Comparison of the Main Metabolites in Different Maturation Stages of Camelliavietnamensis Huang Seeds.

Camellia vietnamensis Huang is an important woody oil crop in China, which has attracted much attention because of its abundant nutritional components and pharmaceutical value. Its seeds undergo a complex series of physiological and biochemical changes during maturation, with consequent alterations in metabolites. In order to investigate the endogenous metabolism of C. vietnamensis on Hainan Island during seed development, in this study, ultra-high-performance liquid tandem chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF-MS) and multivariate statistical analysis (MSA) were used to analyze the differences in the chemical compounds of C. vietnamensis seeds among the four maturation stages. A total of 293 metabolites were identified from the methanol extract of the seeds of C. vietnamensis. Five metabolites, belonging to benzene and substituted derivatives, 5'-deoxyribonucleosides and linear 1,3-diarylpropanoids, were found in all three comparison groups, with consistently down-regulated trends. The Kyoto Encyclopedia of Genes and Genomes (KEGG) results showed that phloretin and 5'-methylthioadenosine were the differentially expressed metabolites when seeds were in the growth periods of S2 and S3, and indole and L-tryptophan were the differentially expressed metabolites when seeds were in the growth periods of S3 and S4. In addition, 34 flavonoid metabolites were detected, of which 4 were differentially expressed. It was indicated that flavonoids dynamically change during all the oil-tea camellia seed development stages. The findings provide data for the better understanding of endogenous metabolic pathways during C. vietnamensis seed development.

Integrated Analysis of Physiological, mRNA Sequencing, and miRNA Sequencing Data Reveals a Specific Mechanism for the Response to Continuous Cropping Obstacles in Pogostemon cablin Roots.

Pogostemon cablin (patchouli) is a commercially important medicinal and industrial crop grown worldwide for its medicinal and aromatic properties. Patchoulol and pogostone, derived from the essential oil of patchouli, are considered valuable components in the cosmetic and pharmaceutical industries. Due to its high application value in the clinic and industry, the demand for patchouli is constantly growing. Unfortunately, patchouli cultivation has suffered due to severe continuous cropping obstacles, resulting in a significant decline in yield and quality. Moreover, the physiological and transcriptional changes in patchouli in response to continuous cropping obstacles remain unclear. This has greatly restricted the development of the patchouli industry. To explore the mechanism underlying the rapid response of patchouli roots to continuous cropping stress, integrated analysis of the transcriptome and miRNA profiles of patchouli roots under continuous and noncontinuous cropping conditions in different growth periods was conducted using RNA sequencing (RNA-seq) and miRNA-seq and complemented with physiological data. The physiological and biochemical results showed that continuous cropping significantly inhibited root growth, decreased root activity, and increased the activity of antioxidant enzymes (superoxide dismutase, peroxidase, and catalase) and the levels of osmoregulators (malondialdehyde, soluble protein, soluble sugar, and proline). Subsequently, we found 4,238, 3,494, and 7,290 upregulated and 4,176, 3,202, and 8,599 downregulated differentially expressed genes (DEGs) in the three growth periods of continuously cropped patchouli, many of which were associated with primary carbon and nitrogen metabolism, defense responses, secondary metabolite biosynthesis, and transcription factors. Based on miRNA-seq, 927 known miRNAs and 130 novel miRNAs were identified, among which 67 differentially expressed miRNAs (DEMIs) belonging to 24 miRNA families were induced or repressed by continuous cropping. By combining transcriptome and miRNA profiling, we obtained 47 miRNA-target gene pairs, consisting of 18 DEMIs and 43 DEGs, that likely play important roles in the continuous cropping response of patchouli. The information provided in this study will contribute to clarifying the intricate mechanism underlying the patchouli response to continuous cropping obstacles. In addition, the candidate miRNAs and genes can provide a new strategy for breeding continuous cropping-tolerant patchouli.

Preparation of Active Peptides from Camellia vietnamensis and Their Metabolic Effects in Alcohol-Induced Liver Injury Cells.

Liver damage seriously affects human health. Over 35% of cases of acute liver damage are caused by alcohol damage. Thus, finding drugs that can inhibit and effectively treat this disease is necessary. This article mainly focuses on the effect of the metabolome physical activity of active peptides in Camellia vietnamensis active peptide (CMAP) and improving liver protection. DEAE Sepharose FF ion-exchange column chromatography was used in separating and purifying crude peptides from Camellia vietnamensis Two components, A1 and A2, were obtained, and the most active A1 was selected. Sephadex G-100 gel column chromatography was used in A1 separation and purification. Three components, Al-1, Al-2, and Al-3, were obtained. Through antioxidant activity in vitro as an index of inspection, the relatively active component A1-2 was removed. Reverse-phase high-performance liquid chromatography showed that the purity of component A1-2 was 93.45%. The extracted CMAPs acted on alcoholic liver injury cells. Metabolomics studies revealed that the up-regulated metabolites were ribothymidine and xanthine; the down-regulated metabolites were hydroxyphenyllactic acid, creatinine, stearoylcarnitine, and inosine. This study provides an effective theoretical support for subsequent research.

Transcriptome analysis of two Pogostemon cablin chemotypes reveals genes related to patchouli alcohol biosynthesis.

Pogostemon cablin, a medicinally and economically important perennial herb, is cultivated around the world due to its medicinal and aromatic properties. Different P. cablin cultivars exhibit different morphological traits and patchouli oil components and contents (especially patchouli alcohol (PA) and pogostone (PO)). According to the signature constituent of the leaf, P. cablin was classified into two different chemotypes, including PA-type and PO-type. To better understand the molecular mechanisms of PA biosynthesis, the transcriptomes of Chinese-cultivated P. cablin cv. PA-type "Nanxiang" (NX) and PO-type "Paixiang" (PX) were analyzed and compared with ribonucleic acid sequencing (RNA-Seq) technology. We obtained a total of 36.83 G clean bases from the two chemotypes, compared them with seven databases and revealed 45,394 annotated unigenes. Thirty-six candidate unigenes participating in the biosynthesis of PA were found in the P. cablin transcriptomes. Overall, 8,390 differentially expressed unigenes were identified between the chemotypes, including 2,467 upregulated and 5,923 downregulated unigenes. Furthermore, six and nine differentially expressed genes (DEGs) were mapped to the terpenoid backbone biosynthetic and sesquiterpenoid and triterpenoid biosynthetic pathways, respectively. One key sesquiterpene synthase gene involved in the sesquiterpenoid and triterpenoid biosynthetic pathways, encoding patchoulol synthase variant 1, was significantly upregulated in NX. Additionally, GC-MS analysis of the two chemotypes in this study showed that the content of PA in NX was significantly higher than that of PX, while the content of PO showed the opposite phenotype. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that the DEG expression tendency was consistent with the transcriptome sequencing results. Overall, 23 AP2/ERF, 13 bHLH, 11 MYB, 11 NAC, three Trihelix, 10 WRKY and three bZIP genes that were differentially expressed may act as regulators of terpenoid biosynthesis. Altogether, 8,314 SSRs were recognized within 6,825 unigenes, with a distribution frequency of 18.32%, among which 1,202 unigenes contained more than one SSR. The transcriptomic characteristics of the two P. cablin chemotypes are comprehensively reported in this study, and these results will contribute to a better understanding of the molecular mechanism of PA biosynthesis. Our transcriptome data also provide a valuable genetic resource for further studies on P. cablin.

Integrative iTRAQ-based proteomic and transcriptomic analysis reveals the accumulation patterns of key metabolites associated with oil quality during seed ripening of Camellia oleifera.

Camellia oleifera (C. oleifera) is one of the four major woody oil-bearing crops in the world and has relatively high ecological, economic, and medicinal value. Its seeds undergo a series of complex physiological and biochemical changes during ripening, which is mainly manifested as the accumulation and transformation of certain metabolites closely related to oil quality, especially flavonoids and fatty acids. To obtain new insights into the underlying molecular mechanisms, a parallel analysis of the transcriptome and proteome profiles of C. oleifera seeds at different maturity levels was conducted using RNA sequencing (RNA-seq) and isobaric tags for relative and absolute quantification (iTRAQ) complemented with gas chromatography-mass spectrometry (GC-MS) data. A total of 16,530 transcripts and 1228 proteins were recognized with significant differential abundances in pairwise comparisons of samples at various developmental stages. Among these, 317 were coexpressed with a poor correlation, and most were involved in metabolic processes, including fatty acid metabolism, α-linolenic acid metabolism, and glutathione metabolism. In addition, the content of total flavonoids decreased gradually with seed maturity, and the levels of fatty acids generally peaked at the fat accumulation stage; these results basically agreed with the regulation patterns of genes or proteins in the corresponding pathways. The expression levels of proteins annotated as upstream candidates of phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) as well as their cognate transcripts were positively correlated with the variation in the flavonoid content, while shikimate O-hydroxycinnamoyltransferase (HCT)-encoding genes had the opposite pattern. The increase in the abundance of proteins and mRNAs corresponding to alcohol dehydrogenase (ADH) was associated with a reduction in linoleic acid synthesis. Using weighted gene coexpression network analysis (WGCNA), we further identified six unique modules related to flavonoid, oil, and fatty acid anabolism that contained hub genes or proteins similar to transcription factors (TFs), such as MADS intervening keratin-like and C-terminal (MIKC_MADS), type-B authentic response regulator (ARR-B), and basic helix-loop-helix (bHLH). Finally, based on the known metabolic pathways and WGCNA combined with the correlation analysis, five coexpressed transcripts and proteins composed of cinnamyl-alcohol dehydrogenases (CADs), caffeic acid 3-O-methyltransferase (COMT), flavonol synthase (FLS), and 4-coumarate: CoA ligase (4CL) were screened out. With this exploratory multiomics dataset, our results presented a dynamic picture regarding the maturation process of C. oleifera seeds on Hainan Island, not only revealing the temporal specific expression of key candidate genes and proteins but also providing a scientific basis for the genetic improvement of this tree species.

Complementary transcriptome and proteome profiling in the mature seeds of Camellia oleifera from Hainan Island.

Camellia oleifera Abel. (C. oleifera), as an important woody tree species producing edible oils in China, has attracted enormous attention due to its abundant unsaturated fatty acids and their associated benefits to human health. To reveal novel insights into the characters during the maturation period of this plant as well as the molecular basis of fatty acid biosynthesis and degradation, we conducted a conjoint analysis of the transcriptome and proteome of C. oleifera seeds from Hainan Island. Using RNA sequencing (RNA-seq) technology and shotgun proteomic method, 59,391 transcripts and 40,500 unigenes were obtained by TIGR Gene Indices Clustering Tools (TGICL), while 1691 protein species were identified from Mass Spectrometry (MS). Subsequently, all genes and proteins were employed in euKaryotic Orthologous Groups (KOG) classification, Gene Ontology (GO) annotation, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to investigate their essential functions. The results indicated that the most abundant pathways were biological metabolic processes. There were 946 unigenes associated with lipid metabolism at the transcriptome level, with 116 proteins at the proteome level; among these, 38 specific proteins were involved in protein-protein interactions, with the majority being related to fatty acid catabolic process. The expression levels of 21 candidate unigenes encoding target proteins were further detected by quantitative real-time polymerase chain reaction (qRT-PCR). Finally, Gas Chromatography Mass Spectrometry (GC-MS) was carried out to determine the fatty acid composition of C. oleifera oil. These findings not only deepened our understanding about the molecular mechanisms of fatty acid metabolism but also offered new evidence concerning the roles of relevant proteins in oil-bearing crops. Furthermore, the lipid-associated proteins recognized in this research might be helpful in providing a reference for the synthetic regulation of C. oleifera oil quality by genetic engineering techniques, thus resulting in potential application in agriculture.

Comparative proteomic analysis of Pogostemon cablin leaves after continuous cropping.

A proteomic approach was used to understand the molecular mechanisms underlying obstacles to the continuous cropping of Pogostemon cablin. We examined differences in protein abundance between control (CK) and continuously cropped (TR) P. cablin leaves at different time points (90, 150, and 210 days after culture). Comparative analysis by two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) revealed 183 differentially expressed protein spots, of which 87 proteins or isoforms were identified using mass spectrometry. Among these differentially expressed proteins (DEPs), 50 proteins or isoforms showed increased abundance and 37 proteins or isoforms showed decreased abundance in the TR sample compared with the abundance in the CK sample. Bioinformatic tools were used to analyze the DEPs. These proteins were classified into 12 categories according to clusters of orthologous groups (COG) analysis, with the majority being involved in post-translational modification, protein turnover, and chaperones, followed by carbohydrate transport and metabolism, and finally, energy production and conversion. Protein-protein interactions revealed that 18 DEPs were involved in energy metabolism, 6 DEPs were involved in stress response, and 4 DEPs were involved in amino acid biosynthesis. Continuous cropping altered the expression of proteins related to energy metabolism, carbohydrate metabolism, and amino acid metabolism in P. cablin leaves. Among these processes, the most affected was energy metabolism, which may be pivotal for resistance to continuous cropping.