Transcriptome Analysis Reveals Roles of Anthocyanin- and Jasmonic Acid-Biosynthetic Pathways in Rapeseed in Response to High Light Stress

Rapeseed (Brassica napus) is one of the major important oil crops worldwide and is largely cultivated in the Qinghai-Tibetan plateau (QTP), where long and strong solar-radiation is well-known. However, the molecular mechanisms underlying rapeseed’s response to light stress are largely unknown. In the present study, the color of rapeseed seedlings changed from green to purple under high light (HL) stress conditions. Therefore, changes in anthocyanin metabolism and the transcriptome of rapeseed seedlings cultured under normal light (NL) and HL conditions were analyzed to dissect how rapeseed responds to HL at the molecular level. Results indicated that the contents of anthocyanins, especially glucosides of cyanidin, delphinidin, and petunidin, which were determined by liquid chromatography-mass spectrometry (LC-MS), increased by 9.6-, 4.2-, and 59.7-fold in rapeseed seedlings exposed to HL conditions, respectively. Next, RNA-sequencing analysis identified 7390 differentially expressed genes (DEGs), which included 4393 up-regulated and 2997 down-regulated genes. Among the up-regulated genes, many genes related to the anthocyanin-biosynthetic pathway were enriched. For example, genes encoding dihydroflavonol reductase (BnDFR) and anthocyanin synthase (BnANS) were especially induced by HL conditions, which was also confirmed by RT-qPCR analysis. In addition, two PRODUCTION OF ANTHOCYANIN PIGMENTATION 2 (BnPAP2) and GLABRA3 (BnGL3) genes encoding MYB-type and bHLH-type transcription factors, respectively, whose expression was also up-regulated by HL stress, were found to be associated with the changes in anthocyanin biosynthesis. Many genes involved in the jasmonic acid (JA)-biosynthetic pathway were also up-regulated under HL conditions. This finding, which is in agreement with the well-known positive regulatory role of JA in anthocyanin biosynthesis, suggests that the JA may also play a key role in the responses of rapeseed seedlings to HL. Collectively, these data indicate that anthocyanin biosynthesis-related and JA biosynthesis-related pathways mediate HL responses in rapeseed. These findings collectively provide mechanistic insights into the mechanisms involved in the response of rapeseed to HL stress, and the identified key genes may potentially be used to improve HL tolerance of rapeseed cultivars through genetic engineering or breeding strategies.

FaTT10, a Laccasel-Like Polyphenol Oxidase Involved in the Accumulation of Proanthocyanins Monomerin Strawberry Fruit

Procyanidins (PAs) is the important flavonoids components in strawberry young fruits, whichis synthesizedthrough direct polymerizationof flavanone-3-ol units. Numerous studies have beendone on the synthesis of PAs, while the oxidation/polymerization processes have not been fully explored. AtTT10, a laccasel-like polyphenol oxidase,was recently reported to be participated in the oxidative polymerization of proanthocyanidins. Whether FaTT10hasapositiveeffect onoxidative polymerization of strawberry fruit PAsis unclear. In this study, FaTT10 in strawberries were isolated and analyzed. Transient overexpression of FaTT10 decreased the accumulation of PAs monomer in strawberry fruit,whereas RNA interference increased the level of anthocyanin and PAs monomer. 4-Dimethylaminocinamaldehyde (DMACA)staining results also showed that lighter blue color in the FaTT10-RNAi fruit and concentrated blue color in the FaTT10-overexpression fruit. Meanwhile, PAs monomer showed a drop-and-rise tendency during strawberry fruit development and ripening.Furthermore, the expression of several flavonoids metabolism-related geneswere affected by the altered FaTT10 gene expression levels, such as L-phenylalanine ammonia-lyase(PAL); dihydroflavonol reductase(DFR);chalcone isomerase(CHI); flavanone 3-hydroxylase(F3H); 4-coumarate:CoA ligase(4CL); Cinnamate 4-hydroxylase(C4H); chalcone synthase(CHS); anthocyanin reductase(ANR); lecoanthocyanidins reductase(LAR) andanthocyanin synthase(ANS). In conclusion, these results revealed that FaTT10 is specifically involved in oxidative polymerization of PAsby regulating the expression of flavonoids metabolism-related genes in strawberry fruits. © 2021 Friends Science Publishers

The MIR-Domain of PbbHLH2 Is Involved in Regulation of the Anthocyanin Biosynthetic Pathway in ”Red Zaosu” (PyrusBretschneideri Rehd.) Pear Fruit

The N-terminal of Myc-like basic helix-loop-helix transcription factors (bHLH TFs) contains an interaction domain, namely the MYB-interacting region (MIR), which interacts with the R2R3-MYB proteins to regulate genes involved in the anthocyanin biosynthetic pathway. However, the functions of MIR-domain bHLHs in this pathway are not fully understood. In this study, PbbHLH2 containing the MIR-domain was identified and its function investigated. The overexpression of PbbHLH2 in ”Zaosu” pear peel increased the anthocyanin content and the expression levels of late biosynthetic genes. Bimolecular fluorescence complementation showed that PbbHLH2 interacted with R2R3-MYB TFs PbMYB9, 10, and 10b in onion epidermal cells and confirmed that MIR-domain plays important roles in the interaction between the MIR-domain bHLH and R2R3-MYB TFs. Moreover, PbbHLH2 bound and activated the dihydroflavonol reductase promoter in yeast one-hybrid (Y1H) and dual-luciferase assays. Taken together these results suggested that the MIR domain of PbbHLH2 regulated anthocyanin biosynthesis in pear fruit peel.

Transcriptome analysis identified genes involved in anthocyanin biosynthesis in Rainbow bamboo (Indosasa hispida MeClure cv. ‘Rainbow’)

Rainbow bamboo (Indosasa hispida) is an ornamental plant, which contains unique red to purple anthocyanin in its culm. However, the biosynthesis and function of anthocyanin in bamboo remains unclear. In this study, RNA-seq was used to investigate the transcriptome of the species and compare the gene expression profiles of red and white culms. The expression levels of genes involved in the anthocyanin biosynthesis pathway were detected using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). In total, 5.92 billion reads were obtained from the culm of Rainbow bamboo, which were assembled into 60,716 unigenes. qRT-PCR showed that the expression levels of anthocyanin biosynthesis-related genes in the red and white culms were higher than that in green leaves and that their levels in the red culm without sheath were higher than that in the white culm with sheath. Transcriptome analysis and qRT-PCR showed that the differences in the expression of genes encoding chalcone isomerase (CHI), dihydroflavonol reductase (DFR), flavonoid 3'-hydroxylase (F3'H), and anthocyanidin 3-O-glycosyltransferase (A3GT) between the culm and leaf were significant. This implies that CHI, DFR, F3'H, and A3GT play important roles in anthocyanin synthesis and accumulation in the culm of Rainbow bamboo.

Leaf:fruit Ratio Affects the Proteomic Profile of Grape Berry Skins

The most obvious effects of a low leaf:fruit (LF) ratio [two leaves for one cluster per shoot (LF2)] on grape (Vitis vinifera) berries are suppressed anthocyanin biosynthesis in the berry skin, decreased berry weight and soluble solids concentration, and increased titratable acidity. In this study, proteins isolated from berry skins grown under low and high LF ratio conditions, LF2 and LF12, respectively, were characterized by two-dimensional gel electrophoresis coupled to mass spectrometry. A survey of ≈600 to 700 spots from berry skin yielded 77 proteins with differential expression between LF12 and LF2 treatments. Of these, the 59 proteins that were identified consisted of 47 proteins that were down-regulated and 12 that were up-regulated under LF2 conditions compared with LF12 conditions. Most proteins involved in metabolism, energy, transcription, protein synthesis, binding function, signal transduction, and cell defense were down-regulated in LF2 berries, whereas two important enzymes of anthocyanin biosynthesis, chalcone synthase and dihydroflavonol reductase, were not detected. Only a few proteins (e.g., two heat shock proteins related to protein fate and nutrient reservoir storage protein) were found to be up-regulated in LF2 berries. This suggested that, with the exception of secondary metabolism, many proteomic events may have an effect on anthocyanin synthesis in the skins responding to LF.

Engineering the Production of Major Catechins byEscherichia coliCarrying Metabolite Genes ofCamellia sinensis

A mimicked biosynthetic pathway of catechin metabolite genes fromC. sinensis, consisting of flavanone 3 hydroxylase (F3H), dihydroflavonol reductase (DFR), and leucoanthocyanidin reductase (LCR), was designed and arranged in two sets of constructs: (a) single promoter in front of F3H and ribosome-binding sequences both in front of DFR and LCR; (b) three different promoters with each in the front of the three genes and ribosome-binding sequences at appropriate positions. RecombinantE. coliBL (DE3) harbouring the constructs were cultivated for 65 h at 26°C in M9 medium consisting of 40 g/L glucose, 1 mM IPTG, and 3 mM eriodictyol. Compounds produced were extracted in ethyl acetate in alkaline conditions after 1 h at room temperature and identified by HPLC. Two of the four major catechins, namely, (−)-epicatechin (0.01 ) and (−)-epicatechin gallate (0.36 mg/L), and two other types ((+)-catechin hydrate (0.13 mg/L) and (−)-catechin gallate (0.04 mg/L)) were successfully produced.