Impact of Light and Dark Treatment on Phenylpropanoid Pathway Genes, Primary and Secondary Metabolites in Agastache rugosa Transgenic Hairy Root Cultures by Overexpressing Arabidopsis Transcription Factor AtMYB12.

Agastache rugosa, otherwise called Korean mint, has a wide range of medicinal benefits. In addition, it is a rich source of several medicinally valuable compounds such as acacetin, tilianin, and some phenolic compounds. The present study aimed to investigate how the Tartary buckwheat transcription factor AtMYB12 increased the primary and secondary metabolites in Korean mint hairy roots cultured under light and dark conditions. A total of 50 metabolites were detected by using high-performance liquid chromatography (HPLC) and gas chromatography-time-of-flight mass spectrometry (GC-TOFMS). The result showed that the AtMYB12 transcription factor upregulated the phenylpropanoid biosynthesis pathway genes, which leads to the highest accumulation of primary and secondary metabolites in the AtMYB12-overexpressing hairy root lines (transgenic) than that of the GUS-overexpressing hairy root line (control) when grown under the light and dark conditions. However, when the transgenic hairy root lines were grown under dark conditions, the phenolic and flavone content was not significantly different from that of the control hairy root lines. Similarly, the heat map and hierarchical clustering analysis (HCA) result showed that most of the metabolites were significantly abundant in the transgenic hairy root cultures grown under light conditions. Principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) showed that the identified metabolites were separated far based on the primary and secondary metabolite contents present in the control and transgenic hairy root lines grown under light and dark conditions. Metabolic pathway analysis of the detected metabolites showed 54 pathways were identified, among these 30 were found to be affected. From these results, the AtMYB12 transcription factor activity might be light-responsive in the transgenic hairy root cultures, triggering the activation of the primary and secondary metabolic pathways in Korean mint.

Metabolic Profiling of Primary and Secondary Metabolites in Kohlrabi (Brassica oleracea var. gongylodes) Sprouts Exposed to Different Light-Emitting Diodes.

Light-emitting diode (LED) technology is one of the most important light sources in the plant industry for enhancing growth and specific metabolites in plants. In this study, we analyzed the growth, primary and secondary metabolites of 10 days old kohlrabi (Brassica oleracea var. gongylodes) sprouts exposed to different LED light conditions. The results showed that the highest fresh weight was achieved under red LED light, whereas the highest shoot and root lengths were recorded below the blue LED light. Furthermore, high-performance liquid chromatography (HPLC) analysis revealed the presence of 13 phenylpropanoid compounds, 8 glucosinolates (GSLs), and 5 different carotenoids. The phenylpropanoid and GSL contents were highest under blue LED light. In contrast, the carotenoid content was found to be maximum beneath white LED light. Principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) of the 71 identified metabolites using HPLC and gas chromatography-time-of-flight mass spectrometry (GC-TOF-MS) showed a clear separation, indicating that different LEDs exhibited variation in the accumulation of primary and secondary metabolites. A heat map and hierarchical clustering analysis revealed that blue LED light accumulated the highest amount of primary and secondary metabolites. Overall, our results demonstrate that exposure of kohlrabi sprouts to blue LED light is the most suitable condition for the highest growth and is effective in increasing the phenylpropanoid and GSL content, whereas white light might be used to enhance carotenoid compounds in kohlrabi sprouts.

Effects of various Agrobacterium rhizogenes strains on hairy root induction and analyses of primary and secondary metabolites in Ocimum basilicum.

The hairy root (HR) culture system is an excellent alternative strategy to the whole plant system for producing valuable compounds. However, selection of suitable Agrobacterium strain for the successful induction of HR is an essential step for enhanced production of beneficial secondary metabolites. In this study, we examined the transformation efficiency of various A. rhizogenes strains (ATCC 13333, ATCC 15834, A4, R1000, R1200, and R1601) for transgenic HRs induction in Ocimum basilicum. Among the tested strains, the R1601 was found to be one of the most promising strain for mass production of HR in terms of transformation efficiency (94%) and the number and length of HR (8.4 ± 0.52 and 1.68 ± 0.14 cm). The HR induced by the same strain exhibited highest levels of rosmarinic acid level (62.05 ± 4.94 µg/g DW) and total phenolic content (62.3 ± 4.95 µg/g DW). A total of 55 metabolites were identified using high-performance liquid chromatography (HPLC) and gas chromatography-time-of-flight mass spectrometry (GC-TOFMS). The PCA and PLS-DA plot of the identified metabolites showed that HR induced by A4 and ATCC 15834 displayed variation in primary and secondary metabolite contents. Analysis of the metabolic pathway identified a total of 56 pathways, among which 35 were found to be impacted. A heat map and hierarchical clustering analysis indicated that HR induced by different Agrobacterium strains exhibited differential metabolites profiles. In conclusion, Agrobacterium strains R1601 is one of the best and most promising strains for inducing mass HR production and enhanced levels of secondary metabolites in O. basilicum.

Effects of Chilling Treatment on Baicalin, Baicalein, and Wogonin Biosynthesis in Scutellaria baicalensis Plantlets.

When plants are exposed to stressful conditions, they modulate their nutrient balance by regulating their primary and secondary metabolisms to adapt. In this study, changes in primary and secondary metabolites elicited by chilling stress treatment and the effects of treatment duration were examined in roots of Scutellaria baicalensis (S. baicalensis) plantlets. The concentrations of most sugars (maltose, glucose, sucrose, and fructose) and of several amino acids (proline and GABA), which are crucial regarding plant defense mechanisms, increased with increasing duration of chilling stress. Furthermore, salicylic acid levels increased after two-day chilling treatments, which may enhance plant tolerance to cold temperatures. The concentrations of flavones (baicalin, baicalein, and wogonin) increased during chilling stress, and those of phenolic acids (ferulic acid and sinapic acid) increased after two-day chilling treatments. The concentrations of these flavones were positively correlated with sucrose levels which acted as energy sources.

Non-destructive measurement of total phenolic compounds in Arabidopsis under various stress conditions.

Quantifying the phenolic compounds in plants is essential for maintaining the beneficial effects of plants on human health. Existing measurement methods are destructive and/or time consuming. To overcome these issues, research was conducted to develop a non-destructive and rapid measurement of phenolic compounds using hyperspectral imaging (HSI) and machine learning. In this study, the Arabidopsis was used since it is a model plant. They were grown in controlled and various stress conditions (LED lights and drought). Images were captured using HSI in the range of 400-1,000 nm (VIS/NIR) and 900-2,500 nm (SWIR). Initially, the plant region was segmented, and the spectra were extracted from the segmented region. These spectra were synchronized with plants' total phenolic content reference value, which was obtained from high-performance liquid chromatography (HPLC). The partial least square regression (PLSR) model was applied for total phenolic compound prediction. The best prediction values were achieved with SWIR spectra in comparison with VIS/NIR. Hence, SWIR spectra were further used. Spectral dimensionality reduction was performed based on discrete cosine transform (DCT) coefficients and the prediction was performed. The results were better than that of obtained with original spectra. The proposed model performance yielded R 2-values of 0.97 and 0.96 for calibration and validation, respectively. The lowest standard errors of predictions (SEP) were 0.05 and 0.07 mg/g. The proposed model out-performed different state-of-the-art methods. These demonstrate the efficiency of the model in quantifying the total phenolic compounds that are present in plants and opens a way to develop a rapid measurement system.

An update on polyethylene and biodegradable plastic mulch films and their impact on the environment.

Many ways are being developed in the realm of agriculture to increase crop yield while inflicting minimal damage to the soil and environment. One among them is the application of agricultural, biodegradable mulch (BDM) films. Organic substances or synthetic materials are used for making mulches. Also, bio-based polymers derived from bacteria, microorganisms, or fossil fuels are used to make BDM films. BDM films are used in crop production because of their high agronomical advantages which lead to sustainable agriculture. These films are placed on the soil's surface, around the plants. Mulches help in conserving the moisture, control the temperature of the soil, control the growth of the weed, and help in the utilization of soil nutrients thus, overall enhancing the crop yield. The usage of plastic mulch in agriculture has expanded substantially all around the world, over the past ten years, this is because polyethylene films are cheaper, easily produced, highly flexible, and durable. However, the improper disposal of used plastic films has resulted in soil pollution and environmental contamination. Traditional mulches are replaced by BDM, which is a more environmentally friendly alternative. After being used, degradable mulch films could be tilled into the soil and are expected to disintegrate over time. This review focuses on the BDMs, their history, plastic mulches, how BDMs became an alternative source for plastic mulches, their composition, and also addresses their significance. In addition, we discuss the environmental impact of films, including how it alters the climate, soil, temperature, and weed management.

Metabolic Analyses and Evaluation of Antioxidant Activity in Purple Kohlrabi Sprouts after Exposed to UVB Radiation.

Various metabolites act as plant defense molecules due to their antioxidant abilities. This study aimed to investigate the influence of UVB irradiation on the accumulation of metabolites, including primary metabolites (sugar, sugar alcohols, amino acids, organic acids, and an amine) and secondary metabolites (anthocyanins, fatty acids, and phenolic acids), and its synergistic antioxidant ability, in purple kohlrabi sprouts. Metabolite analyses revealed a total of 92 metabolites in the sprouts. Specifically, the levels of most amino acids increased after 24 h of UVB treatment, and then slightly decreased in the kohlrabi sprouts. The levels of most sugars and sugar alcohols increased after 24 h of UVB treatment and then decreased. The levels of TCA cycle intermediates and phenolic acids gradually increased during the UVB treatment. Furthermore, the levels of some fatty acids gradually increased during the UVB treatment, and the levels of the other fatty acids increased after 6 h of UVB treatment and then decreased. In particular, the levels of most anthocyanins, known to be strong antioxidants, gradually increased after 24 h of UVB treatment. In the in vitro ABTS scavenging assay, UVB-treated purple kohlrabi sprouts showed increased scavenging ability. This may be attributed to the increased accumulation of metabolites acting as antioxidants, in response to UVB treatment. This study confirmed that UVB irradiation induced the alteration of primary and secondary metabolism in the kohlrabi sprouts.

Identification, In Silico Characterization, and Differential Expression Profiles of Carotenoid, Xanthophyll, Apocarotenoid Biosynthetic Pathways Genes, and Analysis of Carotenoid and Xanthophyll Accumulation in Heracleum moellendorffii Hance.

Heracleum moellendorffii Hance is a non-woody forest plant widely used in China, Korea, and Japan because of its various therapeutic properties. However, the genetic details of the carotenoid pathway (CP), xanthophyll pathway (XP), and apocarotenoid pathway (AP) genes have not been studied. Thus, the CP, XP, and AP genes of H. moellendorffii were detected and analyzed. A total of fifteen genes were identified, of which eight, four, and three belonged to CP, XP, and AP, respectively. All identified genes possessed full open reading frames. Phylogenetic characterization of the identified gene sequences showed the highest similarity with other higher plants. Multiple alignments and 3D dimensional structures showed several diverse conserved motifs, such as the carotene-binding motif, dinucleotide-binding motif, and aspartate or glutamate residues. The results of real-time PCR showed that the CP, XP, and AP genes were highly expressed in leaves, followed by the stems and roots. In total, eight different individual carotenoids were identified using HPLC analysis. The highest individual and total carotenoid content were achieved in the leaves, followed by the stems and roots. This study will provide more information on the gene structure of the CP, XP, and AP genes, which may help to increase the accumulation of carotenoids in H. moellendorffii through genetic engineering. These results could be helpful for further molecular and functional studies of CP, XP, and AP genes.

Comparative Determination of Phenolic Compounds in Arabidopsis thaliana Leaf Powder under Distinct Stress Conditions Using Fourier-Transform Infrared (FT-IR) and Near-Infrared (FT-NIR) Spectroscopy.

The increasing interest in plant phenolic compounds in the past few years has become necessary because of their several important physicochemical properties. Thus, their identification through non-destructive methods has become crucial. This study carried out comparative non-destructive measurements of Arabidopsis thaliana leaf powder sample phenolic compounds using Fourier-transform infrared and near-infrared spectroscopic techniques under six distinct stress conditions. The prediction analysis of 600 leaf powder samples under different stress conditions (LED lights and drought) was performed using PLSR, PCR, and NAS-based HLA/GO regression analysis methods. The results obtained through FT-NIR spectroscopy yielded the highest correlation coefficient (Rp2) value of 0.999, with a minimum error (RMSEP) value of 0.003 mg/g, based on the PLSR model using the MSC preprocessing method, which was slightly better than the correlation coefficient (Rp2) value of 0.980 with an error (RMSEP) value of 0.055 mg/g for FT-IR spectroscopy. Additionally, beta coefficient plots present spectral differences and the identification of important spectral signatures sensitive to the phenolic compounds in the measured powdered samples. Thus, the obtained results demonstrated that FT-NIR spectroscopy combined with partial least squares regression (PLSR) and suitable preprocessing method has a solid potential for non-destructively predicting phenolic compounds in Arabidopsis thaliana leaf powder samples.

Identification, Characterization, and Expression Analysis of Carotenoid Biosynthesis Genes and Carotenoid Accumulation in Watercress (Nasturtium officinale R. Br.).

Watercress (Nasturtium officinale R. Br.) is an important aquatic herb species belonging to the Brassicaceae family. It has various medicinal properties and has been utilized for the treatment of cancer and other diseases; however, currently available genomic information regarding this species is limited. Here, we performed the first comprehensive analysis of the carotenoid biosynthesis pathway (CBP) genes of N. officinale, which were identified from next-generation sequencing data. We identified and characterized 11 putative carotenoid pathway genes; among these, nine full and two partial open reading frames were determined. These genes were closely related to CBP genes of the other higher plants in the phylogenetic tree. Three-dimensional structure analysis and multiple alignments revealed several distinct conserved motifs, including aspartate or glutamate residues, carotene-binding motifs, and dinucleotide-binding motifs. Quantitative reverse transcription-polymerase chain reaction results showed that the CBP was expressed in a tissue-specific manner: expression levels of NoPSY, NoPDS, NoZDS-p, NoCrtISO, NoLCYE, NoCHXE-p, and NoCCD were highest in the flower, whereas NoLCYB, NoCHXB, NoZEP, and NoNCED were highest in the leaves. Stems, roots, and seeds did not show a significant change in the expression compared to the leaves and flowers. High-performance liquid chromatography analysis of the same organs showed the presence of seven distinct carotenoid compounds. The total carotenoid content was highest in the leaves followed by flowers, seeds, stems, and roots. Among the seven individual carotenoids, the levels of six carotenoids (i.e., 13-Z-ß-carotene, 9-Z-ß-carotene, E-ß-carotene, lutein, violaxanthin, and ß-cryptoxanthin) were highest in the leaves. The highest content was observed for lutein, followed by E-ß-carotene, and 9-Z-ß-carotene; these carotenoids were much higher in the leaves compared to the other organs. The results will be useful references for further molecular genetics and functional studies involving this species and other closely related species.

Impact of copper treatment on phenylpropanoid biosynthesis in adventitious root culture of Althaea officinalis L.

Althaea officinalis has been widely used in various pharmaceutical applications. The biological effects and significance of phenylpropanoids in numerous industries are well studied. However, fulfilling consumer demand for these commercially important compounds is difficult. The effect of heavy-metal toxic influence on plants is primarily due to a strong and rapid suppression of growth processes, as well as the decline in activity of the photosynthetic apparatus, also associated with progressing senescence processes. Some of the secondary metabolite production was triggered by the application of heavy metals, but there was not a stress response. In the adventitious root culture of A. officinalis, copper-mediated phenylpropanoid biosynthesis has been investigated in both concentration-and duration-dependent manners. High-performance liquid chromatography (HPLC) analysis revealed a total of nine different phenolic compounds in response to different concentrations of copper chloride. In this study, high productivity of phenolic compounds was observed in the copper chloride treated-adventitious root culture of A. officianalis. In particular, a low concentration of copper chloride led to a significant accumulation of phenolic compounds under optimal conditions. Moreover, all genes responsible for phenylpropanoid biosynthesis may be sensitive to phenolic compound production following copper treatment. Especially, the highest change in transcript level was observed from AoANS at 6 h. According to our findings, treatment with copper chloride (0.5 mM) for 48 or 96 h can be an appropriate method to maximize phenylpropanoid levels in A. officinalis adventitious root culture.

Targeted Metabolic and In-Silico Analyses Highlight Distinct Glucosinolates and Phenolics Signatures in Korean Rapeseed Cultivars.

Rapeseed is an economically important oilseed crop throughout the world. We examined the content and composition of glucosinolates (GSLs) and phenolics in the sprouts of seven Korean cultivars. A total of eight GSLs that include four aliphatic GSLs (AGSLs) (progoitrin, gluconapin, gluconapoleiferin, and glucobrassicanapin) and four indole GSLs (IGSLs) (4-methoxyglucobrassicin, 4-hydroxyglucobrassicin, neoglucobrassicin, and glucobrassicin) were identified in these cultivars. Of the total GSLs, the highest level was detected for progoitrin, while the lowest level was identified for glucobrassicanapin in all the cultivars. Phenolics that include chlorogenic acid, catechin hydrate, 4-hydroxybenzoic acid, gallic acid, ferulic acid, p-coumaric acid, epicatechin, caffeic acid, rutin, quercetin, trans-cinnamic acid, benzoic acid, and kaempferol were present in all the cultivars. Of these, rutin was identified with the highest level while trans-cinnamic acid was identified with the lowest level in all the cultivars. Cluster analysis revealed the unique metabolic signature of eight GSLs and thirteen phenolics for the seven cultivars of rapeseed, which implies that genomic commonality and variability resulted from the previous breeding program. Further, gene expression and cis-regulatory elements suggest that the biosynthesis of GSLs and phenolics of these cultivars appears to be regulated through transcription factors associated with stress responses, phytohormones, and cellular growth.

Evaluation of Growth, Yield, and Biochemical Attributes of Bitter Gourd (Momordica charantia L.) Cultivars under Karaj Conditions in Iran.

Vegetative and reproductive characteristics, fruit yield, and biochemical compounds of six bitter melon cultivars (Iranshahr, Mestisa, No. 486, Local Japanese, Isfahan, and Ilocano) were evaluated under Karaj conditions in Iran. The phytochemical properties of the cultivars were evaluated using both shade-dried and freeze-dried samples at three fruit developmental stages (unripe, semi-ripe, and ripe). There were significant differences in the vegetative and reproductive characteristics among cultivars, where cv. No. 486 was superior to most vegetative attributes. The fruit yield of cultivars varied from 2.98-5.22 kg/plant. The number of days to male and female flower appearance ranged from 19.00-25.33 and from 25-33 days, respectively. The leaf charantin content was in the range of 4.83-11.08 µg/g. Fruit charantin content varied with developmental stage, drying method, and cultivar. The highest charantin content (13.84 ± 3.55 µg/g) was observed at the semi-ripe fruit stage, and it was much higher in the freeze-dried samples than the shade-dried samples. Cultivar No. 486 had the highest (15.43 ± 2.4 µg/g) charantin content, whereas the lowest charantin content (8.51 ± 1.15 µg/g) was recorded in cultivar cv. Local Japanese. The highest total phenol content (25.17 ± 2.27 mg GAE/g) was recorded in freeze-dried samples of ripe fruits of cv. No. 486, whereas the lowest phenol content was detected in the shade-dried samples of semi-ripe fruits of Isfahan. cv. Flavonoid content was higher with the shade-drying method, irrespective of cultivar. In conclusion, considering the fruit yield and active biological compounds in the studied cultivars, cv. No. 486 should be grown commercially because of its higher yield and production of other secondary metabolites.

Molecular Characterization, Expression Analysis of Carotenoid, Xanthophyll, Apocarotenoid Pathway Genes, and Carotenoid and Xanthophyll Accumulation in Chelidonium majus L.

Chelidonium majus L. is a perennial herbaceous plant that has various medicinal properties. However, the genomic information about its carotenoid biosynthesis pathway (CBP), xanthophyll biosynthesis pathway (XBP), and apocarotenoid biosynthesis pathway (ABP) genes were limited. Thus, the CBP, XBP, and ABP genes of C. majus were identified and analyzed. Among the 15 carotenoid pathway genes identified, 11 full and 4 partial open reading frames were determined. Phylogenetic analysis of these gene sequences showed higher similarity with higher plants. Through 3D structural analysis and multiple alignments, several distinct conserved motifs were identified, including dinucleotide binding motif, carotene binding motif, and aspartate or glutamate residues. Quantitative RT-PCR showed that CBP, XBP, and ABP genes were expressed in a tissue-specific manner; the highest expression levels were achieved in flowers, followed by those in leaves, roots, and stems. The HPLC analysis of the different organs showed the presence of eight different carotenoids. The highest total carotenoid content was found in leaves, followed by that in flowers, stems, and roots. This study provides information on the molecular mechanisms involved in CBP, XBP, and ABP genes, which might help optimize the carotenoid production in C. majus. The results could also be a basis of further studies on the molecular genetics and functional analysis of CBP, XBP, and ABP genes.

Overexpression of Ginkgo biloba Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase 2 gene (GbHDR2) in Nicotiana tabacum cv. Xanthi.

2-C-Methyl-d-erythrol-4-phosphate (MEP) pathway in plant supplies isoprene building blocks for carotenoids and chlorophylls essential in photosynthesis as well as plant hormones such as gibberellin and abscisic acid. To assess the effect of overexpression of the terminal enzyme of the MEP pathway, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR), transgenic Nicotiana tabacum overexpressing class 2 HDR from Ginkgo biloba (GbHDR2) under the control of 35S promoter was constructed. Contents of chlorophylls a and b in transgenic tobacco were enhanced by 19 and 7%, respectively, compared to those of the wild type. The carotenoid level was also 18% higher than that in the control plant. As a result, photosynthetic rate of the transgenic tobacco was increased by up to 51%. Diterepenoid duvatrienediol content of transgenic tobacco was also elevated by at least sixfold. To explore the molecular basis of the enhanced isoprenoid accumulation, transcript levels of the key genes involved in the isoprenoid biosynthesis were measured. Transcript levels of geranylgeranyl diphosphate synthase (GGPP), kaurene synthase (KS), gibberellic acid 20 oxidase (GA20ox), and phytoene desaturase (PD) genes in the transgenic tobacco leaves were about twofold higher compared to the wild type. Therefore, upregulation of down-stream genes involved in biosynthesis of di- and tetraterpenoids due to GbHDR2 overexpression was responsible for elevated production of isoprenoids and enhanced photosynthetic rate. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02887-5.

Transcriptomic Analysis, Cloning, Characterization, and Expression Analysis of Triterpene Biosynthetic Genes and Triterpene Accumulation in the Hairy Roots of Platycodon grandiflorum Exposed to Methyl Jasmonate.

Platycodon grandiflorum is a perennial plant that has been used for medicinal purposes. Specifically, it is widely used in Northern China and Korea for the treatment of various diseases. Terpenoids belong to a group called secondary metabolites and have attracted a wide range of interest. Here, we determined the expressed sequence tag (EST) library of the methyl jasmonate (MeJA)-treated hairy root of P. grandiflorum. In total, 5760 ESTs were obtained, but after deleting the vector sequences and removing poor-quality sequences, a total of 2536 ESTs were attained. Of these, 811 contigs and 1725 singletons were annotated. The data were further analyzed with a focus on the gene families of the terpenoid biosynthetic pathway (TBP). We identified and characterized four TBP genes; among these were three full-length cDNAs encoding PgHMGS, PgMK, and PgMVD, whereas PgHMGR had a partial sequence based on the EST library database. Phylogenetic analysis and a pairwise identity matrix showed that these identified genes were closely related to those of other higher plants. Moreover, the tertiary structure and multiple alignment analysis showed that several distinct conserved motifs were present. Quantitative reverse transcription-polymerase chain reaction results revealed that TBP genes were constitutively expressed in all organs of P. grandiflorum, while the expression of transcript levels of these genes varied distinctly among the organs. Additionally, the total amount of platycosides was highly detected in the root, accumulating in concentrations 7.8 and 2.6 times higher than in the hairy root and stem, respectively, and 1.4 times higher than in the leaf and flower. The highest amount of total phytosterols was found to accumulate in the leaves at 9.3, 9.1, 1.8, and 1.6 times higher than that of the stem, root, hairy root, and flower, respectively. After the hairy root was exposed to the MeJA treatment, the transcript levels of PgHMGS, PgHMGR, PgMK, and PgMVD had significantly increased. The highest level of transcript accumulation occurred at 3 h after initial exposure for most of the genes. Meanwhile, triterpene saponin accumulation increased with the increase in the time of exposure, and at 48 h after initial exposure, the total saponin content was the highest recorded.

Profiles of Secondary Metabolites (Phenolic Acids, Carotenoids, Anthocyanins, and Galantamine) and Primary Metabolites (Carbohydrates, Amino Acids, and Organic Acids) during Flower Development in Lycoris radiata.

This study aimed to elucidate the variations in primary and secondary metabolites during Lycorisradiata flower development using high performance liquid chromatography (HPLC) and gas chromatography time-of-flight mass spectrometry (GC-TOFMS). The result showed that seven carotenoids, seven phenolic acids, three anthocyanins, and galantamine were identified in the L. radiata flowers. Most secondary metabolite levels gradually decreased according to the flower developmental stages. A total of 51 metabolites, including amines, sugars, sugar intermediates, sugar alcohols, amino acids, organic acids, phenolic acids, and tricarboxylic acid (TCA) cycle intermediates, were identified and quantified using GC-TOFMS. Among the hydrophilic compounds, most amino acids increased during flower development; in contrast, TCA cycle intermediates and sugars decreased. In particular, glutamine, asparagine, glutamic acid, and aspartic acid, which represent the main inter- and intracellular nitrogen carriers, were positively correlated with the other amino acids and were negatively correlated with the TCA cycle intermediates. Furthermore, quantitation data of the 51 hydrophilic compounds were subjected to partial least-squares discriminant analyses (PLS-DA) to assess significant differences in the metabolites of L. radiata flowers from stages 1 to 4. Therefore, this study will serve as the foundation for a biochemical approach to understand both primary and secondary metabolism in L. radiata flower development.

Expression Analysis of Phenylpropanoid Pathway Genes and Metabolomic Analysis of Phenylpropanoid Compounds in Adventitious, Hairy, and Seedling Roots of Tartary Buckwheat.

Tartary buckwheat (Fagopyrum tataricum) is an important crop that belongs to the Polygonaceae family, whose roots have received considerable attention due to the presence of compounds with high nutritional and medicinal value. In this study, we aimed to develop an efficient protocol for the culture of adventitious (ARs) and hairy (HRs) roots on a half-strength Schenk and Hildebrandt (SH) medium containing different concentrations of the auxins, α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), and indole-3-acetic acid (IAA). The highest percentage of root induction (91.67%) was achieved with 0.5 mg/L IAA, whereas the greatest number of roots was found in 1 mg/L IAA. In contrast, 0.1 mg/L IBA returned the longest roots. As expected, HRs were obtained from in vitro leaf explants infected with Agrobacterium rhizogenes R1000. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of 11 phenolic pathway genes revealed that five genes (FtPAL, FtC3H, FtHQT, FtCHS, and FtANS) were highly expressed in HRs, whereas only four (FtC4H, FtFLS2, FtDFR, and FtANR), and three (Ft4CL, FtCHI, and FtF3H) were recognized in the ARs and seedling roots (SRs), respectively. HPLC analysis of phenolic compounds in different root cultures showed that the majority of the phenolic compounds (both individual and total) were significantly accumulated in the HRs. Principal component analysis (PCA) identified differences among the three root types, whereby HRs were separated from ARs and SRs based on the amount of phenolic compounds present. Analysis of the metabolic pathway revealed that among the identified metabolites, the 3, 2, and 1 pathways were associated with flavonoid, flavone and flavonol, and phenylpropanoid biosynthesis, respectively. Hierarchical clustering analysis and the heat map showed that the different root cultures presented unique metabolites.

Elevated Ozone Levels Affect Metabolites and Related Biosynthetic Genes in Tartary Buckwheat.

Global climate change and the industrial revolution have increased the concentration of tropospheric ozone, a photochemical air pollutant that can negatively affect plant growth and crop production. In the present study, we investigated the effects of O3 on the metabolites and transcripts of tartary buckwheat. A total of 36 metabolites were identified by gas chromatography coupled with time-of-flight mass spectrometry, and principal component analysis was performed to verify the metabolic differences between nontreated and O3-treated tartary buckwheat. The content of threonic acid increased after 2 days of the O3 treatment, whereas it decreased after 4 days of exposure, after which it gradually increased until the eighth day of exposure. In addition, the levels of most metabolites decreased significantly after the O3 treatment. On the contrary, the levels of two anthocyanins, cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside, increased more than 11.36- and 11.43-fold, respectively, after the O3 treatment. To assess the effect of O3 on the genomic level, we analyzed the expression of anthocyanin biosynthesis pathway genes in O3-treated and nontreated buckwheat using quantitative real-time reverse transcription polymerase chain reaction (PCR). We found that the expression of all anthocyanin pathway genes increased significantly in the O3-treated buckwheat compared to that in the nontreated buckwheat. Altogether, our results suggested that O3 affected the transcripts and metabolites of tartary buckwheat, which would eventually cause phenotypic changes in plants.

Metabolic Profiling of Primary Metabolites and Galantamine Biosynthesis in Wounded Lycoris radiata Callus.

Plants are continuously exposed to abiotic and biotic factors that lead to wounding stress. Different plants exhibit diverse defense mechanisms through which various important metabolites are synthesized. Humans can exploit these mechanisms to improve the efficacy of existing drugs and to develop new ones. Most previous studies have focused on the effects of wounding stress on the different plant parts, such as leaves, stems, and roots. To date, however, no study has investigated the accumulation of primary and galantamine content following the exposure of a callus to wounding stress. Therefore, in the present study, we exposed Lycoris radiata calli to wounding stress and assessed the expression levels of several genes involved in metabolic pathways at various time points (0, 3, 6, 12, 24, 48, 72, and 96 h of exposure). Furthermore, we quantify the primary and galantamine content using gas chromatography-time-of-flight mass spectrometry and the high-performance liquid chromatography qRT-PCR analysis of eight galantamine pathway genes (LrPAL-2, LrPAL-3, LrC4H-2, LrC3H, LrTYDC2, LrN4OMT, LrNNR, and LrCYP96T) revealed that seven genes, except LrN4OMT, were significantly expressed following exposure to wounding stress. Galantamine contents of calli after 3, 6, 12, 24, 48, 72, and 96 h of exposure were respectively 2.5, 2.5, 3.5, 3.5, 5.0, 5.0, and 8.5 times higher than that after 0 h of exposure. Furthermore, a total of 48 hydrophilic metabolites were detected in the 0 h exposed callus and 96 h exposed callus using GC-TOFMS. In particular, a strong positive correlation between galantamine and initial precursors, such as phenylalanine and tyrosine, was observed.