Nitrogenous compounds from Aesculus wilsonii seeds.

Eight nitrogenous compounds including five undescribed ones, aeswilnitrousol A (1), aeswilnitrousosides BD (2-4), and 6-(2-hydroxy-3-methylbutylamino)-8-oxoadenine (5) were isolated from the seeds of Aesculus wilsonii. Their structures and absolute configurations were established based on spectroscopic determination, calculated electronic circular dichroism (ECD) analysis, as well as chemical reaction methods. Among the three known compounds, 7 and 8 were obtained from the Aesculus genus for the first time, and 6 was gained from this plant initially. The 13C NMR data of 7 and 8 were reported for the first time. Moreover, the inhibitory effect of all the isolates against LPS-induced nitric oxide production in RAW264.7 macrophages was evaluated. As a result, compounds 2 and 8 exhibited anti-inflammatory activity in a concentration-dependent manner at 10, 25, and 50 µM.

Expression Patterns and Functional Analysis of Three SmTAT Genes Encoding Tyrosine Aminotransferases in Salvia miltiorrhiza.

Tyrosine aminotransferase (TAT, E.C. 2.6.1.5) is a pyridoxal phosphate-dependent aminotransferase that is widely found in living organisms. It catalyzes the transfer of the amino group on tyrosine to α-ketoglutarate to produce 4-hydroxyphenylpyruvic acid (4-HPP) and is the first enzyme for tyrosine degradation. Three SmTATs have been identified in the genome of Salvia miltiorrhiza (a model medicinal plant), but their information is very limited. Here, the expression profiles of the three SmTAT genes (SmTAT1, SmTAT2, and SmTAT3) were studied. All three genes expressed in different tissues and responded to methyl jasmonate stimuli. SmTAT proteins are localized in the cytoplasm. The recombinant SmTATs were subjected to in vitro biochemical properties. All three recombinant enzymes had TAT activities and SmTAT1 had the highest catalytic activity for tyrosine, followed by SmTAT3. Also, SmTAT1 preferred the direction of tyrosine deamination to 4-HPP, while SmTAT2 preferred transamination of 4-HPP to tyrosine. In parallel, transient overexpression of SmTATs in tobacco leaves revealed that all three SmTAT proteins catalyzed tyrosine to 4-HPP in vivo, with SmTAT1 exhibiting the highest enzymatic activity. Overall, our results lay a foundation for the production of tyrosine-derived secondary metabolites via metabolic engineering or synthetic biology in the future.

Aromatic compounds from the seeds of Dolichos lablab L. with anti-inflammatory activity.

Twenty-four aromatic compounds including five novel ones, dolilabphenosides A (1), B1 (2), B2 (3), C1 (4), and C2 (5) were obtained from the seeds of Dolichos lablab L. Their structures were established based on spectroscopic analyses and chemical reactions. Among the known compounds, 9, 10, 14, 17, 19, and 22-24 were gained from the family Leguminosae for the first time, and 6, 8, 11-13, 15, 16, 18, 20, as well as 21 were firstly identified from Dolichos genus. Moreover, the inhibitory effect evaluation of all the isolates against LPS-induced nitric oxide (NO) production in RAW264.7 macrophages suggested that compounds 1-3, 6, 7, 11-15, 17, 20, 21, 23, 24 exhibited anti-inflammatory activity in a concentration-dependent manner. Moreover, the novel compounds, dolilabphenosides A (1), B1 (2), B2 (3) were found to inhibit the secretion of inflammatory cytokine IL-1ß.

The Dynamic Changes in the Main Substances in Codonopsis pilosula Root Provide Insights into the Carbon Flux between Primary and Secondary Metabolism during Different Growth Stages.

The dried root of Codonopsis pilosula (Franch.) Nannf., referred to as Dangshen in Chinese, is a famous traditional Chinese medicine. Polysaccharides, lobetyolin, and atractylenolide III are the major bioactive components contributing to its medicinal properties. Here, we investigated the dynamic changes of the main substances in annual Dangshen harvested at 12 time points from 20 May to 20 November 2020 (from early summer to early winter). Although the root biomass increased continuously, the crude polysaccharides content increased and then declined as the temperature fell, and so did the content of soluble proteins. However, the content of total phenolics and flavonoids showed an opposite trend, indicating that the carbon flux was changed between primary metabolism and secondary metabolism as the temperature and growth stages changed. The changes in the contents of lobetyolin and atractylenolide III indicated that autumn might be a suitable harvest time for Dangshen. The antioxidant capacity in Dangshen might be correlated with vitamin C. Furthermore, we analyzed the expression profiles of a few enzyme genes involved in the polysaccharide biosynthesis pathways at different growth stages, showing that CpUGpase and CPPs exhibited a highly positive correlation. These results might lay a foundation for choosing cultivars using gene expression levels as markers.

SmJAZ4 interacts with SmMYB111 or SmMYC2 to inhibit the synthesis of phenolic acids in Salvia miltiorrhiza.

Jasmonic acid (JA), as an important plant hormone, can induce the synthesis of phenolic acids in Salvia miltiorrhiza Bunge, a model medicinal plant, but the specific mechanism remains to be further elucidated. JA-responsive SmMYB111 positively regulates the biosynthesis of salvianolic acid B (SalB), but the molecular mechanism is unclear. Here, we found that SmMYB111 directly binds to the promoters of SmTAT1 and SmCYP98A14 and activates their transcription. Yeast two hybrid and bimolecular fluorescent complementation assay indicated that SmMYB111 interacts with SmJAZ4. Furthermore, we systematically characterized the function of SmJAZ4, which was highly expressed in flowers and roots and located in the nucleus and cell membrane. The contents of phenolic acids in the SmJAZ4-overexpressed transgenic plantlets and SmJAZ4-overexpressed transgenic hairy roots decreased significantly. SmJAZ4 interacts with SmMYC2 or SmMYB111 to repress their transcriptional activation activity on target enzyme genes of the biosynthesis pathway of phenolic acids. Overall, the molecular mechanism of SmJAZ4-SmMYC2/SmMYB111 module participating in JA signaling regulation of SalB biosynthesis was elucidated, which give a clue for the molecular regulation of phenolic acids biosynthesis in S. miltiorrhiza.

A Novel R2R3-MYB Transcription Factor SbMYB12 Positively Regulates Baicalin Biosynthesis in Scutellaria baicalensis Georgi.

Scutellaria baicalensis Georgi is an annual herb from the Scutellaria genus that has been extensively used as a traditional medicine for over 2000 years in China. Baicalin and other flavonoids have been identified as the principal bioactive ingredients. The biosynthetic pathway of baicalin in S. baicalensis has been elucidated; however, the specific functions of R2R3-MYB TF, which regulates baicalin synthesis, has not been well characterized in S. baicalensis to date. Here, a S20 R2R3-MYB TF (SbMYB12), which encodes 263 amino acids with a length of 792 bp, was expressed in all tested tissues (mainly in leaves) and responded to exogenous hormone methyl jasmonate (MeJA) treatment. The overexpression of SbMYB12 significantly promoted the accumulation of flavonoids such as baicalin and wogonoside in S. baicalensis hairy roots. Furthermore, biochemical experiments revealed that SbMYB12 is a nuclear-localized transcription activator that binds to the SbCCL7-4, SbCHI-2, and SbF6H-1 promoters to activate their expression. These results illustrate that SbMYB12 positively regulates the generation of baicalin and wogonoside. In summary, this work revealed a novel S20 R2R3-MYB regulator and enhances our understanding of the transcriptional and regulatory mechanisms of baicalin biosynthesis, as well as sheds new light on metabolic engineering in S. baicalensis.

Whole genome and transcriptome reveal flavone accumulation in Scutellaria baicalensis roots.

Scutellaria baicalensis Georgi is a medicinal plant in the Lamiaceae family that contains high levels of 4'-deoxyflavone and other flavonoids in its roots. Therefore, it has strong potential as a plant resource for researching the biosynthesis of specific flavonoids. In this study, we report on a chromosome-level S. baicalensis genome assembled to nine chromosomes (376.81M) using PacBio, HiSeq XTen, and Hi-C assisted assembly. The assembly ratio was 99.22%, the contig N50 was 1.80 million bases, and the scaffold N50 was 40.57 million bases, with 31896 genes being annotated. Comparative genome analysis revealed that S. baicalensis and Salvia miltiorrhiza belonged to the same branch, and diverged 36.3 million years ago. Other typically correlated species were Boea hygrometrica and Sesamum indicum. We investigated the structural genes involved in flavonoid synthesis in combination with transcriptome sequencing analysis for different tissues (roots, stems, flowers, leaves) of purple, pink, and white flowers. The results revealed that S.baiF6H is involved in the accumulation of baicalein and was significantly increased in both purple roots vs. pink roots and white roots vs. pink roots. S.baiMYB gene family expression pattern analysis and co-expression network analysis revealed that S.baiMYB transcription factors primarily regulated the production of flavonoids in S. baicalensis. S.baiMYB serves as a major factor regulating flavonoid synthesis in the roots, where yeast one-hybrid assays revealed that these transcription factors could bind to the promoter regions of structural genes to control the accumulation of flavonoids. Genome and transcriptome sequencing, co-expression analysis, and yeast one-hybrid experiments provided valuable genetic resources for understanding flavonoid biosynthesis in S. baicalensis. These findings contribute to a better understanding of the accumulation of metabolites in Lamiaceae.

Overexpression of SmLAC25 promotes lignin accumulation and decreases salvianolic acid content in Salvia miltiorrhiza.

Laccase (LAC) is a blue multicopper oxidase that contains four copper ions, which is involved in lignin polymerization and flavonoid biosynthesis in plants. Although dozens of LAC genes have been identified in Salvia miltiorrhiza Bunge (a model medicinal plant), most have not been functionally characterized. Here, we explored the expression patterns and the functionality of SmLAC25 in S. miltiorrhiza. SmLAC25 has a higher expression level in roots and responds to methyl jasmonate, auxin, abscisic acid, and gibberellin stimuli. The SmLAC25 protein is localized in the cytoplasm and chloroplasts. Recombinant SmLAC25 protein could oxidize coniferyl alcohol and sinapyl alcohol, two monomers of G-lignin and S-lignin. To investigate its function, we generated SmLAC25-overexpressed S. miltiorrhiza plantlets and hairy roots. The lignin content increased significantly in all SmLAC25-overexpressed plantlets and hairy roots, compared with the controls. However, the concentrations of rosmarinic acid and salvianolic acid B decreased significantly in all the SmLAC25-overexpressed lines. Further studies revealed that the transcription levels of some key enzyme genes in the lignin synthesis pathway (e.g., SmCCR and SmCOMT) were significantly improved in the SmLAC25-overexpressed lines, while the expression levels of multiple enzyme genes in the salvianolic acid biosynthesis pathway were inhibited. We speculated that the overexpression of SmLAC25 promoted the metabolic flux of lignin synthesis, which resulted in a decreased metabolic flux to the salvianolic acid biosynthesis pathway.

Systematic Analysis and Functional Characterization of R2R3-MYB Genes in Scutellaria baicalensis Georgi.

R2R3-MYB transcription factors participate in multiple critical biological processes, particularly as relates to the regulation of secondary metabolites. The dried root of Scutellaria baicalensis Georgi is a traditional Chinese medicine and possesses various bioactive attributes including anti-inflammation, anti-HIV, and anti-COVID-19 properties due to its flavonoids. In the current study, a total of 95 R2R3-MYB genes were identified in S. baicalensis and classified into 34 subgroups, as supported by similar exon-intron structures and conserved motifs. Among them, 93 R2R3-SbMYBs were mapped onto nine chromosomes. Collinear analysis revealed that segmental duplications were primarily responsible for driving the evolution and expansion of the R2R3-SbMYB gene family. Synteny analyses showed that the ortholog numbers of the R2R3-MYB genes between S. baicalensis and other dicotyledons had a higher proportion compared to that which is found from the monocotyledons. RNA-seq data indicated that the expression patterns of R2R3-SbMYBs in different tissues were different. Quantitative reverse transcriptase-PCR (qRT-PCR) analysis showed that 36 R2R3-SbMYBs from different subgroups exhibited specific expression profiles under various conditions, including hormone stimuli treatments (methyl jasmonate and abscisic acid) and abiotic stresses (drought and cold shock treatments). Further investigation revealed that SbMYB18/32/46/60/70/74 localized in the nucleus, and SbMYB18/32/60/70 possessed transcriptional activation activity, implying their potential roles in the regulatory mechanisms of various biological processes. This study provides a comprehensive understanding of the R2R3-SbMYBs gene family and lays the foundation for further investigation of their biological function.

Identification and Characterization of Jasmonic Acid Biosynthetic Genes in Salvia miltiorrhiza Bunge.

Jasmonic acid (JA) is a vital plant hormone that performs a variety of critical functions for plants. Salvia miltiorrhiza Bunge (S. miltiorrhiza), also known as Danshen, is a renowned traditional Chinese medicinal herb. However, no thorough and systematic analysis of JA biosynthesis genes in S. miltiorrhiza exists. Through genome-wide prediction and molecular cloning, 23 candidate genes related to JA biosynthesis were identified in S. miltiorrhiza. These genes belong to four families that encode lipoxygenase (LOX), allene oxide synthase (AOS), allene oxide cyclase (AOC), and 12-OPDA reductase3 (OPR3). It was discovered that the candidate genes for JA synthesis of S. miltiorrhiza were distinct and conserved, in contrast to related genes in other plants, by evaluating their genetic structures, protein characteristics, and phylogenetic trees. These genes displayed tissue-specific expression patterns concerning to methyl jasmonate (MeJA) and wound tests. Overall, the results of this study provide valuable information for elucidating the JA biosynthesis pathway in S. miltiorrhiza by comprehensive and methodical examination.

Sorption and distribution performance of organophosphorus compound (Adenosine 5'-monophosphate)on marine sediments.

In this paper, the kinetics and thermodynamics of Adenosine 5'-monophosphate (AMP) sorption on the sediments obtained from the Yangtze River Estuary and adjacent areas were studied, in combination with the effects of the sediments' properties and media conditions. The kinetics curves could be described by a two-compartment first-order equation, and the equilibrium isotherms fitted well with the modified Langmuir and Freundlich models. The analysis of organic phosphorus (OP) fractions changes after sorption indicated that the contents of exchangeable or loosely sorbed PO increased most significantly. Higher organic matter (OM) of the sediments were favorable for the sorption ability. It was also found that the content of OP and OM in the sediments showed an obvious positive correlation, indicating that organic matter rather than Fe/Al oxides played an important role in the migration of OP in the Yangtze River estuary and its adjacent area. Temperature, salinity and pH of the media influenced the sorption of AMP significantly. Increase of temperature was of benefit to the sorption of AMP, which was a spontaneous and exothermic process according to the calculations of the thermodynamic parameters. The sorption capacity was higher at a moderate salinity in the range of our study. With the pH changing from 3 to 10, the sorption capacity exhibited as a "U-trend" curve.

FvWRKY48 binds to the pectate lyase FvPLA promoter to control fruit softening in Fragaria vesca.

The regulatory mechanisms that link WRKY gene expression to fruit ripening are largely unknown. Using transgenic approaches, we showed that a WRKY gene from wild strawberry (Fragaria vesca), FvWRKY48, may be involved in fruit softening and ripening. We showed that FvWRKY48 is localized to the nucleus and that degradation of the pectin cell wall polymer homogalacturonan, which is present in the middle lamella and tricellular junction zones of the fruit, was greater in FvWRKY48-OE (overexpressing) fruits than in empty vector (EV)-transformed fruits and less substantial in FvWRKY48-RNAi (RNA interference) fruits. Transcriptomic analysis indicated that the expression of pectate lyase A (FvPLA) was significantly downregulated in the FvWRKY48-RNAi receptacle. We determined that FvWRKY48 bound to the FvPLA promoter via a W-box element through yeast one-hybrid, electrophoretic mobility shift, and chromatin immunoprecipitation quantitative polymerase chain reaction experiments, and ß-glucosidase activity assays suggested that this binding promotes pectate lyase activity. In addition, softening and pectin degradation were more intense in FvPLA-OE fruit than in EV fruit, and the middle lamella and tricellular junction zones were denser in FvPLA-RNAi fruit than in EV fruit. We speculated that FvWRKY48 maybe increase the expression of FvPLA, resulting in pectin degradation and fruit softening.

Genome-Wide Characterization of SPL Gene Family in Codonopsis pilosula Reveals the Functions of CpSPL2 and CpSPL10 in Promoting the Accumulation of Secondary Metabolites and Growth of C. pilosula Hairy Root.

SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors play critical roles in regulating diverse aspects of plant growth and development, including vegetative phase change, plant architecture, anthocyanin accumulation, lateral root growth, etc. In the present study, 15 SPL genes were identified based on the genome data of Codonopsis pilosula, a well-known medicinal plant. Phylogenetic analysis clustered CpSPLs into eight groups (G1-G8) along with SPLs from Arabidopsis thaliana, Solanum lycopersicum, Oryza sativa and Physcomitrella patens. CpSPLs in the same group share similar gene structure and conserved motif composition. Cis-acting elements responding to light, stress and phytohormone widely exist in their promoter regions. Our qRT-PCR results indicated that 15 CpSPLs were differentially expressed in different tissues (root, stem, leaf, flower and calyx), different developmental periods (1, 2 and 3 months after germination) and various conditions (NaCl, MeJA and ABA treatment). Compared with the control, overexpression of CpSPL2 or CpSPL10 significantly promoted not only the growth of hairy roots, but also the accumulation of total saponins and lobetyolin. Our results established a foundation for further investigation of CpSPLs and provided novel insights into their biological functions. As far as we know, this is the first experimental research on gene function in C. pilosula.

R2R3-MYB Transcription Factor SmMYB52 Positively Regulates Biosynthesis of Salvianolic Acid B and Inhibits Root Growth in Salvia miltiorrhiza.

The dried root of Salvia miltiorrhiza is a renowned traditional Chinese medicine that was used for over 1000 years in China. Salvianolic acid B (SalB) is the main natural bioactive product of S. miltiorrhiza. Although many publications described the regulation mechanism of SalB biosynthesis, few reports simultaneously focused on S. miltiorrhiza root development. For this study, an R2R3-MYB transcription factor gene (SmMYB52) was overexpressed and silenced, respectively, in S. miltiorrhiza sterile seedlings. We found that SmMYB52 significantly inhibited root growth and indole-3-acetic acid (IAA) accumulation, whereas it activated phenolic acid biosynthesis and the jasmonate acid (JA) signaling pathway. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses revealed that SmMYB52 suppressed the transcription levels of key enzyme-encoding genes involved in the IAA biosynthetic pathway and activated key enzyme-encoding genes involved in the JA and phenolic acid biosynthesis pathways. In addition, yeast one-hybrid (Y1H) and dual-luciferase assay showed that SmMYB52 directly binds to and activates the promoters of several key enzyme genes for SalB biosynthesis, including SmTAT1, Sm4CL9, SmC4H1, and SmHPPR1, to promote the accumulation of SalB. This is the first report of a regulator that simultaneously affects root growth and the production of phenolic acids in S. miltiorrhiza.

SmSPL6 Induces Phenolic Acid Biosynthesis and Affects Root Development in Salvia miltiorrhiza.

Salvia miltiorrhiza is a renowned model medicinal plant species for which 15 SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) family genes have been identified; however, the specific functions of SmSPLs have not been well characterized as of yet. For this study, the expression patterns of SmSPL6 were determined through its responses to treatments of exogenous hormones, including indole acetic acid (IAA), gibberellic acid (GA3), methyl jasmonic acid (MeJA), and abscisic acid (ABA). To characterize its functionality, we obtained SmSPL6-ovexpressed transgenic S. miltiorrhiza plants and found that overexpressed SmSPL6 promoted the accumulation of phenolic acids and repressed the biosynthesis of anthocyanin. Meanwhile, the root lengths of the SmSPL6-overexpressed lines were significantly longer than the control; however, both the fresh weights and lateral root numbers decreased. Further investigations indicated that SmSPL6 regulated the biosynthesis of phenolic acid by directly binding to the promoter regions of the enzyme genes Sm4CL9 and SmCYP98A14 and activated their expression. We concluded that SmSPL6 regulates not only the biosynthesis of phenolic acids, but also the development of roots in S. miltiorrhiza.

Transcription factor SmSPL7 promotes anthocyanin accumulation and negatively regulates phenolic acid biosynthesis in Salvia miltiorrhiza.

Plant-specific SQUAMOSA promoter-binding protein-like (SPL) transcription factors play critical regulatory roles during plant growth and development. However, the functions of SPLs in Salvia miltiorrhiza (SmSPLs; a model medicinal plant) have not been reported. Here, the expression patterns and functions of SmSPL7 were characterized in S. miltiorrhiza. SmSPL7 was expressed in all parts of S. miltiorrhiza, with the highest expression level in the leaves, and could be inhibited by multiple hormones, including methyl jasmonate, auxin, abscisic acid, and gibberellin. SmSPL7 is localized within the nucleus and exhibits robust transcriptional activation activity. Transgenic lines overexpressing SmSPL7 demonstrated pronounced growth inhibition, accompanied by increased anthocyanin accumulation via the genetic activation of the anthocyanin biosynthesis pathway. However, SmSPL7 overexpression significantly decreased salvianolic acid B (SalB) production by inhibiting the transcripts of genes implicated in its biosynthesis pathway. Further analysis indicated that SmSPL7 directly binds to SmTAT1 and Sm4CL9 promoters and blocks their expression to inhibit the biosynthesis of SalB. Taken together, these results indicate that SmSPL7 is a negative regulator of SalB biosynthesis but positively regulates anthocyanin accumulation in S. miltiorrhiza. These findings provide new insights into the functionality of the SPL family while establishing an important foundation for further uncovering the crucial roles of SmSPL7 in the growth of S. miltiorrhiza.

MiR408-SmLAC3 Module Participates in Salvianolic Acid B Synthesis in Salvia miltiorrhiza.

MicroRNAs (miRNAs) are important regulators of gene expression involved in plant development and abiotic stress responses. Recently, miRNAs have also been reported to be engaged in the regulation of secondary plant metabolism. However, there are few functional studies of miRNAs in medicinal plants. For this study, we obtained Sm-miR408 interference lines to investigate the function of Sm-miR408 in a medicinal model plant (Salvia miltiorrhiza). It was found that inhibiting the expression of Sm-miR408 could increase the content of salvianolic acid B and rosmarinic acid in the roots. The SmLAC3 and Sm-miR408 expression patterns were analyzed by qRT-PCR. A 5' RLM-RACE assay confirmed that Sm-miR408 targets and negatively regulates SmLAC3. Moreover, the overexpression of SmLAC3 in S. miltiorrhiza promoted the accumulation of salvianolic acids in the roots. Furthermore, the lignin content of the roots in overexpressed SmLAC3 lines was decreased. Taken together, these findings indicated that Sm-miR408 modulates the accumulation of phenolic acids in S. miltiorrhiza by targeting SmLAC3 expression levels.

Genome-Wide Identification of the TIFY Family in Salvia miltiorrhiza Reveals That SmJAZ3 Interacts With SmWD40-170, a Relevant Protein That Modulates Secondary Metabolism and Development.

Salvia miltiorrhiza Bunge (S. miltiorrhiza), a traditional Chinese medicinal herb, contains numerous bioactive components with broad range of pharmacological properties. By increasing the levels of endogenous jasmonate (JA) in plants or treating them with methyl jasmonate (MeJA), the level of tanshinones and salvianolic acids can be greatly enhanced. The jasmonate ZIM (JAZ) proteins belong to the TIFY family, and act as repressors, releasing targeted transcriptional factors in the JA signaling pathway. Herein, we identified and characterized 15 TIFY proteins present in S. miltiorrhiza. Quantitative reverse transcription PCR analysis indicated that the JAZ genes were all constitutively expressed in different tissues and were induced by MeJA treatments. SmJAZ3, which negatively regulates the tanshinones biosynthesis pathway in S. miltiorrhiza and the detailed molecular mechanism is poorly understood. SmJAZ3 acts as a bait protein to capture and identify a WD-repeat containing the protein SmWD40-170. Further molecular and genetic analysis revealed that SmWD40-170 is a positive regulator, promoting the accumulation of secondary metabolites in S. miltiorrhiza. Our study systematically analyzed the TIFY family and speculated a module of the JAZ-WD40 complex provides new insights into the mechanisms regulating the biosynthesis of secondary metabolites in S. miltiorrhiza.

Selection and evaluation of reference genes for qRT-PCR of Scutellaria baicalensis Georgi under different experimental conditions.

Scutellaria baicalensis Georgi is a famous medicinal plant with its dried roots having been used as a traditional Chinese medicinal for more than 2000 years. Although its genome sequence has previously been published and molecular biology methods have been used to study this species, no suitable internal reference genes have been investigated for standardization of gene expression via quantitative real-time polymerase chain reaction (qRT-PCR). Here, the stabilities of 10 candidate reference genes, ACT11, ACT7, α-TUB, ß-TUB, GAPDH, UBC, RPL, SAM, HSP70, and PP2A, were analyzed by four different procedures of GeNorm, NormFinder, BestKeeper, and RefFinder. Their expression stabilities were evaluated under various conditions, including different tissue types (root, stem, leaf, and flower), hormone stimuli treatments (methyl jasmonate, salicylic acid, and abscisic acid), and abiotic stresses (heavy metal, salt, drought, cold, and wounding). The results indicated that ß-TUB was the most stable gene for all tested samples, while ACT11 was the most unstable. The most stable reference gene was not consistent under different conditions. ß-TUB exhibited the highest stability for different tissue types and abiotic stresses, while for hormone stimuli treatments, ACT7 showed the highest stability. To confirm the applicability of suitable reference genes, we selected to SbF6H and SbF8H as target genes to analyze their expression levels in different tissues. This study helps to the accurate quantification of the relative expression levels of interest genes in S. baicalensis via qRT-PCR analysis.

Hydrocarbons to carboxyl-rich alicyclic molecules: A continuum model to describe biodegradation of petroleum-derived dissolved organic matter in contaminated groundwater plumes.

Relationships between dissolved organic matter (DOM) reactivity and chemical composition in a groundwater plume containing petroleum-derived DOM (DOMHC) were examined by quantitative and qualitative measurements to determine the source and chemical composition of the compounds that persist downgradient. Samples were collected from a transect down the core of the plume in the direction of groundwater flow. An exponential decrease in dissolved organic carbon concentration resulting from biodegradation along the transect correlated with a continuous shift in fluorescent DOMHC from shorter to longer wavelengths. Moreover, ultrahigh resolution mass spectrometry showed a shift from low molecular weight (MW) aliphatic, reduced compounds to high MW, unsaturated (alicyclic/aromatic), high oxygen compounds that are consistent with carboxyl-rich alicyclic molecules. The degree of condensed aromaticity increased downgradient, indicating that compounds with larger, conjugated aromatic core structures were less susceptible to biodegradation. Nuclear magnetic resonance spectroscopy showed a decrease in alkyl (particularly methyl) and an increase in aromatic/olefinic structural motifs. Collectively, data obtained from the combination of these complementary analytical techniques indicated that changes in the DOMHC composition of a groundwater plume are gradual, as relatively low molecular weight (MW), reduced, aliphatic compounds from the oil source were selectively degraded and high MW, alicyclic/aromatic, oxidized compounds persisted.