Identification and Functional Verification of the Glycosyltransferase Gene Family Involved in Flavonoid Synthesis in Rubus chingii Hu.

Glycosylation is catalyzed by UDP-glycosyltransferase (UGT) and plays an important role in enriching the diversity of flavonoids. Rubus plants contain a lot of natural flavonoid glycosides, which are important plants with a homology of medicine and food. However, information about the Rubus UGT gene family is very limited. In this study, we carried out genome-wide analysis and identified the 172, 121, 130, 121 UGT genes in R. chingii, R. corchorifolius, R. idaeus, and R. occidentalis, respectively, and divided them into 18 groups. The analysis of the protein motif and gene structure showed that there were structural and functional conservations in the same group, but there were differences among different groups. Gene replication analysis showed that raspberry and dicotyledons had a higher homology. The expansion of the UGTs gene family was mainly driven by tandem replication events, and experienced purified selection during the long evolution of the raspberry. Cis-acting element analysis showed that they were related to plant growth and development, hormone regulation, and stress response. In addition, according to a comprehensive analysis of the co-expression network constructed by transcriptome data and phylogenetic homology, RchUGT169 was identified as a flavonoid glucosyltransferase. Through the transient expression in tobacco, it was verified that RchUGT169 could catalyze the conversion of kaempferol and quercetin to the corresponding flavonoid glycosides. In conclusion, this research enriched the understanding of the diversity of UGTs in Rubus and determined that RcUGT169 can catalyze flavonoids.

Comparative Analysis of Chloroplast Genomes of "Tiantai Wu-Yao" (Lindera aggregata) and Taxa of the Same Genus and Different Genera.

Lindera aggregata is a species of the Lauraceae family, which has important medicinal, economic and ornamental values. In this study, we sequenced, assembled and annotated the chloroplast genome of L. aggregata and reannotated and corrected eight unverified annotations in the same genus. The chloroplast genomes taxa from Lindera and from different genera of Lauraceae were compared and analyzed, and their phylogenetic relationship and divergence time were speculated. All the 36 chloroplast genomes had typical quadripartite structures that ranged from 150,749 to 154,736 bp in total length. These genomes encoded 111-112 unique genes, including 78-79 protein-coding genes, 29-30 tRNA and 4 rRNA. Furthermore, there were 78-97 SSRs loci in these genomes, in which mononucleotide repeats were the most abundant; there were 24-49 interspersed repeats, and forward repeat types were the most frequent. The codon bias patterns of all species tended to use codons ending with A or U. Five and six highly variable regions were identified within genus and between genera, respectively, and three common regions (ycf1, ndhF-rpl32 and rpl32-trnL) were identified, which can be used as important DNA markers for phylogeny and species identification. According to the evaluation of the Ka/Ks ratio, most of the genes were under purifying selection, and only 10 genes were under positive selection. Finally, through the construction of the evolutionary tree of 39 chloroplast genomes, the phylogenetic relationship of Lauraceae was clarified and the evolutionary relationship of Lindera was revealed. The species of genus Lindera experienced rapid adaptive radiation from Miocene to Pleistocene. The results provided valuable insights for the study of chloroplast genomes in the Lauraceae family, especially in the genus Lindera.

Genome-Wide Identification of Expansins in Rubus chingii and Profiling Analysis during Fruit Ripening and Softening.

Improving fruit size or weight, firmness, and shelf life is a major target for horticultural crop breeding. It is associated with the depolymerization and rearrangement of cell components, including pectin, hemicellulose, cellulose, and other structural (glyco)proteins. Expansins are structural proteins to loosen plant cell wall polysaccharides in a pH-dependent manner and play pivotal roles in the process of fruit development, ripening, and softening. Rubus chingii Hu, a unique Chinese red raspberry, is a prestigious pharmaceutical and nutraceutical dual-function food with great economic value. Thirty-three RchEXPs were predicted by genome-wide identification in this study, containing twenty-seven α-expansins (EXPAs), three ß-expansins (EXPBs), one expansin-like A (EXPLA), and two expansin-like B (EXPLBs). Subsequently, molecular characteristics, gene structure and motif compositions, phylogenetic relationships, chromosomal location, collinearity, and regulatory elements were further profiled. Furthermore, transcriptome sequencing (RNA-seq) and real-time quantitative PCR assays of fruits from different developmental stages and lineages showed that the group of RchEXPA5, RchEXPA7, and RchEXPA15 were synergistically involved in fruit expanding and ripening, while another group of RchEXPA6 and RchEXPA26 might be essential for fruit ripening and softening. They were regulated by both abscisic acid and ethylene and were collinear with phylogenetic relationships in the same group. Our new findings laid the molecular foundation for improving the fruit texture and shelf life of R. chingii medicinal and edible fruit.

Pseudomonas bijieensis Strain XL17 within the P. corrugata Subgroup Producing 2,4-Diacetylphloroglucinol and Lipopeptides Controls Bacterial Canker and Gray Mold Pathogens of Kiwifruit.

Kiwifruit worldwide suffers from the devastating diseases of bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) and gray mold caused by Botrytis cinerea. Here, an endophytic bacterium XL17 isolated from a rape crown gall was screened out for its potent antagonistic activities against Psa and B. cinerea. Strain XL17 and its cell-free culture filtrate (CF) inhibited the growth of Psa and B. cinerea, Psa-associated leaf necrosis, and B. cinerea-associated kiwifruit necrosis. Electron microscopy showed that XL17 CF could damage the cell structures of Psa and B. cinerea. Genome-based taxonomy revealed that strain XL17 belongs to Pseudomonas bijieensis within the P. corrugata subgroup of the P. fluorescens species complex. Among the P. corrugata subgroup containing 31 genomospecies, the presence of the phl operon responsible for the biosynthesis of the phenolic polyketide 2,4-diacetylphloroglucinol (DAPG) and the absence of the lipopeptide/quorum sensing island can serve as the genetic marker for the determination of a plant-protection life style. HPLC detected DAPG in extracts from XL17 CF. MALDI-TOF-MS analysis revealed that strain XL17 produced cyclic lipopeptides of the viscosin family and orfamide family. Together, phenotypic, genomic, and metabolic analyses identified that P. bijieensis XL17 producing DAPG and cyclic lipopeptides can be used to control bacterial canker and gray mold pathogens of kiwifruit.

Development of fruit color in Rubus chingii Hu (Chinese raspberry): A story about novel offshoots of anthocyanin and carotenoid biosynthesis.

Rubus chingii, is widely distributed in many Asian countries and well known for its medicinal and dietary properties. Diversity of fruit color in raspberry has been attributed to the presence of either anthocyanins or carotenoids. In this study, we investigated anthocyanins and carotenoids, and their biosynthesis by LC-MS/MS. Six anthocyanins mainly consisted of flavanol-anthocyanins while five carotenoids mainly consisted of ß-citraurin esters. Flavanol-anthocyanins were produced from an offshoot of the anthocyanin biosynthesis, which started with biosynthesis of flavanols and anthocyanidin by leucoanthocyanidin reductase (LAR)/anthocyanidin reductase (ANR) and anthocyanidin synthase (ANS/LDOX) respectively. ß-citraurin esters were produced from cleavage of zeaxanthin and esterification by organic acid, which was an offshoot of the carotenoid biosynthesis. The offshoot started with biosynthesis of zeaxanthin and ß-citraurin by carotene ß-hydroxylase (CHYB/LUT5) and carotenoid cleavage dioxygenase (CCD) respectively. During fruit ripening, biosynthesis of flavanols and anthocyanins was down-regulated by genes/proteins involved in phenylpropanoid and flavonoid biosynthesis, while biosynthesis of ß-citraurin esters was up-regulated by imbalanced expression of genes/proteins involved in ß,ß-ring and ß, ε-ring hydroxylation. Thus, ß-citraurin esters, instead of anthocyanins imparted reddish color to the ripe fruit. These pigments and their biosynthesis in R. chingii are totally different from what occurs in other raspberry species.

Combined transcriptomic and metabolic analyses reveal potential mechanism for fruit development and quality control of Chinese raspberry (Rubus chingii Hu).

KEY MESSAGE: Combined transcriptomic and metabolic analyses reveal that fruit of Rubus chingii Hu launches biosynthesis of phenolic acids and flavonols at beginning of fruit set and then coordinately accumulated or converted to their derivatives. Rubus chingii Hu (Chinese raspberry) is an important dual functional food with nutraceutical and pharmaceutical values. Comprehensively understanding the mechanisms of fruit development and bioactive components synthesis and regulation could accelerate genetic analysis and molecular breeding for the unique species. Combined transcriptomic and metabolic analyses of R. chingii fruits from different developmental stages, including big green, green-to-yellow, yellow-to-orange, and red stages, were conducted. A total of 89,188 unigenes were generated and 57,545 unigenes (64.52%) were annotated. Differential expression genes (DEGs) and differentially accumulated metabolites (DAMs) were mainly involved in the biosynthesis of secondary metabolites. The fruit launched the biosynthesis of phenolic acids and flavonols at the very beginning of fruit set and then coordinately accumulated or converted to their derivatives. This was tightly regulated by expressions of the related genes and MYB and bHLH transcription factors. The core genes products participated in the biosynthesis of ellagic acid (EA) and kaempferol-3-O-rutinoside (K-3-R), such as DAHPS, DQD/SDH, PAL, 4CL, CHS, CHI, F3H, F3'H, FLS, and UGT78D2, and their corresponding metabolites were elaborately characterized. Our research reveals the molecular and chemical mechanisms of the fruit development of R. chingii. The results provide a solid foundation for the genetic analysis, functional genes isolation, fruit quality improvement and modifiable breeding of R. chingii.

Transcriptomic, Proteomic and Metabolomic Analysis of Flavonoid Biosynthesis During Fruit Maturation in Rubus chingii Hu.

Rubus chingii HU, is a medicinal and nutritious fruit, which is very rich in flavonoids. However, the biosynthesis of its flavonoids is poorly understood. This study examined flavonoids and the genes/proteins at four fruit ripening phases using LC-MS/MS and qPCR. Six major kinds of anthocyanins, primarily consisted of flavanol-anthocyanins, which differed in form or concentration from other Rubus species. In contrast to other known raspberries species, R. chingii had a decline in flavonoids during fruit ripening, which was due to down-regulation of genes and proteins involved in phenylpropanoid and flavonoid biosynthesis. Unexpectedly, anthocyanin also continuously decreased during fruit maturation. This suggests that anthocyanins are not responsible for the fruit's reddish coloration. Flavanol-anthocyanins were derived from the proanthocyanidin pathway, which consumed two flavonoid units both produced through the same upstream pathway. Their presence indicates a reduction in the potential biosynthesis of anthocyanin production. Also, the constantly low expression of RchANS gene resulted in low levels of anthocyanin biosynthesis. The lack of RchF3'5'H gene/protein hindered the production of delphinidin glycosides. Flavonoids primarily comprising of quercetin/kaempferol-glycosides were predominately located at fruit epidermal-hair and placentae. The proportion of receptacle/drupelets changes with the maturity of the fruit and may be related to a decrease in the content of flavonoids per unit mass as the fruit matures. The profile and biosynthesis of R. chingii flavonoids are unique to Rubus. The unique flavonol pathways of R. chingii could be used to broaden the genetic diversity of raspberry cultivars and to improve their fruit quality.

Metabolite profile and genes/proteins expression in ß-citraturin biosynthesis during fruit ripening in Chinese raspberry (Rubus chingii Hu).

Carotenoids are one of the most abundant pigments in raspberries. Rubus chingii Hu, indigenous to China, is traditionally consumed for health benefits. However, the carotenoid composition and pathways of R. chingii have not yet been studied. In this study, the components of carotenoids and genes/proteins involved in their biosynthesis were investigated during four fruit ripening phases via LC-MS/MS. Zeaxanthin, ß-citraurin and its esters, first identified in Rubus, gradually accumulated during fruit maturation. These compounds, rather than anthocyanins, were responsible for the ripe fruit coloration. In carotenoid metabolism, upstream synthesis genes of RcPSY2 (CL1406.Contig2), RcPDS1 (CL7625.Contig2), RcZDS1 (CL590.Contig6) and RcCRTISO1 (CL6919.Contig2) were up-regulated in gene/protein expression to accelerate carotene biosynthesis. Downstream genes of RcLUT5CHYB/CYP97A (CL8884.Contig3) and RcCHYB/BCH (CL7966.Contig1) were up-regulated in gene/protein expression, while RcCHYE/CYP97C (CL9380.Contig1/2) were maintained at low levels. RcLCYE (Unigene19570) was down-regulated while RcLCYB (CL7586.Contig1) was up-regulated and then down-regulated. These differential gene/protein expressions between LCYB and LCYE, and between CHYE and CHYB led to zeaxanthin accumulation by elevating its biosynthetic enzymes and lowering enzymes for lutein biosynthesis. In apocarotenoid biosynthesis, RcCCD (CL1310.Contig3) was up-regulated in gene/protein expression, which raised the content of ß-citraurin and its esters. Additionally, these genes/proteins diverged into different subgroups with distinct pattens of expression, suggesting their difference in function. For example, RcPSY1/3, RcZDS2, and RcCRTISO2/3 genes were expressed at very low levels, suggesting that they may be active in other tissues rather than in fruit. The mechanism of zeaxanthin and ß-citraurin biosynthesis is responsible for fruit coloration, which is completely novel to Rubus.

Characterization of carotenoids and phenolics during fruit ripening of Chinese raspberry (Rubus chingii Hu).

Chinese raspberry (Rubus chingii Hu) is a fruit valued for it's health benefits, which is indigenous to China. It is a great source of antioxidants. However, the fruit phytochemicals are poorly understood. Phenolics and carotenoids attract much attention for their antioxidant capability, and they dramatically change during fruit ripening, leading to the difference in color, flavor and medicinal components. In this study, we investigated the change of carotenoids, phenolics and antioxidant activity using spectrophotometry during four different ripening phases i.e. mature green (MG), green yellow (GY), yellow orange (YO) and red (RE). The major components of carotenoids, anthocyanins, ellagitannins and flavonols were identified and quantified by LC-MS/MS. As a result, five carotenoids (mainly ß-Citraurin and its esters), six anthocyanins (mainly anthocyanins covalently linked to another flavonoid unit), methyl (S)-flavogallonate and rourinoside were first identified in Rubus. In contrast to other known raspberries, R. chingii had a continuous decrease in total phenolics during fruit ripening, which was due to a continuous decrease in flavonoids (including anthocyanin). Total anthocyanin and flavonoid respectively declined from 19.5 to 6.9 mg/100 g FW, and 646.2 to 128.5 mg/100 g FW during fruit maturation and coloration. Accordingly, the components of anthocyanins, ellagitannins and flavonols also declined, thus resulting in a decrease in antioxidant activity (from 41.2 to 10.1 TEAC/100 g FW in ABTS and from 35.3 to 7.7 mmol TEAC/100 g FW in FRAP). In contrast, total carotenoid increased from 184.2 to 305.4 mg/100 g FW. Accordingly, the components of carotenoids also increased, with the exception of lutein. Additionally, kaempferol and quercetin were the main flavonoid aglycones, which were linked to a variety of glycosides. These kaempferol- and quercetin-glycosides mainly accumulated in epidermal hair and placentae. Notably, carotenoids (i.e. ß-citraurin esters), instead of anthocyanins, gradually accumulated during fruit ripening, imparting the reddish color to ripe fruit.