Transcriptome and Metabolome Analysis Reveal the Flavonoid Biosynthesis Mechanism of Abelmoschus manihot L. at Different Anthesis Stages.

Abelmoschus manihot L. (HSK) is a rare and endangered species in the wild that grows on the cliffs of deep mountains. As a natural plant, the chemical composition of HSK is relatively complex, which mainly includes flavonoids, organic acids, polysaccharides, and various trace elements with good effects of clearing away heat, anti-inflammatory, analgesic, and calming nerves, and inhibiting tumor cells. In this experiment, different developmental stages of HSK flowers were used for optimization of the flavonoid extraction and determining method. The antioxidant activities, flavonoid accumulation pattern, and synthesis regulatory network were analyzed using biochemistry, RNA-seq, and UPLC-MS/MS. The total content of flavonoids, vitexin rhamnoside, hyperoside, and rutin in HSK flowers at T3 stage (flower wilting) was significantly higher than in T2 (full flowering) and T1 (bud) stages. Compared with T1 and T2, the antioxidant capacity of the T3 flower alcohol extract was also the strongest, including the total reducing ability, DPPH clearance, OH clearance, O2- clearance, and total antioxidant capacity. A total of 156 flavonoids and 47,179 unigenes were detected by UPLC-MS/MS and RNA-Seq, respectively. The candidate genes and key metabolites involved in flavonoid biosynthesis were identified and the regulatory networks were also analyzed in this study. qRT-PCR test further proved that the gene expression level was consistent with the results of RNA sequence data. The relationship between the gene expression and flavonoid accumulation network provides a theoretical basis for the mining and regulation of functional genes related to the flavonoid biosynthesis and metabolism in Abelmoschus manihot L.

Accumulation and physicochemical properties of starch in relation to eating quality in different parts of taro (Colocasia esculenta) corm.

The accumulation and physicochemical properties of starch affect the eating quality of taro corm. This study aims to investigate the accumulation, morphology, and physicochemical properties of starch from inner and outer tissues in the top, middle, and basal parts of taro corm. Structural and morphological observations showed that the inner tissues of the taro corm accumulated more starch, and the middle tissue had moderate amylose content and the largest granule diameter. Starch from different tissues exhibited A-type orthorhombic structure and similar nuclear magnetic resonance spectrum. The relative crystallinity of starch in the middle tissue was higher than that in the top and basal tissues. Compared with middle and basal tissues, starch from top tissue showed higher peak viscosity, pasting time, swelling power and solubility. Compared with the top and basal tissues, the middle tissue of taro corm exhibited higher index of eating quality including smell, texture, and total evaluation score. The results indicated that starches in various spatial parts of taro corm exhibit differences in accumulation, morphology, structure and physicochemical properties that lead to diverse eating qualities.

Analysis of development, accumulation and structural characteristics of starch granule in wheat grain under nitrogen application.

Nitrogen is one of the most important nutrients for wheat growth and has a critical influence on yield and quality. This study aims to examine how medium nitrogen level (240 kg/hm2) affects the starch granule development, starch accumulation, and structural characteristics of wheat starch. The results showed that nitrogen treatment could reduce the biosynthesis of starch and amylose, delay the degradation of starch in pericarp, and promote the proportion of B-type small starch granule in endosperm compared with those in the N0. In addition, the composition and distribution of starch granules were changed, the crystal structure in the inner lamellae and ordered structure in the external region of starch granules were affected, and the swelling power and solubility of starch during wheat development were increased. The effect of nitrogen treatment on the mRNA expression of enzymes related to starch biosynthesis or degradation varied in different developmental stages. During middle and later grain filling stages, AGPase, GBSSI, and GBSSII were lower, and SSS, SBE, and DBE were higher in N240 than in N0. This study indicated that nitrogen application at booting stage significantly affected the structural characteristics of starch, and ultimately determines its quality.

New insights into the mechanism of storage protein biosynthesis in wheat caryopsis under different nitrogen levels.

Effect of different nitrogen levels (0, 150, and 300 kg hm-2) at booting stage on storage protein biosynthesis and processing quality of wheat was investigated using microstructural and ultrastructural observation, RNA sequencing, and quality analysis in this study. The results showed that the storage protein genes encoding ω- and γ-gliadin and low molecular weight glutenin subunit were upregulated at N150, and the genes encoding α- or ß-gliadin and avenin-like protein were upregulated at N300. Two nitrogen levels induced expression of some interesting regulating genes, such as USE1, STX1B_2_3, SEC23, SEC24, SEC61A, HSP A1_8, HSP20, and HSP90B/TRA1. These regulatory genes were enriched in the KEGG pathway protein export, SNARE interactions in vesicular transport, and protein processing in endoplasmic reticulum. The amount, morphology, and accumulation pattern of protein body in four different endosperm regions in developing caryopsis show different response to N150 and N300, of which N300 had greater influence than N150. N150 and N300 both enhanced the contents of protein components, endosperm fullness, grain hardness, and parameters of processing quality, with the latter showing a greater degree of influence. Contrary to the accumulation pattern of protein body, N300 reduced the ratio of the amount of starch granules to the area ratio of protein body to starch granule. Results suggested that the difference of different nitrogen levels affecting storage protein biosynthesis might be through affecting the expression of the encoding and regulating gene of storage protein.

Characterization of a Family IV uracil DNA glycosylase from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5.

The hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 encodes two uracil DNA glycosylases (UDGs): Tba UDG247 and Tba UDG194. Herein, we characterized biochemically Tba UDG194. Compared with Tba UDG247, Tba UDG194 exhibits different biochemical characteristics. At >85 °C, >90 cleavage percentage was observed, suggesting that Tba UDG194 can remove uracil from DNA at physiological temperature of its host. Thus, the enzyme is the most thermophilic glycosylase among all the reported UDGs. Furthermore, the optimal pH of the enzyme activity was estimated to be 10, which is higher than that of Tba UDG247. Similar to Tba UDG247, Tba UDG194 activity is independent on a divalent metal ion. Mn2+, Zn2+ and Cu2+ display inhibitory effect on the enzyme activity at varied degreed whereas Mg2+ and Ca2+ have no detectable effect on the enzyme activity. In addition, Tba UDG194 is a salt-tolerant enzyme that retains compromised activity at 600 mM NaCl. Furthermore, Tba UDG 194 displays the following substrate preference: U ≈ U/G > U/T > U/A > U/C. The Arrhenius activation energy was estimated to be 20.1 ± 3.4 kcal/mol, theoretically representing the energy barrier for uracil removal from DNA by Tba UDG194. Overall, our observations suggest that Tba UDG194 might be involved in removal of uracil in DNA in Thermococcus cells.

Homoeolog expression bias and expression level dominance in resynthesized allopolyploid Brassica napus.

BACKGROUND: Allopolyploids require rapid genetic and epigenetic modifications to reconcile two or more sets of divergent genomes. To better understand the fate of duplicate genes following genomic mergers and doubling during allopolyploid formation, in this study, we explored the global gene expression patterns in resynthesized allotetraploid Brassica napus (AACC) and its diploid parents B. rapa (AA) and B. oleracea (CC) using RNA sequencing of leaf transcriptomes. RESULTS: We found that allopolyploid B. napus formation was accompanied by extensive changes (approximately one-third of the expressed genes) in the parental gene expression patterns ('transcriptome shock'). Interestingly, the majority (85%) of differentially expressed genes (DEGs) were downregulated in the allotetraploid. Moreover, the homoeolog expression bias (relative contribution of homoeologs to the transcriptome) and expression level dominance (total expression level of both homoeologs) were thoroughly investigated by monitoring the expression of 23,766 B. oleracea-B. rapa orthologous gene pairs. Approximately 36.5% of the expressed gene pairs displayed expression bias with a slight preference toward the A-genome. In addition, 39.6, 4.9 and 9.0% of the expressed gene pairs exhibited expression level dominance (ELD), additivity expression and transgressive expression, respectively. The genome-wide ELD was also biased toward the A-genome in the resynthesized B. napus. To explain the ELD phenomenon, we compared the individual homoeolog expression levels relative to those of the diploid parents and found that ELD in the direction of the higher-expression parent can be explained by the downregulation of homoeologs from the dominant parent or upregulation of homoeologs from the nondominant parent; however, ELD in the direction of the lower-expression parent can be explained only by the downregulation of the nondominant parent or both homoeologs. Furthermore, Gene Ontology (GO) enrichment analysis suggested that the alteration in the gene expression patterns could be a prominent cause of the phenotypic variation between the newly formed B. napus and its parental species. CONCLUSIONS: Collectively, our data provide insight into the rapid repatterning of gene expression at the beginning of Brassica allopolyploidization and enhance our knowledge of allopolyploidization processes.

Novel Insights into miRNA Regulation of Storage Protein Biosynthesis during Wheat Caryopsis Development under Drought Stress.

Drought stress is a significant abiotic stress factor that affects wheat yield and quality. MicroRNA (miRNA) plays an important role in regulating caryopsis development in response to drought stress. However, little is known about the expression characteristics of miRNAs and how they regulate protein accumulation in wheat caryopsis under drought stress. To address this, two small RNA libraries of wheat caryopsis under control and drought stress conditions were constructed and sequenced. A total of 125 miRNAs were identified in the two samples, of which 110 were known and 15 were novel. A total of 1,981 miRNA target genes were predicted and functional annotations were obtained from various databases for 1,641 of them. Four miRNAs were identified as differential expression under drought stress, and the expression patterns of three of them were consistent with results obtained by reverse transcription polymerase chain reaction (RT-PCR) and reverse transcription quantitative polymerase chain reaction (RT-qPCR). Moreover, three miRNA-target pairs showed negative regulation tendency, as revealed by RT-qPCR. Functional enrichment and pathway analysis revealed that four pathways might be involved in storage protein biosynthesis. Furthermore, drought stress significantly increased the accumulation of protein bodies and protein content in wheat endosperm. In summary, our findings suggest that drought stress may enhance storage protein by regulating the expression of miRNAs and their target genes.

A comparative study of the seed structure between resynthesized allotetraploid and their diploid parents.

Brassicaceae is at the forefront of evolution because of its frequent hybridization. Hybridization is responsible for the induction of widespread genetic and phenotype changes, making it important in agricultural production. In this study, we obtained resynthesized allotetraploid Brassica napus by performing interspecific crossing of B. rapa × B. oleracea combined with embryo rescue. We applied light microscopy and electronic microscopy to analyze the microstructure and ultrastructure of seeds of diploid parents and their allotetraploid progeny. Results showed that pigments in the seed coat were mainly distributed in the palisade layer. B. rapa presented the highest amount of pigment followed by B. napus and B. oleracea. B. napus had the thickest palisade layer followed by B. rapa and B. oleracea. The seed coat microsculpturing in B. rapa and B. napus was characterized as reticulate or reticulate-foveate, whereas that in B. oleracea was observed to be rugose and sulcate. The area index of the protein body was higher in central meristematic cells than in parenchyma cells. By contrast, the area index of the oil body was the lowest in central meristematic cells. Protein bodies were found to be heterogeneous with crystal globoids in two diploid parents and resynthesized allotetraploid progenies. Oil bodies consisted of large and small oil bodies, the sizes of which differed between two parents and allotetraploid progenies. Small oil bodies were spheroid, whereas large oil bodies were ovoid in shape. The quantity of oil bodies indicated that oil bodies were spheroid in two parents, ranging in size from 0.12 to 1.18 µm. In comparison, the size of large oil bodies in allotetraploid progenies exceeds 2.0 µm. These findings suggest that the anatomy of resynthesized allotetraploid seeds remarkably differs from that of two diploid parents, and these differences definitely affect the nutritional components of rapeseeds.

Analysis of flavonoids and hydroxycinnamic acid derivatives in rapeseeds (Brassica napus L. var. napus) by HPLC-PDA--ESI(--)-MS(n)/HRMS.

A comprehensive description of flavonoids and hydroxycinnamic acid derivatives in Brassica napus L. var. napus seeds is important to improve rapeseed quality. HPLC-PDA-ESI(-)-MS(n)/HRMS has been broadly applied to study phenolic compounds in plants. In the present study, crude phenolic compounds extracted from rapeseed were subjected to column chromatography, alkaline hydrolysis, and HPLC-PDA-ESI(-)-MS(n)/HRMS analysis. A total of 91 flavonoids and hydroxycinnamic acid derivatives were detected, including 39 kaempferol derivatives, 11 isorhamnetin derivatives, 5 quercetin derivatives, 6 flavanols and their oligomers, and 30 hydroxycinnamic acid derivatives. A total of 78 of these compounds were tentatively identified; of these, 55 were reported for the first time in B. napus L. var. napus and 24 were detected for the first time in the genus Brassica. This research enriches our knowledge of the phenolic composition of rapeseed and provides a reliable guide for the selection of rapeseed with valuable breeding potential.

Use of digital gene expression to discriminate gene expression differences in early generations of resynthesized Brassica napus and its diploid progenitors.

BACKGROUND: Polyploidy is an important evolutionary mechanism in flowering plants that often induces immediate extensive changes in gene expression through genomic merging and doubling. Brassica napus L. is one of the most economically important polyploid oil crops and has been broadly studied as an example of polyploid crop. RNA-seq is a recently developed technique for transcriptome study, which could be in choice for profiling gene expression pattern in polyploids. RESULTS: We examined the global gene expression patterns of the first four generations of resynthesized B. napus (F1-F4), its diploid progenitors B. rapa and B. oleracea, and natural B. napus using digital gene expression analysis. Almost 42 million clean tags were generated using Illumina technology to produce the expression data for 25959 genes, which account for 63% of the annotated B. rapa genome. More than 56% of the genes were transcribed from both strands, which indicate the importance of RNA-mediated gene regulation in polyploidization. Tag mapping of the B. rapa genome generated 19023, 18547, 24383, 20659, 18881, 20692, and 19955 annotated genes for the B. rapa, B. oleracea, F1-F4 of synthesized B. napus, and natural B. napus libraries, respectively. The unambiguous tag-mapped genes in the libraries were functionally categorized via gene ontological analysis. Thousands of differentially expressed genes (DEGs) were identified and revealed the substantial changes in F1-F4. Among the 20 most DEGs are DNA binding/transcription factor, cyclin-dependent protein kinase, epoxycarotenoid dioxygenase, and glycine-rich protein. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the DEGs suggested approximately 120 biological pathways. CONCLUSIONS: The systematic deep sequencing analysis provided a comprehensive understanding of the transcriptome complexity of early generations of synthesized B. napus. This information broadens our understanding of the mechanisms of B. napus polyploidization and contributes to molecular and genetic research by enriching the Brassica database.