Cytochrome P450 CYP716A254 catalyzes the formation of oleanolic acid from ß-amyrin during oleanane-type triterpenoid saponins biosynthesis in Anemone flaccida.

Anemone flaccida Fr. Shmidt (Ranunculaceae), known as 'Di Wu' in China, is a perennial herb which has long been used to treat arthritis. The rhizome of A. flaccida contains pharmacologically active components i.e. oleanane-type triterpenoid saponins. Oleanolic acid is natural triterpenoid in plants with diverse biological activities. The biosynthesis of oleanolic acid involves cyclization of 2,3-oxidosqualene to the oleanane-type triterpenoid skeleton, followed by a series of oxidation reactions catalyzed by cytochrome P450 monooxygenase (CYP450). Previously, we identified four possible cytochrome P450 genes belonging to CYP716A subfamily from the transcriptome of A. flaccida. In this study, we identified one of those genes "CYP716A254" encoding a cytochrome P450 monooxygenase from A. flaccida that catalyzes the conversion of the ß-amyrin into oleanolic acid. The heterologous expression of CYP716A254 in yeast resulted in oxidation of ß-amyrin at the C-18 position to oleanolic acid production. These results provide an important basis for further studies of oleanane-type triterpenoid saponins synthesis in A. flaccida.

Methane emission, methanogenic and methanotrophic communities during rice-growing seasons differ in diversified rice rotation systems.

Appropriate crop rotation in rice field is an important measure to maintain soil fertility and rice productivity. However, the effects of different rice rotation systems on methane (CH4) emission and the underlying mechanisms, as well as rice grain yields have not been well assessed. Here, a 2-year field study involving three rice rotation systems (Wh-PR: wheat-flooded rice rotation, Ra-PR: rapeseed-flooded rice rotation, Ra-UR: rapeseed-aerobic rice rotation) was conducted. CH4 emissions, methanogenic and methanotrophic communities and rice grain yields were measured during rice growing seasons to determine which rice rotation pattern can reduce CH4 emissions and improve rice grain yields. The average cumulative CH4 emission was 136.19 kg C ha-1 in Ra-PR system, which was significantly higher than that in Wh-PR and Ra-UR systems by 60.6 % and 14.6-fold, respectively. These results were mainly attributed to the low soil dissolved organic carbon in Wh-PR system and the well aerated soil condition in Ra-UR system, as compared with Ra-PR system. Rice grain yields exhibited no significant differences among the three rotation systems in 2019 and 2020. The abundances of methanogens in Ra-PR system were obviously higher than those in Wh-PR and Ra-UR systems. While the abundances of methanotrophs were comparable between Ra-PR and Wh-PR systems, which exhibited significantly lower abundances than that in Ra-UR system. CH4 fluxes showed markedly positive relations to the abundances of methanogens, while exhibited no relationship with the abundances of methanotrophs. Both methanogenic and methanotrophic community compositions differed considerably in Wh-PR and Ra-UR systems in comparison with Ra-PR system. Specifically, the relative low abundances of Methanothrix and Type I methanotrophs occurred in Wh-PR and Ra-UR systems, whereas Methanosarcina, Methanocella, Methanomassiliicoccus and type II methanotrophs (Methylocystis and Methylosinus) were found in higher relative abundances in Wh-PR and Ra-UR systems. Overall, changing the preceding upland crop types or introducing aerobic rice to substitute flooded rice in rice-based rotation systems could diminish CH4 emissions, mainly by regulating soil properties and eventually changing soil methanogenic and methanotrophic communities.

Appropriate Reference Genes for RT-qPCR Normalization in Various Organs of Anemone flaccida Fr. Schmidt at Different Growing Stages.

Anemone flaccida Fr. Schmidt is a traditional medicinal herb in southwestern China and has multiple pharmacological effects on bruise injuries and rheumatoid arthritis (RA). A new drug with a good curative effect on RA has recently been developed from the extract of A. flaccida rhizomes, of which the main medicinal ingredients are triterpenoid saponins. Due to excessive exploitation, the wild population has been scarce and endangered in a few of its natural habitats and research on the cultivation of the plant commenced. Studies on the gene expressions related to the biosynthesis of triterpenoid saponins are not only helpful for understanding the effects of environmental factors on the medicinal ingredient accumulations but also necessary for monitoring the herb quality of the cultivated plants. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) as a sensitive and powerful technique has been widely used to detect gene expression across tissues in plants at different stages; however, its accuracy and reliability depend largely on the reference gene selection. In this study, the expressions of 10 candidate reference genes were evaluated in various organs of the wild and cultivated plants at different stages, using the algorithms of geNorm, NormFinder and BestKeeper, respectively. The purpose of this study was to identify the suitable reference genes for RT-qPCR detection in A. flaccida. The results showed that two reference genes were sufficient for RT-qPCR data normalization in A. flaccida. PUBQ and ETIF1a can be used as suitable reference genes in most organs at various stages because of their expression stabilitywhereas the PUBQ and EF1Α genes were desirable in the rhizomes of the plant at the vegetative stage.

Correction: Identification of soil P fractions that are associated with P loss from surface runoff under various cropping systems and fertilizer rates on sloped farmland.

[This corrects the article DOI: 10.1371/journal.pone.0179275.].

Enhancement of gama-aminobutyric acid (GABA) and other health-related metabolites in germinated red rice (Oryza sativa L.) by ultrasonication.

Red rice (Oryza sativa L.) that has a red (reddish brown) bran layer in de-hulled rice is known to contain rich biofunctional components. Germination is an effective technique to improve the nutritional quality, digestibility, and flavor of de-hulled rice. Ultrasonication, a form of physical stimulation, has been documented as a novel approach to improve the nutritional quality of plant-based food. This study was undertaken to test the use of ultrasound to enhance the nutritional value of red rice. Ultrasonication (5min, 16W/L) was applied to rice during soaking or after 66h germination. Changes of metabolites (amino acids, sugars, and organic acids) in red rice treated by ultrasonication were determined using a GC/MS plant primary metabolomics analysis platform. Differential expressed metabolites were identified through multivariate statistical analysis. Results showed that γ-aminobutyric acid (GABA) and riboflavin (vitamin B2) in red rice significantly increased after germination for 72h, and then experienced a further increase after treatment by ultrasound at different stages during germination. The metabolomics analysis showed that some plant metabolites, i.e. GABA, O-phosphoethanolamine, and glucose-6-phosphate were significantly increased after the ultrasonic treatment (VIP>1.5) in comparison with the untreated germinated rice. The findings of this study showed that controlled germination with ultrasonic stress is an effective method to enhance GABA and other health-promoted components in de-hulled rice.

Phytoextraction and biodegradation of atrazine by Myriophyllum spicatum and evaluation of bacterial communities involved in atrazine degradation in lake sediment.

The accumulation of atrazine in lake sediments leads to persistent contamination, which may damage the succeeding submerged plants and create potential threats to the lake eco-environment. In this study, the degradation characteristics of atrazine and its detoxication by Myriophyllum spicatum and the associated bacterial community in lake sediments were evaluated. M. spicatum absorbed more than 18-fold the amount of atrazine in sediments and degraded atrazine to hydroxyatrazine (HA), deelthylatrazine (DEA), didealkylatrazine (DDA), cyanuric acid (CYA) and biuret. The formation of biuret suggested for the first time, the ring opening of atrazine in an aquatic plant. The residual rate of atrazine was 6.5 ±â€¯2.0% in M. spicatum-grown sediment, which was significantly lower than the 18.0 ±â€¯2.5% in unplanted sediments on day 60 (P < 0.05). Moreover, on day 15, the increase in contents of HA, CYA and biuret in M. spicatum-grown sediment indicated that M. spicatum promoted the degradation and removal of atrazine following rapid dechlorination. The colonization of M. spicatum and the addition of atrazine altered the structure of the dominant bacterial community in sediments, including effects on Nitrospirae and Acidobacteria. Based on the maximum amount among the genera of atrazine-degrading bacteria, Acetobacter was most likely responsible for the degradation of atrazine. Our findings reveal the natural attenuation of atrazine by aquatic organisms.

Identification of soil P fractions that are associated with P loss from surface runoff under various cropping systems and fertilizer rates on sloped farmland.

Soil phosphorus (P) fractions and runoff P concentration were measured to understand the fate of soil P entering surface runoff water during summer cropping season of different double cropping systems under two fertilizer regimes. The dominant form of runoff P was particulate P (PP). Runoff total P (TP) was higher at the vegetative growth stage and lower at the crop reproductive stage. TP and PP were derived mainly from soil Olsen-P, Al-P and Fe-P and amounts increased with sediment content in runoff water. Runoff P discharge was closely related to the changes in soil P forms. Soil Olsen-P, mainly consisting of some Ca2-P and Al-P, was increased by elevating fertilizer rate. Along with crop growth, there were active interconversions among Olsen-P, Org-P, Fe-P and O-Al-P in the soil, and some available P converted into Ca10-P, with O-Fe-P possibly being a transitional form for this conversion. The oilseed rape/corn system had less runoff TP at the early stage, and wheat/sweet potato system had a lower runoff P at the late stage. Intercropping corn with sweet potato in the field with oilseed rape as a previous crop may be helpful for alleviating runoff P load during the summer in this region.

Occurrence and characterization of CaCO3-P coprecipitation on the leaf surface of Potamogeton crispus in water.

In this paper, the characterization of CaCO3-P coprecipitation on the leaf surface of Potamogeton crispus at various temperatures in pot experiments was investigated. White precipitates occurred on the leaf surfaces during the P. crispus growth period, and the chemical analysis demonstrates that the white precipitates contain Ca and P. The primary constituent of the white precipitates on the leaf of P. crispus was octacalcium phosphate (OCP) and hydroxyapatite. XRD characterization showed that the precipitates mostly consisted of crystals formed by calcium carbonate and hydroxyapatite, and the high calcium/phosphorus ratio indicated that the white coprecipitates were CaCO3-P. The scanning electron microscopy (SEM) and energy-dispersive X-ray spectrometry (EDX) results confirmed that the precipitates on the surface of P. crispus leaves were carbonate-containing hydroxylapatite. In addition, no significant differences was observed in the structure of CaCO3-P coprecipitation between room temperature and consistent temperature treatments, which means that a little change in the temperature cannot change the process of Ca-P coprecipitation. Finally, coprecipitation of CaCO3-P on the leaf surface of P. crispus was proposed based on the morphology and structure analysis of CaCO3-P coprecipitation.

Enhancing Contents of γ-Aminobutyric Acid (GABA) and Other Micronutrients in Dehulled Rice during Germination under Normoxic and Hypoxic Conditions.

Biofortification of staple grains with high contents of essential micronutrients is an important strategy to overcome micronutrient malnutrition. However, few attempts have targeted at γ-aminobutyric acid (GABA), a functional nutrient for aging populations. In this study, two rice cultivars, Heinuo and Xianhui 207, were used to investigate changes in GABA and other nutritional compounds of dehulled rice after germination under normoxic and hypoxic conditions. Forty-one metabolites were identified in both cultivars treated by normoxic germination, whereas the germinated dehulled rice of Heinuo and Xianhui 207 under hypoxic treatment had 43 and 41 metabolites identified, respectively. GABA increased in dehulled rice after germination, especially under hypoxia. Meanwhile, a number of other health-beneficial and/or flavor-related compounds such as lysine and d-mannose increased after the hypoxic treatment. The accumulation of GABA exhibited genotype-specific modes in both normoxic and hypoxic treatments. With regard to GABA production, Xianhui 207 was more responsive to the germination process than Heinuo, whereas Heinuo was more responsive to hypoxia than Xianhui 207. This study provides a promising approach to biofortify dehulled rice with increased GABA and other nutrients through metabolomic-based regulation.

Comprehensive Analysis of the Triterpenoid Saponins Biosynthetic Pathway in Anemone flaccida by Transcriptome and Proteome Profiling.

BACKGROUND: Anemone flaccida Fr. Shmidt (Ranunculaceae), commonly known as 'Di Wu' in China, is a perennial herb with limited distribution. The rhizome of A. flaccida has long been used to treat arthritis as a tradition in China. Studies disclosed that the plant contains a rich source of triterpenoid saponins. However, little is known about triterpenoid saponins biosynthesis in A. flaccida. RESULTS: In this study, we conducted the tandem transcriptome and proteome profiling of a non-model medicinal plant, A. flaccida. Using Illumina HiSeq 2000 sequencing and iTRAQ technique, a total of 46,962 high-quality unigenes were obtained with an average sequence length of 1,310 bp, along with 1473 unique proteins from A. flaccida. Among the A. flaccida transcripts, 36,617 (77.97%) showed significant similarity (E-value < 1e (-5)) to the known proteins in the public database. Of the total 46,962 unigenes, 36,617 open reading frame (ORFs) were predicted. By the fragments per kilobases per million reads (FPKM) statistics, 14,004 isoforms/unigenes were found to be upregulated, and 14,090 isoforms/unigenes were down-regulated in the rhizomes as compared to those in the leaves. Based on the bioinformatics analysis, all possible enzymes involved in the triterpenoid saponins biosynthetic pathway of A. flaccida were identified, including cytosolic mevalonate pathway (MVA) and the plastidial methylerythritol pathway (MEP). Additionally, a total of 126 putative cytochrome P450 (CYP450) and 32 putative UDP glycosyltransferases were selected as the candidates of triterpenoid saponins modifiers. Among them, four of them were annotated as the gene of CYP716A subfamily, the key enzyme in the oleanane-type triterpenoid saponins biosynthetic pathway. Furthermore, based on RNA-Seq and proteome analysis, as well as quantitative RT-PCR verification, the expression level of gene and protein committed to triterpenoids biosynthesis in the leaf versus the rhizome was compared. CONCLUSION: A combination of the de novo transcriptome and proteome profiling based on the Illumina HiSeq 2000 sequencing platform and iTRAQ technique was shown to be a powerful method for the discovery of candidate genes, which encoded enzymes that were responsible for the biosynthesis of novel secondary metabolites in a non-model plant. The transcriptome data of our study provides a very important resource for the understanding of the triterpenoid saponins biosynthesis of A. flaccida.

The Transcript Profile of a Traditional Chinese Medicine, Atractylodes lancea, Revealing Its Sesquiterpenoid Biosynthesis of the Major Active Components.

Atractylodes lancea (Thunb.) DC., named "Cangzhu" in China, which belongs to the Asteraceae family. In some countries of Southeast Asia (China, Thailand, Korea, Japan etc.) its rhizome, commonly called rhizoma atractylodis, is used to treat many diseases as it contains a variety of sesquiterpenoids and other components of medicinal importance. Despite its medicinal value, the information of the sesquiterpenoid biosynthesis is largely unknown. In this study, we investigated the transcriptome analysis of different tissues of non-model plant A. lancea by using short read sequencing technology (Illumina). We found 62,352 high quality unigenes with an average sequence length of 913 bp in the transcripts of A. Lancea. Among these, 43,049 (69.04%), 30,264 (48.53%), 26,233 (42.07%), 17,881 (28.67%) and 29,057(46.60%) unigenes showed significant similarity (E-value<1e(-5)) to known proteins in Nr, KEGG, SWISS-PROT, GO, and COG databases, respectively. Of the total 62,352 unigenes, 43,049 (Nr Database) open reading frames were predicted. On the basis of different bioinformatics tools we identify all the enzymes that take part in the terpenoid biosynthesis as well as five different known sesquiterpenoids via cytosolic mevalonic acid (MVA) pathway and plastidal methylerythritol phosphate (MEP) pathways. In our study, 6, 864 Simple Sequence Repeats (SSRs) were also found as great potential markers in A. lancea. This transcriptomic resource of A. lancea provides a great contribution in advancement of research for this specific medicinal plant and more specifically for the gene mining of different classes of terpenoids and other chemical compounds that have medicinal as well as economic importance.