Chemical characterization and discovery of novel quality markers in Citrus aurantium L. fruit from traditional cultivation areas in China using GC-MS-based cuticular waxes analysis.

Citrus aurantium L. fruit is a commonly used Chinese medicine whose therapeutic effects tends to be affected by growing conditions. In order to gain insights into the effects of growing location on the cuticular wax composition of C. aurantium L. fruit, we analyzed the differences in the wax composition of its fruits collected from different regions. The findings showed that the cuticular waxes in the fruit peels were mainly composed of fatty acids, which differed quantitatively in the chemical profiles of C. aurantium L. samples from different geographical conditions. Particularly, the concentrations of linoleic acid and stearic acid in the total component content of the fruit peel were above 1%, with a greater level in the geo-authentic samples. Thus, GC-MS-based wax analysis was first used for the chemical characterization and quantification of cuticular waxes, which could be considered as a rapid way for evaluating the quality of medicinal fruits.

Natural variation and artificial selection at the BnaC2.MYB28 locus modulate Brassica napus seed glucosinolate.

The degradation products of glucosinolates (GSLs) greatly lower the nutritional value of rapeseed (Brassica napus) meal; thus, reduction of seed GSL content (SGC) has become an important objective of rapeseed breeding. In our previous study, we finely mapped a major QTL (qGSL-C2) for SGC to a 49-kb collinear region on B. rapa chromosome A2. Here, we experimentally validated that BnaC2.MYB28, encoding an R2R3-MYB transcription factor, is the causal gene of qGSL-C2. BnaC2.MYB28 is a nucleus-localized protein mainly expressed in vegetative tissues. Knockout of BnaC2.MYB28 in the high-SGC parent G120 reduced SGC to a value lower than that in the low-SGC parent ZY50, while overexpression of BnaC2.MYB28 in both parental lines (G120 and ZY50) led to extremely high SGC, indicating that BnaC2.MYB28 acts as a positive regulator of SGC in both parents. Molecular characterization revealed that BnaC2.MYB28 forms a homodimer and specifically interacts with BnaMYC3. Moreover, BnaC2.MYB28 can directly activate the expression of GSL biosynthesis genes. Differential expression abundance resulting from the polymorphic promoter sequences, in combination with the different capability in activating downstream genes involved in aliphatic GSL biosynthesis, caused the functional divergence of BnaC2.MYB28 in SGC regulation between the parents. Natural variation of BnaC2.MYB28 was highly associated with SGC in natural germplasm and has undergone artificial selection in modern low-GSL breeding. This study provides important insights into the core function of BnaC2.MYB28 in regulating SGC and a promising strategy for manipulating SGC in rapeseed.

DNA barcoding and comparative RNA-Seq analysis provide new insights into leaf formation using a novel resource of high-yielding Epimedium koreanum.

Epimedium koreanum Nakai, a well-known traditional Chinese medicinal herb, has been widely used to treat osteoporosis and sexual dysfunction for thousands of years. However, due to the decreasing population of East Asian natural resources, yearly output of Epimedium crude herb has been in low supply year by year. In this study, an unusual variety of E. koreanum was discovered in Dunhua, Jilin Province, the northernmost area where this variety was found containing 6 individuals, with three branches that had 27 leaflets, which is much more than the typical leaflet number of 9. Firstly, the novel E. koreanum varety was identified using DNA barcodes. Then, 1171 differentially expressed genes (DEGs) were discovered through parallel RNA-seq analysis between the newly discovered variety and wild type (WT) E. koreanum plant. Furthermore, the results of bioinformatics investigation revealed that 914 positively and 619 negatively correlated genes associated with the number of leaflets. Additionally, based on RNA-Seq and qRT-PCR analysis, two homologous hub TCP genes, which were commonly implicated in plant leaf development, and shown to be up regulated and down regulated in the discovered newly variety, respectively. Thus, our study discovered a novel wild resource for leaf yield rewarding medicinal Epimedium plant breeding, provided insights into the relationship between plant compound leaf formation and gene expression of TCPs transcription factors and other gene candidates, providing bases for creating high yield cultivated Epimedium variety by using further molecular selection and breeding techniques in the future.

Genome- and transcriptome-wide association studies reveal the genetic basis and the breeding history of seed glucosinolate content in Brassica napus.

A high content of seed glucosinolates and their degradation products imposes anti-nutritional effects on livestock; therefore, persistent efforts are made to reduce the seed GSL content to increase the commercial value of rapeseed meal. Here, we dissected the genetic structure of SGC by genome-wide association studies (GWAS) combined with transcriptome-wide association studies (TWAS). Fifteen reliable quantitative trait loci (QTLs) were identified to be associated with the reduced SGC in modern B. napus cultivars by GWAS. Analysis of the selection strength and haplotypes at these QTLs revealed that low SGC was predominantly generated by the co-selection of qGSL.A02.2, qGSL.C02.1, qGSL.A09.2, and qGSL.C09.1. Integration of the results from TWAS, comprehensive bioinformatics, and POCKET algorithm analyses indicated that BnaC02.GTR2 (BnaC02g42260D) is a candidate gene underlying qGSL.C02.1. Using CRISPR/Cas9-derived Bna.gtr2s knockout mutants, we experimentally verified that both BnaC02.GTR2 and its three paralogs positively regulate seed GSL accumulation but negatively regulated vegetative tissue GSL contents. In addition, we observed smaller seeds with higher seed oil content in these Bna.gtr2 mutants. Furthermore, both RNA-seq and correlation analyses suggested that Bna.GTR2s might play a comprehensive role in seed development, such as amino acid accumulation, GSL synthesis, sugar assimilation, and oil accumulation. This study unravels the breeding selection history of low-SGC improvement and provides new insights into the molecular function of Bna.GTR2s in both seed GSL accumulation and seed development in B. napus.

Structural analysis of the meiosis-related protein MS5 reveals non-canonical papain enhancement by cystatin-like folds.

MS5 is a meiosis-related protein belonging to the Brassicaceae-specific domain of unknown function family and characterized by the MS5 superfamily domain (MSD). In this study, we elucidated the three-dimensional crystal structure and potential biochemical function of the MSD. It was observed that the MSD adopts a cystatin-like fold, mainly consisting of a central α-helix and four- or five-stranded antiparallel ß-sheets that wrap around it. However, unlike cystatins, which inhibit cysteine proteases, the MSD displayed allosteric activation of papain. We believe that our study provides insight into novel mechanisms of proteolytic enzyme regulation and may serve as a basis for functional studies of the MS5 family proteins in plants.

Molecular mechanisms underpinning the multiallelic inheritance of MS5 in Brassica napus.

The Brassica-specific gene MS5 mediates early meiotic progression, and its allelic variants contribute to a valuable genic male sterility three-line hybrid production system in rapeseed (Brassica napus L.). However, the underlying mechanisms of its triallelic inheritance are poorly understood. Herein, we show that the restorer allele MS5a and the maintainer allele MS5c are both necessary for male fertility in B. napus. The functional divergence of MS5a and MS5c is strongly related to sequence variations in their coding regions and less strongly to their promoter regions. The male-sterile allele MS5b encodes a chimeric protein containing only the complete MS5 coiled-coil (CC) domain, having lost the MS5 superfamily domain. Both MS5a and MS5c can form homodimers in the nucleus via the CC domain. MS5b can interact competitively with MS5a or MS5c to form non-functional heterodimers. Owing to the close transcript levels of MS5b and MS5c in MS5b MS5c , these heterodimers induced a dominant-negative effect of MS5b on MS5c , resulting in a male-sterile phenotype. The extremely high transcript abundance of MS5a maintains sufficient MS5a homodimers in MS5a MS5b , causing the recovery of male sterility. These findings provide substantial genetic and molecular evidence to improve our understanding of the mechanisms underlying the multiallelic inheritance of MS5, and enable the construction of a solid foundation for improved use of the MS5-controlled GMS system in Brassica species.