substances. In this study, we found that the heat shock transcription factor PtHSF1 positively regulated the synthesis of triacylglycerol and fucoxanthin in Phaeodactylum tricornutum. Overexpression of PtHSF1 could increase the contents of triacylglycerol and fucoxanthin and upregulate key enzyme genes involved in the triacylglycerol and fucoxanthin biosynthesis pathways. On the other hand, gene silencing of PtHSF1 reduced the contents of triacylglycerol and fucoxanthin and the expression of the key enzyme genes involved in the triacylglycerol and fucoxanthin biosynthesis pathways. Further biochemical analysis revealed that PtHSF1 upregulated glycerol-2-phosphate acyltransferase 3 (GPAT3) and 1-deoxy-d-xylulose 5-phosphate synthase (DXS) by directly binding to their promoters, while genetic analysis demonstrated that PtHSF1 acted upstream of GPAT3 and DXS to regulate triacylglycerol and fucoxanthin synthesis. Therefore, in addition to elucidating the regulation mechanisms underlying PtHSF1-mediated triacylglycerol and fucoxanthin synthesis, this study also provided a candidate target for metabolic engineering of triacylglycerol and fucoxanthin in P. tricornutum.

Insight of microbial degradation of n-hexadecane and n-heneicosane in soil during natural attenuation and bioaugmentation by Compound-specific Stable Isotope Analysis (CSIA)

Microbial degradation of n-hexadecane (n-C16) and n-heneicosane (n-C21) in soil during natural attenuation (NA) and bioaugmentation (BA) were investigated by compound-specific stable isotope analysis (CSIA), microbial community and key enzyme genes analysis. In both NA and BA treatments, more than 95% of n-C16 and n-C21 were degraded within 3 and 6 days, respectively, with a slight faster degradation in BA than NA. According to CSIA, n-C16 showed a reverse hydrogen isotope fractionation effect, while n-C21 had a normal hydrogen isotope fractionation effect, and the degree of hydrogen isotope fractionation in BA were greater than NA. Isotope enrichment factors (εH) of n-C16 and n-C21were 3.50 ± 0.60‰ and − 2.11 ± 0.57‰ in NA, and 5.18 ± 0.46‰ and − 2.73 ± 0.39‰ in BA, respectively. The dominant genera in BA and NA were significantly different for n-C16 and n-C21 degradation and few strains added during BA remained active, such as Rhodococcus in both n-C16 and n-C21, and Brevundimonas only in n-C21, which showing high application potential in alkanes contamination remediation. Both alkB and almA genes played more key roles than Cytochromes P450 in n-C16 and n-C21 biodegradation, but alkB was much more promoted by n-C16 while almA was more motivated by n-C21. The different types of alkane-degrading bacteria with different enzyme reaction kinetics might be the reason of opposite hydrogen isotope fractionation of n-C16 and n-C21 during their biodegradation. Above all, CSIA can be a proper technology to quantitatively evaluate the biodegradation of different alkane during NA and BA bioremediation process in contamination sites.

High-quality Fagopyrum esculentum genome provides insights into the flavonoid accumulation among different tissues and self-incompatibility.

Common buckwheat (Fagopyrum esculentum) and Tartary buckwheat (Fagopyrum tataricum), the two most widely cultivated buckwheat species, differ greatly in flavonoid content and reproductive mode. Here, we report the first high-quality and chromosome-level genome assembly of common buckwheat with 1.2 Gb. Comparative genomic analysis revealed that common buckwheat underwent a burst of long terminal repeat retrotransposons insertion accompanied by numerous large chromosome rearrangements after divergence from Tartary buckwheat. Moreover, multiple gene families involved in stress tolerance and flavonoid biosynthesis such as multidrug and toxic compound extrusion (MATE) and chalcone synthase (CHS) underwent significant expansion in buckwheat, especially in common buckwheat. Integrated multi-omics analysis identified high expression of catechin biosynthesis-related genes in flower and seed in common buckwheat and high expression of rutin biosynthesis-related genes in seed in Tartary buckwheat as being important for the differences in flavonoid type and content between these buckwheat species. We also identified a candidate key rutin-degrading enzyme gene (Ft8.2377) that was highly expressed in Tartary buckwheat seed. In addition, we identified a haplotype-resolved candidate locus containing many genes reportedly associated with the development of flower and pollen, which was potentially related to self-incompatibility in common buckwheat. Our study provides important resources facilitating future functional genomics-related research of flavonoid biosynthesis and self-incompatibility in buckwheat.

Full-length transcriptome combined with RNA sequence analysis of Fraxinus chinensis.

The dry root or stem bark of Fraxinus chinensis is a famous herb Qin Pi which is known for its anti-inflammatory, analgesic, anti-tumor, liver protective and diuretic pharmacological effects, the fundamental chemical components are coumarin, phenylethanol glycosides and flavonoids. However, it is difficult to clarify the secondary metabolite synthesis pathway and key genes involved in the pathway because of lack genome information of Fraxinus chinensis. To generate a complete transcriptome of Fraxinus chinensis and to clarify the differentially expressed genes (DEGs) in leaves and stem barks. In this study, full-length transcriptome analysis and RNA-Seq were combined to characterize Fraxinus chinensis transcriptome. A total of 69,145 transcripts were acquired and regarded as reference transcriptome, 67,441 transcripts (97.47%) were annotated to NCBI non-redundant protein (Nr), SwissProt, the Kyoto Encyclopedia of Genes and Genomes (KEGG) and eukaryotic orthologous groups (KOG) databases. A total of 18,917 isoforms were annotated to KEGG database and classified to 138 biological pathways. In total, 10,822 simple sequence repeat (SSRs) and 11,319 resistance (R) gene were classified to 18 types, and 3947 transcription factors (TFs) were identified in full-length transcriptome analysis. Additionally, 15,095 DEGs were detected by RNA-seq in leaves and barks, including 4696 significantly up-regulated and 10,399 significantly down-regulated genes. And 254 transcripts were annotated into phenylpropane metabolism pathway containing 86 DEGs and ten of these enzyme genes were verified by qRT-PCR. It laid the foundation for further exploration of the biosynthetic pathway of phenylpropanoids and related key enzyme genes.

Differential analysis of transcriptomic and metabolomic of free fatty acid rancidity process in oil palm (Elaeis guineensis) fruits of different husk types.

Oil palm is the world's highest yielding oil crop and its palm oil has high nutritional value, making it an oilseed plant with important economic value and application prospects. After picking, oil palm fruits exposed to air will gradually become soft and accelerate the process of fatty acid rancidity, which will not only affect their flavor and nutritional value, but also produce substances harmful to the human body. As a result, studying the dynamic change pattern of free fatty acids and important fatty acid metabolism-related regulatory genes during oil palm fatty acid rancidity can provide a theoretical basis for improving palm oil quality and extending its shelf life. The fruit of two shell types of oil palm, Pisifera (MP) and Tenera (MT), were used to study the changes of fruit souring at different times points of postharvesting, combined with LC-MS/MS metabolomics and RNA-seq transcriptomics techniques to analyze the dynamic changes of free fatty acids during fruit rancidity, and to find out the key enzyme genes and proteins in the process of free fatty acid synthesis and degradation according to metabolic pathways. Metabolomic study revealed that there were 9 different types of free fatty acids at 0 hours of postharvest, 12 different types of free fatty acids at 24 hours of postharvest, and 8 different types of free fatty acids at 36 hours of postharvest. Transcriptomic research revealed substantial changes in gene expression between the three harvest phases of MT and MP. Combined metabolomics and transcriptomics analysis results show that the expression of SDR, FATA, FATB and MFP four key enzyme genes and enzyme proteins in the rancidity of free fatty acids are significantly correlated with Palmitic acid, Stearic acid, Myristic acid and Palmitoleic acid in oil palm fruit. In terms of binding gene expression, the expression of FATA gene and MFP protein in MT and MP was consistent, and both were expressed higher in MP. FATB fluctuates unevenly in MT and MP, with the level of expression growing steadily in MT and decreasing in MP before increasing. The amount of SDR gene expression varies in opposite directions in both shell types. The above findings suggest that these four enzyme genes and enzyme proteins may play an important role in regulating fatty acid rancidity and are the key enzyme genes and enzyme proteins that cause differences in fatty acid rancidity between MT and MP and other fruit shell types. Additionally, differential metabolite and differentially expressed genes were present in the three postharvest times of MT and MP fruits, with the difference occurring 24 hours postharvest being the most notable. As a result, 24 hours postharvest revealed the most obvious difference in fatty acid tranquility between MT and MP shell types of oil palm. The results from this study offer a theoretical underpinning for the gene mining of fatty acid rancidity of various oil palm fruit shell types and the enhancement of oilseed palm acid-resistant germplasm cultivation using molecular biology methods.

Transcriptomics explores the potential of flavonoid in non-medicinal parts of Saposhnikovia divaricata (Turcz.) Schischk.

Saposhnikovia divaricata is a traditional Chinese medicine in China, which is widely used in clinic. The root of S. divaricata is often used as medicine, but little research has been done on its other tissues. In this study, the contents of root and leaf of S. divaricata were determined by HPLC, the differentially expressed genes were screened by transcriptome sequencing at molecular level, and then verified by network pharmacology. The results showed that the content of 4'-O-β-D-glucosyl-5-O-methylvisamminol in the leaves was significantly higher than that in the roots, which was about 3 times higher than that in the roots. In addition, 10 differentially expressed key enzyme genes were screened in plant hormone signal transduction, phenylpropanoid and flavonoid biosynthetic pathways. C4H and CYP98A were up-regulated in root, while F3H was down-regulated in root. They can be used as important candidate genes for the mechanism of quality difference of S. divaricata. Finally, network pharmacological validation showed that 5-O-methylvesamitol plays an important role in the treatment of ulcerative colitis. These findings not only provide insight into flavonoid biosynthesis in S. divaricata associated molecular regulation, but also provide a theoretical basis for the development and utilization of S. divaricata.

Effect and Mechanism of Lepista nuda Mycelia Obtained via In Vitro Culture on the Lifespan of Drosophila melanogaster

To provide a theoretical basis for biogenic fly-killing pesticides, in this study, we sought to examine the lethal effects of Lepista nuda mycelium-supplemented diets in Drosophila melanogaster. In doing so, we also studied the effects of Lepista nuda mycelium-supplemented diets on lifespan, antioxidant enzyme activity, peroxide content, relative transcript amounts of antioxidant enzyme genes, signaling pathways, and lifespan. Lower Lepista nuda mycelium-supplemented diets activated the antioxidant system and prolonged lifespan, while higher mycelium-supplemented diets had a significant toxic effect. After the administration of mycelium-supplemented diets for 24 h, the highest corrected mortality (41.96%) and lifespan inhibition rates (96.50%) were observed. In addition, the antioxidant enzyme activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px); the relative transcript amounts of the key antioxidant enzyme genes SOD, CAT, and TrxR-1; and the lifespan genes Hep and Nrf2 were found to be the lowest, while the contents of the oxidation products malondialdehyde (MDA), protein carbonylation product (PCO), and triglyceride (TG); the relative transcript amounts of the signaling pathway-related genes S6K, TOR, and Keap-1; and the lifespan gene MTH were observed to be the highest after 48 h. Higher Lepista nuda mycelium-supplemented diets significantly inhibited lifespan, acting via the initiation of oxidative stress systems.

Effects of arbuscular mycorrhizal fungus inoculation on the growth and nitrogen metabolism of Catalpa bungei C.A.Mey. under different nitrogen levels.

Evidence suggests that arbuscular mycorrhizal fungi (AMF) may promote the growth of woody plants. However, the effects of AMF on nitrogen (N) metabolism in plants, especially trees, and its regulatory mechanism are rarely reported. Here, the effects of AMF inoculation on the growth and N nutrition status of Catalpa bungei under different N levels were reported. Three N levels (low, medium, high) and two mycorrhizal inoculation treatments (inoculation with Rhizophagus intraradices or not) were used with factorial design. The results showed that medium N could significantly improve the physiological metabolism and growth of C. bungei seedlings. However, when N was excessive, growth was significantly inhibited whether inoculated AMF or not. Compared with non-inoculated treatments, AMF inoculation could promote the absorption of N and P, improve photosynthesis under low to medium N levels, thus promoting the growth of seedlings. AMF changed the biomass allocation in seedlings by reducing the stem mass ratio and root/shoot ratio, and increasing the leaf mass ratio. At medium N levels, compared with non-inoculated treatment, AMF inoculation could significantly promote root growth by changing root hormone levels and improving root architecture and root activity. Under N addition, AMF inoculation could improve the absorption and assimilation of N by regulating the expression of key enzyme genes of N metabolism and nitrate transporter genes (NRT2.4, NRT2.5, NRT2.7) in roots, and enhancing the activities of the key enzyme of N metabolism. This study may provide a reference for the application of AMF in the cultivation and afforestation technology of C. bungei in Northwest China.

Genome-wide characterization of LBD transcription factors in switchgrass (Panicum virgatum L.) and the involvement of PvLBD12 in salt tolerance.

PvLBD12 enhanced the salt tolerance by increasing proline accumulation, improving K+ accumulation, and decreasing reactive oxygen species level in switchgrass. Abiotic stresses are the serious factors which limit plant development and productivity and restrict the agricultural economy. It is important, therefore, to understand the mechanism of abiotic tolerance in plants. Lateral organ boundaries domain (LBD) proteins as plant-specific transcription factors play important function in plant lateral organ development, plant regeneration, and abiotic stress. In our study, we identify 69 LBD members from switchgrass genome-wide sequences and classify them based on their homology with LBD proteins in Arabidopsis. RT-qPCR showed that PvLBD genes had different expression patterns under abiotic stress conditions, indicating that they play important roles in various stress. PvLBD12 was selected as a candidate gene for further functional analysis because it had the highest expression level under salt stress. Overexpression of PvLBD12 enhanced salt tolerance by altering a wide range of physiological responses (like increased proline accumulation, reduced malondialdehyde production, improved K+ accumulation, and reduced Na+ absorption) in switchgrass. Some stress response genes such as proline biosynthesis gene PvP5CS1, vacuolar Na+(K+)/H+ antiporter gene PvNHX1, two key ROS-scavenging enzyme genes PvCAT and PvSOD were all upregulated in PvLBD12 overexpression lines. Taken together, PvLBD12 plays a pivotal role in response to salt stress by increasing proline accumulation, improving K+ accumulation, reducing Na+ absorption, and decreasing reactive oxygen species level. It will be better to understand the potential biological functions of LBD genes in other plants.

Cocultivation of pigeon pea hairy root cultures and Aspergillus for the enhanced production of cajaninstilbene acid.

Pigeon pea hairy root cultures (PPHRCs) have been proven to be a promising alternative for the production of health-beneficial phenolic compounds, such as the most important health-promoting compound, i.e., cajaninstilbene acid (CSA). In this study, PPHRCs were cocultured with live Aspergillus fungi for further improving phenolic productivity via biological elicitation. Aspergillus oryzae CGMCC 3.951 (AO 3.951) was found to be the optimal fungus that could achieve the maximum increment of CSA (10.73-fold increase) in 42-day-old PPHRCs under the inoculum size of mycelia 0.50% and cocultivation time 36h. More precisely, the contents of CSA in hairy roots and culture media after fungal elicitation increased by 9.87- and 62.18-fold over control, respectively. Meanwhile, the contents of flavonoid glycosides decreased, while aglycone yields increased upon AO 3.951 elicitation. Moreover, AO 3.951 could trigger the oxidative stress and pathogen defense response thus activating the expression of biosynthesis- and ABC transporter-related genes, which contributed to the intracellular accumulation and extracellular secretion of phenolic compounds (especially CSA) in PPHRCs. And PAL2, 4CL2, STS1, and I3'H were likely to be the potential key enzyme genes regulating the biosynthesis of CSA, and ABCB11X1-1, ABCB11, and ABCG24X2 were closely related to the transmembrane transport of CSA. Overall, the cocultivation approach could make PPHRCs more commercially attractive for the production of high-value phenolic compounds such as CSA and flavonoid aglycones in nutraceutical/medicinal fields. And the elucidation of crucial biosynthesis and transport genes was important for systematic metabolic engineering aimed at increasing CSA productivity. KEY POINTS: • Cocultivation of PPHRCs and live fungi was to enhance CSA production and secretion. • PPHRCs augmented CSA productivity 10.73-fold when cocultured with AO 3.951 mycelia. • Several biosynthesis and transport genes related to CSA production were clarified.

Transcriptome Analysis Reveals the Central Role of the Transcription Factor MYB in Regulating Anthocyanin Accumulation in Economic Grape Species (Vitis vinifera)

To cultivate different grape varieties according to market needs, it is necessary to study the regulation mechanism of color changes in different development stages of grapes. In this study, RNA-sequencing (RNA-Seq) technology was used to compare and analyze the transcriptome data of four grape varieties at the same development stage. Among the annotated differential genes, the anthocyanin synthesis pathway in the flavonoid pathway was mainly studied. Further RT-qPCR analysis of key enzyme genes, in the flavonoid synthesis pathway of the anthocyanin metabolism pathway, showed that the MYB transcription factor family had binding sites at the start of the four enzyme genes. The relative expression of the MYB transcription factor and enzyme gene in the transcriptome data was verified by reverse transcription polymerase chain reaction. Subcellular localization and gene function verification of the transcription factor MYB2 confirmed its regulatory role in anthocyanins.

Metabolic profile and transcriptome reveal the mystery of petal blotch formation in rose

BackgroundPetal blotch is a unique ornamental trait in angiosperm families, and blotch in rose petal is rare and has great esthetic value. However, the cause of the formation of petal blotch in rose is still unclear. The influence of key enzyme genes and regulatory genes in the pigment synthesis pathways needs to be explored and clarified.ResultsIn this study, the rose cultivar ‘Sunset Babylon Eyes’ with rose-red to dark red blotch at the base of petal was selected as the experimental material. The HPLC-DAD and UPLC-TQ-MS analyses indicated that only cyanidin 3,5-O-diglucoside (Cy3G5G) contributed to the blotch pigmentation of ‘Sunset Babylon Eyes’, and the amounts of Cy3G5G varied at different developmental stages. Only flavonols but no flavone were found in blotch and non-blotch parts. As a consequence, kaempferol and its derivatives as well as quercetin and its derivatives may act as background colors during flower developmental stages. Despite of the differences in composition, the total content of carotenoids in blotch and non-blotch parts were similar, and carotenoids may just make the petals show a brighter color. Transcriptomic data, quantitative real-time PCR and promoter sequence analyses indicated that RC7G0058400 (F3’H), RC6G0470600 (DFR) and RC7G0212200 (ANS) may be the key enzyme genes for the early formation and color deepening of blotch at later stages. As for two transcription factor, RC7G0019000 (MYB) and RC1G0363600 (WRKY) may bind to the promoters of critical enzyme genes, or RC1G0363600 (WRKY) may bind to the promoter of RC7G0019000 (MYB) to activate the anthocyanin accumulation in blotch parts of ‘Sunset Babylon Eyes’.ConclusionsOur findings provide a theoretical basis for the understanding of the chemical and molecular mechanism for the formation of petal blotch in rose.

Identification of the Genome-Wide Expression Patterns of Non-Coding RNAs Associated with Tanshinones Synthesis Pathway in Salvia miltiorrhiza

The red root of Salvia miltiorrhiza Bunge, a famous traditional Chinese medicine (TCM), was caused by tanshinone in epidermis cells. In order to study the biological function of ncRNAs in the tanshinone synthesis, the expression patterns of mRNA and ncRNAs were comprehensively analyzed in red (high tanshinone content) and white root (low tanshinone content) tissues derived from the same plant. A total of 731 differentially expressed genes (DEGs) were mainly enriched in primary metabolic pathways such as galactose and nitrogen, and some secondary metabolic pathways such as phenylpropanoid and terpenoids. A total of 70 miRNAs, 48 lncRNAs, and 26 circRNAs were identified as differentially expressed (DE) ones. The enrichment pathway of the targets of DE-lncRNA were mainly in ribosome, carbon metabolism, plant hormone signal transduction, and glycerophospholipid metabolism. The function of the targets genes of 59 miRNAs combined with DE-circRNAs was mainly involved in plant–pathogen interaction, endocytosis, phenylpropanoid biosynthesis, and sesquiterpenoid and triterpenoid biosynthesis pathways. Most genes of the tanshinone synthesis pathway had a higher expression. Some ncRNAs were predicted to regulate several key enzyme genes of the tanshinone synthesis pathway, such as SmDXS2, SmGGPPS1, SmKSL. Furthermore, most target genes were related to the resistance of pathogens. The present study exhibited the tissue-specific expression patterns of ncRNAs, which would provide a basis for further research into the regulation mechanism of ncRNAs in the tanshinone synthesis process.