Transcriptome and metabolome analyses of lignin biosynthesis mechanism of Platycladus orientalis.

Background: Platycladus orientalis, as an important plant for ecological protection, is a pioneer tree species for afforestation in arid and barren mountainous areas. Lignin has the functions of water and soil conservation, strengthening plant mechanical strength and resisting adverse environmental effects and plays an important role in the ecological protection benefits of P. orientalis. Methods: In this study, annual dynamic observations of the lignin content in roots, stems and leaves of one-year-old seedlings of a P. orientalis half-sib family were carried out, and combined transcriptome and metabolome analyses were carried out during three key stages of P. orientalis stem development. Results: The lignin contents in roots, stems and leaves of P. orientalis showed extremely significant spatiotemporal differences. In the stems, lignin was mainly distributed in the cell walls of the pith, xylem, phloem, pericyte, and epidermis, with differences in different periods. A total of 226 metabolites were detected in the stem of P. orientalis, which were divided into seven categories, including 10 synthetic precursor compounds containing lignin. Among them, the content of coniferyl alcohol was the highest, accounting for 12.27% of the total content, and caffeyl alcohol was the lowest, accounting for 7.05% only. By annotating the KEGG functions, a large number of differentially expressed genes and differential metabolites were obtained for the comparison combinations, and seven key enzymes and 24 related genes involved in the process of lignin synthesis in P. orientalis were selected. Conclusions: Based on the results of the metabolic mechanism of lignin in P. orientalis by biochemical, anatomical and molecular biological analyzes, the key regulatory pathways of lignin in P. orientalis were identified, which will be of great significance for regulating the lignin content of P. orientalis and improving the adaptability and resistance of this plant.

Transcriptome and chemical analyses revealed the mechanism of flower color formation in Rosa rugosa.

Rosa rugosa is a famous Chinese traditional flower with high ornamental value and well environmental adapt ability. The cultivation of new colorful germplasms to improve monotonous flower color could promote its landscape application. However, the mechanism of flower color formation in R. rugosa remains unclear. In this study, combined analyses of the chemical and transcriptome were performed in the R. rugosa germplasms with representative flower colors. Among the identified anthocyanins, cyanidin 3,5-O-diglucoside (Cy3G5G) and peonidin 3,5-O-diglucoside (Pn3G5G) were the two dominant anthocyanins in the petals of R. rugosa. The sum content of Cy3G5G and Pn3G5G was responsible for the petal color intensity, such as pink or purple, light- or dark- red. The ratio of Cy3G5G to Pn3G5G was contributed to the petal color hue, that is, red or pink/purple. Maintaining both high relative and high absolute content of Cy3G5G may be the precondition for forming red-colored petals in R. rugosa. Cyanidin biosynthesis shunt was the dominant pathway for anthocyanin accumulation in R. rugosa, which may be the key reason for the presence of monotonous petal color in R. rugosa, mainly pink/purple. In the upstream pathway of cyanidin biosynthesis, 35 differentially expressed structural genes encoding 12 enzymes co-expressed to regulate the sum contents of Cy3G5G and Pn3G5G, and then determined the color intensity of petals. RrAOMT, involved in the downstream pathway of cyanidin biosynthesis, regulated the ratio of Cy3G5G to Pn3G5G via methylation and then determined the color hue of petals. It was worth mentioning that significantly higher delphinidin-3,5-O-diglucoside content and RrF3'5'H expression were detected from deep purple-red-flowered 8-16 germplasm with somewhat unique and visible blue hue. Three candidate key transcription factors identified by correlation analysis, RrMYB108, RrC1, and RrMYB114, might play critical roles in the control of petal color by regulating the expression of both RrAOMT and other multiple structural genes. These results provided novel insights into anthocyanin accumulation and flower coloration mechanism in R. rugosa, and the candidate key genes involved in anthocyanin biosynthesis could be valuable resources for the breeding of ornamental plants in future.

Transcriptomic Analyses Reveal Key Genes Involved in Pigment Biosynthesis Related to Leaf Color Change of Liquidambar formosana Hance.

Liquidambar formosana Hance has a highly ornamental value as an important urban greening tree species with bright and beautiful leaf color. To gain insights into the physiological and molecular mechanisms of L. formosana leaf color change, the leaves of three different clones were sampled every ten days from October 13, 2019, five times in total, which are S1, S2, S3, S4 and S5. Transcriptome sequencing was performed at S1 and S4. The chlorophyll content of the three clones decreased significantly, while the anthocyanins content of the three clones increased significantly in the coloring stage. The anthocyanins content of clone 2 was far more than that of the other two clones throughout the period of leaf color change. The transcriptome analysis showed that six DEGs related to anthocyanins biosynthesis, including CHS (chalcone synthase), CHI (chalcone isomerase), F3'H (flavonoid 3'-hydroxylase), DFR (dihydroflavonol 4-reductase), ANS (anthocyanidin synthase) and FLS (flavonol synthase), were found in three clones. Clone 2 has another three DEGs related to anthocyanins biosynthesis, including PAL (Phenylalanine ammonia-lyase), F3'5'H (flavonoid 3',5'-hydroxylase) and UFGT (flavonoid 3-O-glucosyltransferase). We lay a foundation for understanding the molecular regulation mechanism of the formation of leaf color by exploring valuable genes, which is helpful for L. formosana breeding.

24-Epibrassinolide Promotes Fatty Acid Accumulation and the Expression of Related Genes in Styrax tonkinensis Seeds.

Styrax tonkinensis, whose seeds are rich in unsaturated fatty acids (UFAs), is a high oil value tree species, and the seed oil has perfect biodiesel properties. Therefore, the elucidation of the effect of 24-epibrassinolide (EBL) on fatty acid (FA) concentration and the expression of FA biosynthesis-related genes is critical for deeply studying the seed oil in S. tonkinensis. In this study, we aimed to investigate the changing trend of FA concentration and composition and identify candidate genes involved in FA biosynthesis under EBL treatment using transcriptome sequencing and GC-MS. The results showed that 5 µmol/L of EBL (EBL5) boosted the accumulation of FA and had the hugest effect on FA concentration at 70 days after flowering (DAF). A total of 20 FAs were identified; among them, palmitic acid, oleic acid, linoleic acid, and linolenic acid were the main components. In total, 117,904 unigenes were detected, and the average length was 1120 bp. Among them, 1205 unigenes were assigned to 'lipid translations and metabolism' in COG categories, while 290 unigenes were assigned to 'biosynthesis of unsaturated fatty acid' in KEGG categories. Twelve important genes related to FA biosynthesis were identified, and their expression levels were confirmed by quantitative real-time PCR. KAR, KASIII, and accA, encoding FA biosynthesis-related enzymes, all expressed the highest at 70 DAF, which was coincident with a rapid rise in FA concentration during seed development. FAD2 and FATB conduced to UFA and saturated fatty acids (SFA) accumulation, respectively. EBL5 induced the expression of FA biosynthesis-related genes. The concentration of FA was increased after EBL5 application, and EBL5 also enhanced the enzyme activity by promoting the expression of genes related to FA biosynthesis. Our research could provide a reference for understanding the FA biosynthesis of S. tonkinensis seeds at physiological and molecular levels.

Transcriptome Analysis of Endogenous Hormone Response Mechanism in Roots of Styrax tonkinensis Under Waterlogging.

As a promising oil species, Styrax tonkinensis has great potential as a biofuel due to an excellent fatty acid composition. However, frequent flooding caused by global warming and the low tolerance of the species to waterlogging largely halted its expansion in waterlogged areas. To explore endogenous hormones and phytohormone-related molecular response mechanism of S. tonkinensis under waterlogging, we determined 1-aminocyclopropane-1-carboxylic acid (ACC) and three phytohormone content (ABA, abscisic acid; SA, salicylic acid; IAA, indole-3-acetic acid) and analyzed the transcriptome of its seedlings under waterlogged condition of 3-5 cm. The sample collecting time was 0, 9, 24, and 72 h, respectively. It was concluded that ACC presented an upward trend, but other plant hormones showed a downward trend from 0 to 72 h under waterlogging stress. A total of 84,601 unigenes were assembled with a total length of 81,389,823 bp through transcriptome analysis. The GO enrichment analysis of total differentially expressed genes (DEGs) revealed that 4,637 DEGs, 8,238 DEGs, and 7,146 DEGs were assigned into three main GO functional categories in 9 vs. 0 h, 24 vs. 0 h, and 72 vs. 0 h, respectively. We also discovered several DEGs involved in phytohormone synthesis pathway and plant hormone signaling pathway. It was concluded that the decreased transcription of PYL resulted in the weak ABA signal transduction pathway. Moreover, decreased SA content caused by the low-expressed PAL might impact the resistance of S. tonkinensis seedlings under waterlogging stress. Our research may provide a scientific basis for the understanding of the endogenous hormone response mechanism of S. tonkinensis to waterlogging and lay a foundation for further exploration of the waterlogging defect resistance genes of S. tonkinensis and improving its resistance to waterlogging stress.

Transcriptomic and Metabolomic Analysis Unravels the Molecular Regulatory Mechanism of Fatty Acid Biosynthesis in Styrax tonkinensis Seeds under Methyl Jasmonate Treatment.

As the germ of a highly productive oil tree species, Styrax tonkinensis seeds have great potential to produce biodiesel and they have marvelous fatty acid (FA) composition. In order to explore the molecular regulatory mechanism of FA biosynthesis in S. tonkinensis seeds after methyl jasmonate (MJ) application, transcriptomic and metabolomic techniques were adopted so as to dissect the genes that are related to FA biosynthesis and their expression levels, as well as to discover the major FA concentration and composition. The results revealed that 200 µmol/L of MJ (MJ200) increased the crude fat (CF) mass fraction and generated the greatest impact on CF accumulation at 70 days after flowering. Twenty FAs were identified, among which palmitic acid, oleic acid, linoleic acid and linolenic acid were the major FAs, and the presence of MJ200 affected their concentrations variously. MJ200 could enhance FA accumulation through elevating the activity of enzymes that are related to FA synthesis. The number of differentially expressed genes increased with the seeds' development in general. Fatty acid biosynthesis, the biosynthesis of unsaturated fatty acid, fatty acid elongation and glycerolipid metabolism were the main lipid metabolism pathways that were found to be involved. The changes in the expression levels of EAR, KAR, accA, accB and SAD2 were consistent with the changes in the CF mass fraction, indicating that they are important genes in the FA biosynthesis of S. tonkinensis seeds and that MJ200 promoted their expression levels. In addition, bZIP (which was screened by weighted correlation network analysis) also created significant impacts on FA biosynthesis. Our research has provided a basis for further studies on FA biosynthesis that is regulated by MJ200 at the molecular level and has helped to clarify the functions of key genes in the FA metabolic pathway in S. tonkinensis seeds.

Effects of Fruit Shading on Gene and Protein Expression During Starch and Oil Accumulation in Developing Styrax tonkinensis Kernels.

Styrax tonkinensis has great potential as a biofuel feedstock source having industrial oilseeds with excellent fatty acids (FAs) composition and good fuel properties. Photosynthesis in the developing pericarp could affect the carbon distribution in kernel. During kernel development, more carbon sources are allocated to starch rather than lipid, when the pericarp photosynthesis is reduced by fruit shading treatment. After shading the fruits at 50 days after flowering (DAF), samples of shaded fruit (FSK) and controls (CK) were collected at 80 DAF and analyzed using the proteomic method. We identified 3,181 proteins, of which 277 were differentially expressed proteins, all downregulated in the FSK group. There were 56 proteins found involved in carbohydrate metabolism and lipid biosynthesis leading to oil accumulation with their iTRAQ ratios of FSK/CK ranging from 0.7123 to 1.1075. According to the qRT-PCR analyses, the key genes related to FA and triacylglycerol (TAG) biosynthesis were significantly downregulated between 60 and 90 DAF especially at 80 DAF, while the key genes involved in starch biosynthesis and FA desaturase had no significant difference between the two groups at 80 DAF. Fruit shading is a negative treatment for lipid accumulation but not starch accumulation by restraining enzymic protein expression involved in FA and TAG biosynthesis during S. tonkinensis kernel development.

Functional Divergence Analysis of AGL6 Genes in Prunus mume.

The AGAMOUS-LIKE6 (AGL6) lineage is an important clade of MADS-box transcription factors that play essential roles in floral organ development. The genome of Prunus mume contains two homoeologous AGL6 genes that are replicated as gene fragments. In this study, two AGL6 homologs, PmAGL6-1 and PmAGL6-2, were cloned from P. mume and then functionally characterized. Sequence alignment and phylogenetic analyses grouped both genes into the AGL6 lineage. The expression patterns and protein-protein interaction patterns showed significant differences between the two genes. However, the ectopic expression of the two genes in Arabidopsis thaliana resulted in similar phenotypes, including the promotion of flowering, alteration of floral organ structure, participation in the formation of the floral meristem and promotion of pod bending. Therefore, gene duplication has led to some functional divergence of PmAGL6-1 and PmAGL6-2 but their functions are similar. We thus speculated that AGL6 genes play a crucial role in flower development in P. mume.

Proteomic analysis of metabolic mechanisms associated with fatty acid biosynthesis during Styrax tonkinensis kernel development.

BACKGROUND: Styrax tonkinensis is a white-flowered tree with considerable potential as a feedstock source for biodiesel production from the oily seed contained within its nutlike drupes. Transcriptome changes during oil accumulation have been previously reported, but not concurrent changes in the proteome. RESULTS: Using proteomic analysis of samples collected at 50, 70, 100 and 130 days after flowering (DAF), we identified 1472 differentially expressed proteins (DEPs). Based on their expression patterns, we grouped the DEPs into nine clusters and analyzed the pathway enrichment. Proteins related to starch and sucrose metabolism were most abundant at 50 DAF. Proteins involved in fatty acid (FA) biosynthesis were mainly grouped into a cluster that peaked at 70 DAF. Proteins related to protein processing in endoplasmic reticulum had two major patterns, trending either upwards or downwards, while proteins involved in amino acid biosynthesis showed more complex relationships. We identified 42 key enzymes involved in lipid accumulation during kernel development, including the acetyl-CoA carboxylase complex (ACC) and the pyruvate dehydrogenase complex (PDC). One oil body membrane protein, oleosin, continuously increased during kernel development. CONCLUSION: A regulatory network of oil accumulation processes was built based on protein and available transcriptome expression data, which were in good temporal agreement. This analysis placed ACC and PDC in the center of the network, suggesting that the glycolytic provision of substrate plays a central regulatory role in FA biosynthesis and oil accumulation. © 2021 Society of Chemical Industry.

Transcriptome analysis of metabolic pathways associated with oil accumulation in developing seed kernels of Styrax tonkinensis, a woody biodiesel species.

BACKGROUND: Styrax tonkinensis (Pierre) Craib ex Hartwich has great potential as a woody biodiesel species having seed kernels with high oil content, excellent fatty acid composition and good fuel properties. However, no transcriptome information is available on the molecular regulatory mechanism of oil accumulation in developing S. tonkinensis kernels. RESULTS: The dynamic patterns of oil content and fatty acid composition at 11 time points from 50 to 150 days after flowering (DAF) were analyzed. The percent oil content showed an up-down-up pattern, with yield and degree of unsaturation peaking on or after 140 DAF. Four time points (50, 70, 100, and 130 DAF) were selected for Illumina transcriptome sequencing. Approximately 73 million high quality clean reads were generated, and then assembled into 168,207 unigenes with a mean length of 854 bp. There were 5916 genes that were differentially expressed between different time points. These differentially expressed genes were grouped into 9 clusters based on their expression patterns. Expression patterns of a subset of 12 unigenes were confirmed by qRT-PCR. Based on their functional annotation through the Basic Local Alignment Search Tool and publicly available protein databases, specific unigenes encoding key enzymes, transmembrane transporters, and transcription factors associated with oil accumulation were determined. Three main patterns of expression were evident. Most unigenes peaked at 70 DAF, coincident with a rapid increase in oil content during kernel development. Unigenes with high expression at 50 DAF were associated with plastid formation and earlier stages of oil synthesis, including pyruvate and acetyl-CoA formation. Unigenes associated with triacylglycerol biosynthesis and oil body development peaked at 100 or 130 DAF. CONCLUSIONS: Transcriptome changes during oil accumulation show a distinct temporal trend with few abrupt transitions. Expression profiles suggest that acetyl-CoA formation for oil biosynthesis is both directly from pyruvate and indirectly via acetaldehyde, and indicate that the main carbon source for fatty acid biosynthesis is triosephosphate originating from phosphohexose outside the plastid. Different sn-glycerol-3-phosphate acyltransferases are implicated in diacylglycerol biosynthesis at early versus late stages of oil accumulation. Triacylglycerol biosynthesis may be accomplished by both diacylglycerol and by phospholipid:diacylglycerol acyltransferases.

Metabolite Profiling and Classification of Developing Styrax tonkinensis Kernels.

BACKGROUND: Styrax tonkinensis is an economic tree species with high timber, medicine, oil, and ornamental value. Its seed, containing a particularly high oil content, are widely studied for their biodiesel properties by nutritional components and oil body ultrastructure. However, their comprehensive biochemical compositions have not been studied. METHODS: During S. tonkinensis kernel development, we collected samples from four time points for metabolite profiling and classification through gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. RESULTS: A total of 187 and 1556 metabolites were obtained, respectively. All of the metabolites were grouped into 19 and 21 classes by their chemical properties and into 8 clusters based on their change trends, respectively. Among all the metabolites, carboxylic acids and derivatives, flavonoids, fatty acyls, glycerophospholipids, organooxygen compounds, prenol lipids, and steroids and steroid derivatives were the main components. Alanine, glutamine, tryptophan, tyrosine and valine were the five most abundant amino acids. Palmitic acid, stearic acid, oleic acid and linoleic acid were the four major free fatty acids. Flavans, flavonoid glycosides and o-methylated flavonoids were the three major flavonoids. The differential metabolites distributions between different time points were identified. A pathway enrichment was performed, which was mainly focused on three groups, amino acids metabolism, carbon flow from sucrose to lipid and secondary metabolites biosynthesis. CONCLUSIONS: It's the first time to analyze the metabolite fingerprinting for developing S. tonkinensis kernels and identify varied kinds of flavonoids. We performed metabolite profiling, classification and pathway enrichment to assess the comprehensive biochemical compositions. Our results described the change in major metabolites and main metabolic processes during S. tonkinensis kernel development and provided a variety of bases for seed applications as biofuel or medicine.

The nutrient distribution in the continuum of the pericarp, seed coat, and kernel during Styrax tonkinensis fruit development.

BACKGROUND: Styrax tonkinensis is a great potential biofuel as the species contains seeds with a particularly high oil content. Understanding the nutrient distribution in different parts of the fruit is imperative for the development and enhancement of S. tonkinensis as a biodiesel feedstock. METHODS: From 30 to 140 days after flowering (DAF), the development of S. tonkinensis fruit was tracked. The morphology change, nutrient content, and activity of associated enzymes in the continuum of the pericarp, seed coat, and kernel were analyzed. RESULTS: Between 30 and 70 DAF, the main locus of dry matter deposition shifted from the seed coat to the kernel. The water content within the pericarp remained high throughout development, but at the end (130 DAF later) decreased rapidly. The water content within both the seed coat and the kernel consistently declined over the course of the fruit development (30-110 DAF). Between 70 and 80 DAF, the deposition centers for sugar, starch, protein, potassium, and magnesium was transferred to the kernel from either the pericarp or the seed coat. The calcium deposition center was transferred first from pericarp to the seed coat and then to the kernel before it was returned to the pericarp. The sucrose to hexose ratio in the seed coat increased between 30 and 80 DAF, correlating with the accumulation of total soluble sugar, starch, and protein. In the pericarp, the sucrose to hexose ratio peaked at 40 and 100 DAF, correlating with the reserve deposition in the following 20-30 days. After 30 DAF, the chlorophyll concentration of both the pericarp and the seed coat dropped. The maternal unit (the pericarp and the seed coat) in fruit showed a significant positive linear relationship between chlorophyll b/a and the concentration of total soluble sugar. The potassium content had significant positive correlation with starch (ρ = 0.673, p = 0.0164), oil (ρ = 0.915, p = 0.000203), and protein content (ρ = 0.814, p = 0.00128), respectively. The concentration of magnesium had significant positive correlation with starch (ρ = 0.705, p = 0.0104), oil (ρ = 0.913, p = 0.000228), and protein content (ρ = 0.896, p = 0.0000786), respectively. Calcium content had a significant correlation with soluble sugar content (ρ = 0.585, p = 0.0457). CONCLUSIONS: During the fruit development of S. tonkinensis, the maternal unit, that is, the pericarp and seed coat, may act a nutrient buffer storage area between the mother tree and the kernel. The stage of 70-80 DAF is an important time in the nutrient distribution in the continuum of the pericarp, seed coat, and kernel. Our results described the metabolic dynamics of the continuum of the pericarp, seed coat, and kernel and the contribution that a seed with high oil content offers to biofuel.

Transcriptome profile analysis reveals the ontogenesis of rooted chichi in Ginkgo biloba L.

The Ginkgo biloba L. chichi is a unique organ. To explore the molecular mechanisms underlying the ontogenesis of G. biloba chichi, we used RNA-seq to analyse the transcriptome profile of rooted chichi at two developmental stages (ch1 and ch2) and nearby tissues (ck), and each sample had three biological replicates. A total of 57.74 Gb of clean bases were generated in nine cDNA libraries. These bases were de novo assembled into 68,277 unigenes with average length of 844 bp, and 51.47% of the unigenes had a match in at least one public database. The differentially expressed genes (DEGs) in ch1 vs. ck and ch2 vs. ck were 2748 and 8594, respectively. The DEGs involved in the auxin signal pathway, auxin polar transport, storage-related proteins, and the cell cycle pathway might play roles in the ontogenesis of chichi. The quantitative real-time PCR results were closely correlated with transcriptome data. The transcriptome resources generated in the current study provide gene expression profiles and differential expression profiles of G. biloba chichi and offer an essential resource to probe the molecular mechanisms underlying the ontogenesis of G. biloba chichi.

Identification and Characterization of MicroRNAs in Ginkgo biloba var. epiphylla Mak.

Ginkgo biloba, a dioecious plant known as a living fossil, is an ancient gymnosperm that stands distinct from other gymnosperms and angiosperms. Ginkgo biloba var. epiphylla (G. biloba var. epiphylla), with ovules borne on the leaf blade, is an unusual germplasm derived from G. biloba. MicroRNAs (miRNAs) are post-transcriptional gene regulators that play critical roles in diverse biological and metabolic processes. Currently, little is known about the miRNAs involved in the key stage of partly epiphyllous ovule germination in G. biloba var. epiphylla. Two small RNA libraries constructed from epiphyllous ovule leaves and normal leaves of G. biloba var. epiphylla were sequenced on an Illumina/Solexa platform. A total of 82 miRNA sequences belonging to 23 families and 53 putative novel miRNAs were identified in the two libraries. Differential expression analysis showed that 25 conserved and 21 novel miRNAs were differentially expressed between epiphyllous ovule leaves and normal leaves. The expression patterns of partially differentially expressed miRNAs and the transcript levels of their predicted target genes were validated by quantitative real time RT-PCR. All the expression profiles of the 21 selected miRNAs were similar to those detected by Solexa deep sequencing. Additionally, the transcript levels of almost all the putative target genes of 9 selected miRNAs were opposite to those of the corresponding miRNAs. The putative target genes of the differentially expressed miRNAs were annotated with Gene Ontology terms related to reproductive process, metabolic process and responding to stimulus. This work presents a broad range of small RNA transcriptome data obtained from epiphyllous ovule and normal leaves of G. biloba var. epiphylla, which may provide insights into the miRNA-mediated regulation in the epiphyllous ovule germination process.