Polyphenol oxidases regulate pollen development through modulating flavonoids homeostasis in tobacco.

Pollen development is critical in plant reproduction. Polyphenol oxidases (PPOs) genes encode defense-related enzymes, but the role of PPOs in pollen development remains largely unexplored. Here, we characterized NtPPO genes, and then investigated their function in pollen via creating NtPPO9/10 double knockout mutant (cas-1), overexpression 35S::NtPPO10 (cosp) line and RNAi lines against all NtPPOs in Nicotiana tabacum. NtPPOs were abundantly expressed in the anther and pollen (especially NtPPO9/10). The pollen germination, polarity ratio and fruit weights were significantly reduced in the NtPPO-RNAi and cosp lines, while they were normal in cas-1 likely due to compensation by other NtPPO isoforms. Comparisons of metabolites and transcripts between the pollen of WT and NtPPO-RNAi, or cosp showed that decreased enzymatic activity of NtPPOs led to hyper-accumulation of flavonoids. This accumulation might reduce the content of ROS. Ca2+ and actin levels also decreased in pollen of the transgenic lines.Thus, the NtPPOs regulate pollen germination through the flavonoid homeostasis and ROS signal pathway. This finding provides novel insights into the native physiological functions of PPOs in pollen during reproduction.

Microbial transformation of osthole by human intestinal fungi and anti-osteoporosis activities of its metabolites.

Osthole is one of the major constituents in Cnidium monnieri (L.) Cuss. and possesses anti-osteoporosis activity. In this work, the biotransformation of osthole was performed based on the human intestinal fungi Mucor circinelloides. Six metabolites including three new metabolites (S2, S3, S4) were obtained, and their chemical structures were elucidated by spectroscopic data analysis. The major biotransformation reactions involved hydroxylation and glycosylation. In addition, all metabolites were evaluated for their anti-osteoporosis activity using MC3T3-E1 cells. The results demonstrated that S4, S5 and S6 could significantly promote MC3T3-E1 cell growth compared to osthole.

Enhanced profiling and quantification of ginsenosides from mountain-cultivated ginseng and comparison with garden-cultivated ginseng.

Panax ginseng can be generally divided into mountain-cultivated ginseng (MCG) and garden-cultivated ginseng (GCG). The market price of MCG is significantly higher than that of GCG. However, the chemical compositions of MCG and the differences from GCG remained unclear. In this study, an integrated strategy combing an offline two-dimensional liquid chromatography separation, LTQ-orbitrap dual mode acquisition, and Q-trap full quantification/quasi-quantification was proposed to explore and compare the chemical compositions of MCG. Consequently, 559 ginsenosides were characterized, among which 437 ginsenosides were in-depth characterized with α-chain and ß-chain annotated. Subsequently, enhanced quantification of 213 ginsenosides was conducted in 57 batches of MCG and GCG. Ginsenosides were found more abundant in MCG than GCG. In addition, 25-year-old MCG could be distinctly differentiated from 15/20-year-old MCG. This strategy facilitated the enhanced profiling and comparison of ginsenosides, improved the quality control tactics of MCG and provided a reference approach for other ginseng related products.

[Identification of chemical constituents of Xiaochengqi Decoction by UPLC-Q-TOF/Fast DDA combined with UNIFI software].

Based on the combination of ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF) and Waters UNIFI software, the chemical constituents of the classic prescription Xiaochengqi Decoction were qualitatively analyzed and identified. The UPLC conditions are as follows: Acquity HSS T3 reverse phase column(2.1 mm ×100 mm, 1.8 µm), column temperature of 30 ℃, mobile phase of 0.1% formic acid aqueous solution(A)-acetonitrile(B), and flow rate of 0.3 mL·min~(-1). High-resolution MS data of Xiaochengqi Decoction were collected in ESI~(+/-) modes by Fast DDA. The structures of the chemical constituents were tentatively characterized or identified by UNIFI software according to the retention time of reference standards and characteristic fragment ions in MS profile, and literature data. A total of 233 components in Xiaochengqi Decoction were identified, with 93 from wine-processed Rhei Radix et Rhizoma, 104 from bran-processed Aurantii Fructus Immaturus, and 36 from ginger-processed Magnoliae Officinalis Cortex. These 233 components included anthraquinones, flavonoids, lignans, alkaloids, coumarins, and phenylethanoid glycosides. The result provided experimental evidence for the further study on establishment of quality standard and product development of the formula.

Software Assisted Multi-Tiered Mass Spectrometry Identification of Compounds in Traditional Chinese Medicine: Dalbergia odorifera as an Example.

The complexity of metabolites in traditional Chinese medicine (TCM) hinders the comprehensive profiling and accurate identification of metabolites. In this study, an approach that integrates enhanced column separation, mass spectrometry post-processing and result verification was proposed and applied in the identification of flavonoids in Dalbergia odorifera. Firstly, column chromatography fractionation, followed by liquid chromatography-tandem mass spectrometry was used for systematic separation and detection. Secondly, a three-level data post-processing method was applied to the identification of flavonoids. Finally, fragmentation rules were used to verify the flavonoid compounds. As a result, a total of 197 flavonoids were characterized in D. odorifera, among which seven compounds were unambiguously identified in level 1, 80 compounds were tentatively identified by MS-DIAL and Compound Discoverer in level 2a, 95 compounds were annotated by Compound discoverer and Peogenesis QI in level 2b, and 15 compounds were exclusively annotated by using SIRIUS software in level 3. This study provides an approach for the rapid and efficient identification of the majority of components in herbal medicines.

An enhanced strategy integrating offline two-dimensional separation with data independent acquisition mode and deconvolution: Characterization of metabolites of Uncaria rhynchophylla in rat plasma as a case.

The importance to clarify the drug metabolites is beyond doubt in view of their potential efficacy and safety. However, due to the complex matrix interference, relatively low content and the co-eluting effect, it is of a great challenge to comprehensively and systematically characterize the metabolites in vivo, especially for the traditional Chinese medicines (TCMs) due to the numerous types of components. In the present study, a comprehensive off-line two-dimensional separation system combining with data independent acquisition (DIA) mode and multi-dimensional data deconvolution method was established for chromatographic separation, data acquisition and data procession of indole alkaloids in rat plasma after intragastrically administrated with the extract of Uncaria rhynchophylla at the dose of 1 g/kg. The orthogonality of the off-line 2D separation system consisting of HILIC for first-dimensional separation and the PRLC for second-dimensional separation was valuated with the "asterisk" equations, and the results showed that off-line 2D separation system had passable orthogonality (A0 = 53.3%). Furthermore, the DIA mode was applied to capture MS/MS spectra in view of its advantage in acquiring MS data, and an effective multi-dimensional deconvolution method integrating the calculation of chemical formula, the extraction of diagnostic ion, the filter of ring double bond (RDB) and the judgement of neutral loss was established to parse the spectra for the complicated DIA data for comprehensive analysis of metabolites in rat plasma. Ultimately, a total of 127 indole alkaloids were tentatively characterized, and the main metabolic pathways were inferred as demethylation, dehydrogenation, hydroxylation and deglycosylation. The off-line two-dimensional separation system was applied for the comprehensive characterization of metabolites in vivo for the first time. This study suggested a new approach to enable the enrichment, separation and analysis of the low content components in vivo.

Identification of pollen and pistil polygalacturonases in Nicotiana tabacum and their function in interspecific stigma compatibility.

KEY MESSAGE: The pollen and pistil polygalacturonases in Nicotiana tabacum were identified and found to regulate pollen tube growth and interspecific compatibility. Polygalacturonase (PG) is one of the enzymes catalyzing the hydrolysis of pectin. This process plays important roles in the pollen and pistil. In this research, the pollen and pistil PGs in Nicotiana tabacum (NtPGs) were identified, and their expression, localization and the potential function in the pollen and interspecific stigma incompatibility were explored. The results showed that 118 NtPGs were retrieved from the genome of N. tabacum. The phylogenetic tree and RT-qPCR analysis led to the identification of 10 pollen PGs; among them, two, seven and one showed specifically higher expression levels in the early development of anthers, during pollen maturation and in mature anthers, respectively, indicating their function difference. Immunofluorescence analysis showed that PGs were located in the cytoplasm of (1) mature pollen and (2) in vitro grown pollen tubes, as well as in the wall of in vivo grown pollen tubes. Four NtPGs in clade A were identified as the pistil PGs, and the pistil PGs were not found in clade E. Significantly higher PGs expression was recorded after incompatible pollination in comparison with the compatible stigma, indicating a potential function of PGs in regulating stigma incompatibility. The influence of PGs on pollen tube growth was explored in vitro and partly in vivo, showing that high PGs activity inhibited pollen tube growth. The application of PGs on the otherwise compatible stigma resulted in pollen tube growth inhibition or failure of germination. These results further supported that increased PGs expression in incompatible stigma might be partially responsible for the interspecific stigma incompatibility in Nicotiana.

Extending the generality of leaf economic design principles in the cycads, an ancient lineage.

Cycads are the most ancient lineage of living seed plants, but the design of their leaves has received little study. We tested whether cycad leaves are governed by the same fundamental design principles previously established for ferns, conifers and angiosperms, and characterized the uniqueness of this relict lineage in foliar trait relationships. Leaf structure, photosynthesis, hydraulics and nutrient composition were studied in 33 cycad species from nine genera and three families growing in two botanical gardens. Cycads varied greatly in leaf structure and physiology. Similarly to other lineages, light-saturated photosynthetic rate per mass (Am ) was related negatively to leaf mass per area and positively to foliar concentrations of chlorophyll, nitrogen (N), phosphorus and iron, but unlike angiosperms, leaf photosynthetic rate was not associated with leaf hydraulic conductance. Cycads had lower photosynthetic N use efficiency and higher photosynthetic performance relative to hydraulic capacity compared with other lineages. These findings extend the relationships shown for foliar traits in angiosperms to the cycads. This functional convergence supports the modern synthetic understanding of leaf design, with common constraints operating across lineages, even as they highlight exceptional aspects of the biology of this key relict lineage.