Comprehensive metabolomic and lipidomic alterations in response to heat stress during seed germination and seedling growth of Arabidopsis.

Temperature affects seed germination and seedling growth, which is a critical and complex stage in plant life cycle. However, comprehensive metabolic basis on temperature implicating seed germination and seedling growth remains less known. Here, we applied the high-throughput untargeted metabolomic and advanced shotgun lipidomic approaches to profile the Arabidopsis 182 metabolites and 149 lipids under moderate (22°C, 28°C) and extreme high (34°C, 40°C) temperatures. Our results showed that a typical feature of the metabolism related to organic acids/derivates and amines was obviously enriched at the moderate temperature, which was implicated in many cellular responses towards tricarboxylic acid cycle (TCA), carbohydrates and amino acids metabolism, peptide biosynthesis, phenylpropanoid biosynthesis and indole 3-acetate (IAA) biosynthetic pathway. Whereas, under extreme high temperatures, there was no seed germination, but 148 out of total 182 metabolites were highly enriched, involving in the galactose metabolism, fatty acid degradation, tryptophan/phenylalanine metabolism, and shikimic acid-mediated pathways especially including alkaloids metabolism and glucosinolate/flavone/flavonol biosynthesis. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) also exhibited the gradually increased tendency from moderate temperatures to extreme high temperatures; whereas phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylglycerol (PG), monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol (SQDG) were contrary to decrease. Another typical feature of the distinguished metabolites between 22°C and 28°C, the TCA, disaccharides, nucleotides, polypeptides, SQDG and the biosynthesis of fatty acids and glucobrassicin-mediated IAA were obviously decreased at 28°C, while amino acids, trisaccharides, PE, PC, PA, PS, MGDG, DGDG and diacylglycerol (DAG) preferred to enrich at 28°C, which characterized the alteration of metabolites and lipids during fast seedling growth. Taking together, our results provided the comprehensive metabolites phenotyping, revealed the characteristics of metabolites necessary for seed germination and/or seedling growth under different temperatures, and provided insights into the different metabolic regulation of metabolites and lipid homeostasis for seed germination and seedling growth.

Rapid quantification of 50 fatty acids in small amounts of biological samples for population molecular phenotyping.

Efficient quantification of fatty-acid (FA) composition (fatty-acidome) in biological samples is crucial for understanding physiology and pathophysiology in large population cohorts. Here, we report a rapid GC-FID/MS method for simultaneous quantification of all FAs in numerous biological matrices. Within eight minutes, this method enabled simultaneous quantification of 50 FAs as fatty-acid methyl esters (FAMEs) in femtomole levels following the efficient transformation of FAs in all lipids including FFAs, cholesterol-esters, glycerides, phospholipids and sphingolipids. The method showed satisfactory inter-day and intra-day precision, stability and linearity (R2 > 0.994) within a concentration range of 2-3 orders of magnitude. FAs were then quantified in typical multiple biological matrices including human biofluids (urine, plasma) and cells, animal intestinal content and tissue samples. We also established a quantitative structure-retention relationship (QSRR) for analytes to accurately predict their retention time and aid their reliable identification. We further developed a novel no-additive retention index (NARI) with endogenous FAMEs reducing inter-batch variations to 15 seconds; such NARI performed better than the alkanes-based classical RI, making meta-analysis possible for data obtained from different batches and platforms. Collectively, this provides an inexpensive high-throughput analytical system for quantitative phenotyping of all FAs in 8-minutes multiple biological matrices in large cohort studies of pathophysiological effects.

Identification of Veratrum Species in Pimacao Based on ITS2 Sequences and Steroidal Alkaloids by a Pseudo-Targeted Metabolomics Method.

Pimacao is a traditional Chinese folk medicine and is the main component of the famous Chinese herbal remedy "Yunnan Baiyao" for its significant analgesic activity in the treatment of wounds. Due to increases in consumption, its wild population is now difficult to find, and adulterant from the same genus has occurred. However, this is challenging to distinguish the species of Veratrum in Pimacao using dried roots and rhizomes or medicinal powder. ITS2 sequences and steroidal alkaloids by the non-targeted and pseudo-targeted metabolomics methods were taken advantage of establishing an effective identification method. Based on the ITS2 sequence, metabolite profiling of steroidal alkaloids and morphological characteristics, the classification of two distinct subspecies in V. mengzeanum has been reinforced. In addition, the new subspecies V. mengzeanum subsp. phuwae was collected in China for the first time. The ITS2 sequence could be used in the identification of V. taliense, V. mengtzeanum, V. stenophyllum, and V. nigrum, but is insufficient for intraspecific identification. Simultaneously, 147 variables were labeled by non-targeted analysis accomplished utilizing an ultra-high-performance liquid chromatography electrospray ionization orbitrap tandem mass spectrometry (UPLC-ESI-QE-Orbitrap-MS) system consisting of an Orbitrap QE HF-X. Followed by a pseudo-targeted analysis method developed for the Qtrap 6500-plus mass spectrometry system coupled with an ESI source, 29 labeled steroidal alkaloids detected by the MRM mode could distinguish between four species. Notably, 25 labeled steroidal alkaloids could distinguish between three closely related species. These have the potential to be used as markers for identification. Furthermore, there were several variables with statistical differences between two subspecies of V. mengtzeanum and populations of V. taliense, V. mengtzeanum, and V. stenophyllum.

Methylation and serum response factor mediated in the regulation of gene ARRDC3 in breast cancer.

Breast cancer poses a serious threat to women's life and health and many factors contribute to breast cancer including gene mutation and epigenetics. Gene ARRDC3 was usually repressed in breast cancer and methylation in promoter was reported to be involved in gene ARRDC3 expression regulation. To this end, the methylation status for gene ARRDC3 promoter was assayed by the Massarray quantitative method. The results indicated that different methylation level CpG sites including CpG_6, CpG_13.14, CpG_17.18, and CpG_25 existed between the tumor tissue and the adjacent normal tissue. In order to further verify whether methylation participated in gene ARRDC3 expression, three cell lines were treated with methylation inhibitor Aza-2'-deoxycytidine including A-375, HepG2, and MDA-MB-231. The results revealed that methylation inhibition observably increased ARRDC3 mRNA expression. Then we confirmed the effective length of promoter through the fluorescence report assay used for further analysis. The results showed that the 1746 bp length promoter produced the maximum fluorescence signal. To obtain the direct evidence that methylation in gene ARRDC3 promoter mediated in ARRDC3 expression regulation, the promoter plasmid was methylated by M.SssI enzyme and subjected to the fluorescence report assay. The results showed that methylation in the promoter markedly suppressed relative luciferase activity. In addition, the ecRNA was also analyzed for the methylation regulation and results illustrated that the ecRNA did not regulate ARRDC3 promoter methylation. However, several methylation CpG sites were found to be around CpG_25 site such as TGCATGG, TTGCAA, TTCGTA, and ATAGTT. These sites provide a good clue for further research in methylation for gene ARRDC3 expression regulation. Furthermore, the possible transcription factors involved in the ARRDC3 regulation were investigated by western blot, luciferase activity analysis and ChiP assay. These results documented that gene ARRDC3 expression was improved by SRF and that the methylation affected the interaction between the promoter and SRF. Lastly, the inhibition role of gene ARRDC3 on breast cancer was probed in vivo and in vitro and our results demonstrated that ARRDC3 could inhibit breast cancer growth through the STAT3 signal pathway. In summary, Gene ARRDC3 was inhibited by promoter methylation and was promoted by transcription factor SRF by binding the promoter region and the inhibition on breast cancer growth was exerted by ARRDC3 through STAT3 signal pathway.