Integrated metabolome and transcriptome analysis of differences in quality of ripe Lycium barbarum L. fruits harvested at different periods.

BACKGROUND: Wolfberry is well-known for its high nutritional value and medicinal benefits. Due to the continuous ripening nature of Goji berries and the fact that they can be commercially harvested within a few weeks, their phytochemical composition may change during the harvesting process at different periods. RESULT: The involved molecular mechanisms of difference in fruit quality of ripe Lycium barbarum L. harvested at four different periods were investigated by transcriptomic and metabolomics analyses for the first time. According to the results we obtained, it was found that the appearance quality of L. barbarum fruits picked at the beginning of the harvesting season was superior, while the accumulation of sugar substances in L. barbarum fruits picked at the end of the harvesting season was better. At the same time the vitamin C and carotenoids content of wolfberry fruits picked during the summer harvesting season were richer. Ascorbic acid, succinic acid, glutamic acid, and phenolic acids have significant changes in transcription and metabolism levels. Through the network metabolic map, we found that ascorbic acid, glutamic acid, glutamine and related enzyme genes were differentially accumulated and expressed in wolfberry fruits at different harvesting periods. Nevertheless, these metabolites played important roles in the ascorbate-glutathione recycling system. Ascorbic acid, phenolic substances and the ascorbate-glutathione recycling system have antioxidant effects, which makes the L. barbarum fruits harvested in the summer more in line with market demand and health care concepts. CONCLUSION: This study laid the foundation for understanding the molecular regulatory mechanisms of quality differences of ripe wolfberry fruits harvested at different periods, and provides a theoretical basis for enhancing the quality of L. barbarum fruits.

Integrative transcriptome and metabolome analysis reveals the discrepancy in the accumulation of active ingredients between Lycium barbarum cultivars.

MAIN CONCLUSION: The combined analysis of transcriptome and metabolome provided molecular insight into the dynamics of multiple active ingredients biosynthesis and accumulation across different cultivars of Lycium barbarum. Lycium barbarum L. has a high concentration of active ingredients and is well known in traditional Chinese herbal medicine for its therapeutic properties. However, there are many Lycium barbarum cultivars, and the content of active components varies, resulting in inconsistent quality between Lycium barbarum cultivars. At present, few research has been conducted to reveal the difference in active ingredient content among different cultivars of Lycium barbarum at the molecular level. Therefore, the transcriptome of 'Ningqi No.1' and 'Qixin No.1' during the three development stages (G, T, and M) was constructed in this study. A total of 797,570,278 clean reads were obtained. Between the two types of wolfberries, a total of 469, 2394, and 1531 differentially expressed genes (DEGs) were obtained in the 'G1 vs. G10,' 'T1 vs. T10,' and 'M1 vs. M10,' respectively, and were annotated with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology identifiers. Using these transcriptome data, most DEGs related to the metabolism of the active ingredients in 'Ningqi No.1' and 'Qixin No.1' were identified. Moreover, a widely targeted metabolome analysis of the metabolites of 'Ningqi 1' and 'Qixin 1' fruits at the maturity stage revealed 1,135 differentially expressed metabolites (DEMs) in 'M1 vs. M10,' and many DEMs were associated with active ingredients such as flavonoids, alkaloids, terpenoids, and so on. We further quantified the flavonoid, lignin, and carotenoid contents of the two Lycium barbarum cultivars during the three developmental stages. The present outcome provided molecular insight into the dynamics of multiple active ingredients biosynthesis and accumulation across different cultivars of Lycium barbarum, which would provide the basic data for the formation of Lycium barbarum fruit quality and the breeding of outstanding strains.

Major quality regulation network of flavonoid synthesis governing the bioactivity of black wolfberry.

Black wolfberry (Lycium ruthenicum Murr.) contains various bioactive metabolites represented by flavonoids, which are quite different among production regions. However, the underlying regulation mechanism of flavonoid biosynthesis governing the bioactivity of black wolfberry remains unclear. Presently, we compared the bioactivity of black wolfberry from five production regions. Multi-omics were performed to construct the regulation network associated with the fruit bioactivity. The detailed regulation mechanisms were identified using genetic and molecular methods. Typically, Qinghai (QH) fruit exhibited higher antioxidant and anti-inflammatory activities. The higher medicinal activity of QH fruit was closely associated with the accumulation of eight flavonoids, especially Kaempferol-3-O-rutinoside (K3R) and Quercetin-3-O-rutinoside (rutin). Flavonoid biosynthesis was found to be more active in QH fruit, and the upregulation of LrFLS, LrCHS, LrF3H and LrCYP75B1 caused the accumulation of K3R and rutin, leading to high medicinal bioactivities of black wolfberry. Importantly, transcription factor LrMYB94 was found to regulate LrFLS, LrCHS and LrF3H, while LrWRKY32 directly triggered LrCYP75B1 expression. Moreover, LrMYB94 interacted with LrWRKY32 to promote LrWRKY32-regulated LrCYP75B1 expression and rutin synthesis in black wolfberry. Transgenic black wolfberry overexpressing LrMYB94/LrWRKY32 contained higher levels of K3R and rutin, and exhibited high medicinal bioactivities. Importantly, the LrMYB94/LrWRKY32-regulated flavonoid biosynthesis was light-responsive, showing the importance of light intensity for the medicinal quality of black wolfberry. Overall, our results elucidated the regulation mechanisms of K3R and rutin synthesis, providing the basis for the genetic breeding of high-quality black wolfberry.

Genomic Analysis of Romanian Lycium Genotypes: Exploring BODYGUARD Genes for Stress Resistance Breeding.

Goji berries, long valued in Traditional Chinese Medicine and Asian cuisine for their wide range of medicinal benefits, are now considered a 'superfruit' and functional food worldwide. Because of growing demand, Europe and North America are increasing their goji berry production, using goji berry varieties that are not originally from these regions. European breeding programs are focusing on producing Lycium varieties adapted to local conditions and market demands. By 2023, seven varieties of goji berries were successfully registered in Romania, developed using germplasm that originated from sources outside the country. A broader project focused on goji berry breeding was initiated in 2014 at USAMV Bucharest. In the present research, five cultivated and three wild L. barbarum genotypes were compared to analyse genetic variation at the whole genome level. In addition, a case study presents the differences in the genomic coding sequences of BODYGUARD (BDG) 3 and 4 genes from chromosomes 4, 8, and 9, which are involved in cuticle-related resistance. All three BDG genes show distinctive differences between the cultivated and wild-type genotypes at the SNP level. In the BDG 4 gene located on chromosome 8, 69% of SNPs differentiate the wild from the cultivated genotypes, while in BDG 3 on chromosome 4, 64% of SNPs could tell the difference between the wild and cultivated goji berry. The research also uncovered significant SNP and InDel differences between cultivated and wild genotypes, in the entire genome, providing crucial insights for goji berry breeders to support the development of goji berry cultivation in Romania.

Genome-wide analysis of the TIFY family in Lycium and the negative regulation of stomatal development by LrJAZ2 gene.

Stomata are ports that facilitate gas and water vapor exchange during plant photosynthesis and transpiration. Stomatal development is strictly regulated by endogenous hormone. Jasmonate, an important signal that modulates multiple physiological processes in plants, has been found to negatively regulate stomatal development in Arabidopsis thaliana, yet the molecular mechanisms underlying stomata development signaling remain to be understood. Jasmonate ZIM-domain (JAZ) proteins are the members of TIFY family and the key component of JA signaling pathway. Its function in stomatal development is unclear to data. Here, we screened out 24 TIFY family members against the genome of Lycium, and identified a JAZ member by combination analyses of evolutionary tree, cis-elements in promoter and gene expression patterns. Overexpression of this gene (LrJAZ2) in Lycium ruthenicum and Arabidopsis thaliana indicated LrJAZ2 negatively regulates stomatal development. Microscopic observations revealed that overexpression of LrJAZ2 negatively regulated stomatal development by decreasing stomatal density and index, which may lead to lower leaf transpiration rates. Transcriptome data indicated the overexpression of LrJAZ2 up-regulated the stomatal related genes such as LrERL2, LrPYL4, and down-regulated the LrSPCH. Collectively, our study found that LrJAZ2 is a key gene in stomatal development regulation in L. ruthenicum and provided new insights into the regulation of stomatal development.

Transcriptional deciphering of the metabolic pathways associated with the bioactive ingredients of wolfberry species with different quality characteristics.

BACKGROUND: Wolfberry is rich in carotenoids, flavonoids, vitamins, alkaloids, betaines and other bioactive ingredients. For over 2,000 years, wolfberry has been used in China as a medicinal and edible plant resource. Nevertheless, the content of bioactive ingredients varies by cultivars, resulting in uneven quality across wolfberry cultivars and species. To date, research has revealed little about the underlying molecular mechanism of the metabolism of flavonoids, carotenoids, and other bioactive ingredients in wolfberry. RESULTS: In this context, the transcriptomes of the Lycium barbarum L. cultivar 'Ningqi No. 1' and Lycium chinense Miller were compared during the fruit maturity stage using the Illumina NovaSeq 6000 sequencing platform, and subsequently, the changes of the gene expression profiles in two types of wolfberries were analysed. In total, 256,228,924 clean reads were obtained, and 8817 differentially expressed genes (DEGs) were identified, then assembled by Basic Local Alignment Search Tool (BLAST) similarity searches and annotated using Gene Ontology (GO), Clusters of Orthologous Groups of proteins (KOG), and the Kyoto Encyclopedia of Genes and Genomes (KEGG). By combining these transcriptome data with data from the PubMed database, 36 DEGs related to the metabolism of bioactive ingredients and implicated in the metabolic pathway of carotenoids, flavonoids, terpenoids, alkaloids, vitamins, etc., were identified. In addition, among the 9 differentially expressed transcription factors, LbAPL, LbPHL11 and LbKAN4 have raised concerns. The protein physicochemical properties, structure prediction and phylogenetic analysis indicated that LbAPL and LbPHL11 may be good candidate genes involved in regulating the flavonoid metabolism pathway in wolfberry. CONCLUSIONS: This study provides preliminary evidence for the differences in bioactive ingredient content at the transcription level among different wolfberry species, as well as a research and theoretical basis for the screening, cloning and functional analysis of key genes involved in the metabolism of bioactive ingredients in wolfberry.

Comparative Analysis of the GATA Transcription Factors in Five Solanaceae Species and Their Responses to Salt Stress in Wolfberry (Lycium barbarum L.).

GATA proteins are a class of zinc-finger DNA-binding proteins that participate in diverse regulatory processes in plants, including the development processes and responses to environmental stresses. However, a comprehensive analysis of the GATA gene family has not been performed in a wolfberry (Lycium barbarum L.) or other Solanaceae species. There are 156 GATA genes identified in five Solanaceae species (Lycium barbarum L., Solanum lycopersicum L., Capsicum annuum L., Solanum tuberosum L., and Solanum melongena L.) in this study. Based on their phylogeny, they can be categorized into four subfamilies (I-IV). Noticeably, synteny analysis revealed that dispersed- and whole-genome duplication contributed to the expansion of the GATA gene family. Purifying selection was a major force driving the evolution of GATA genes. Moreover, the predicted cis-elements revealed the potential roles of wolfberry GATA genes in phytohormone, development, and stress responses. Furthermore, the RNA-seq analysis identified 31 LbaGATA genes with different transcript profiling under salt stress. Nine candidate genes were then selected for further verification using quantitative real-time PCR. The results revealed that four candidate LbaGATA genes (LbaGATA8, LbaGATA19, LbaGATA20, and LbaGATA24) are potentially involved in salt-stress responses. In conclusion, this study contributes significantly to our understanding of the evolution and function of GATA genes among the Solanaceae species, including wolfberry.

Identification and expression analysis of the small auxin-up RNA (SAUR) gene family in Lycium ruthenicum.

The plant hormone auxin regulates numerous aspects of plant growth and development, and small auxin-up RNA (SAUR) is the largest family of early auxin response genes in higher plants. SAUR has been implicated in the regulation of multiple biological processes. However, no comprehensive analysis of SAUR genes has been reported in Lycium ruthenicum. L. ruthenicum is a thorny shrub with very pronounced salt and drought tolerance, and studies have shown that stem thorns are related to drought tolerance in L. ruthenicum. In this study, the identification, phylogenetic analysis, and conserved motif prediction of SAUR genes were extensively explored. Furthermore, the tissue expression patterns of selected SAUR genes were assayed with quantitative real-time polymerase chain reaction (RT-qPCR). A total of 33 putative LrSAURs were identified and divided into three clusters in a phylogenetic tree of L. ruthenicum. MEME analysis identified 10 motifs in L. ruthenicum, and the results suggested that motif 1 and motif 3 were widely distributed. Analyzing the transcriptome data of stem thorns at four developmental stages indicated that LrSAURs were differentially expressed in L. ruthenicum, and could be divided into six expression patterns. The RT-qPCR analysis of 21 genes showed that LrSAUR2, LrSAUR8, LrSAUR9, LrSAUR11, LrSAUR12, and LrSAUR19 were mainly expressed in stems and stem thorns, and may be related to stem thorn development.

Changes in m6A RNA methylation are associated with male sterility in wolfberry.

BACKGROUND: N6-methyladenosine (m6A) modification is the most abundant type of RNA modification in eukaryotic cells, playing pivotal roles in multiple plant growth and development processes. Yet the potential role of m6A in conferring the trait of male sterility in plants remains unknown. RESULTS: In this study, we performed RNA-sequencing (RNA-Seq) and m6A-sequencing (m6A-Seq) of RNAs obtained from the anther tissue of two wolfberry lines: 'Ningqi No.1' (LB1) and its natural male sterile mutant 'Ningqi No.5' (LB5). Based on the newly assembled transcriptome, we established transcriptome-wide m6A maps for LB1 and LB5 at the single nucleus pollen stage. We found that the gene XLOC_021201, a homolog of m6A eraser-related gene ALKBH10 in Arabidopsis thaliana, was significantly differentially expressed between LB1 and LB5. We also identified 1642 and 563 m6A-modified genes with hypermethylated and hypomethylated patterns, respectively, in LB1 compared with LB5. We found the hypermethylated genes significantly enriched in biological processes related to energy metabolism and lipid metabolism, while hypomethylation genes were mainly linked to cell cycle process, gametophyte development, and reproductive process. Among these 2205 differentially m6A methylated genes, 13.74% (303 of 2205) were differentially expressed in LB1 vis-à-vis LB5. CONCLUSIONS: This study constructs the first m6A transcriptome map of wolfberry and establishes an association between m6A and the trait of male sterility in wolfberry.

[Comparative analysis of differentially expressed genes for biosynthesis of active ingredients in fruits of different cultivars of Lycium barbarum L. based on transcriptome sequencing].

To explore the differentially expressed genes (DEGs) related to biosynthesis of active ingredients in wolfberry fruits of different varieties of Lycium barbarum L. and reveal the molecular mechanism of the differences of active ingredients, we utilized Illumina NovaSeq 6000 high-throughput sequencing technology to conduct transcriptome sequencing on the fruits of 'Ningqi No.1' and 'Ningqi No.7' during the green fruit stage, color turning stage and maturity stage. Subsequently, we compared the profiles of related gene expression in the fruits of the two varieties at different development stages. The results showed that a total of 811 818 178 clean reads were obtained, resulting in 121.76 Gb of valid data. There were 2 827, 2 552 and 2 311 DEGs obtained during the green fruit stage, color turning stage and maturity stage of 'Ningqi No. 1' and 'Ningqi No. 7', respectively, among which 2 153, 2 050 and 1 825 genes were annotated in six databases, including gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG) and clusters of orthologous groups of proteins (KOG). In GO database, 1 307, 865 and 624 DEGs of green fruit stage, color turning stage and maturity stage were found to be enriched in biological processes, cell components and molecular functions, respectively. In the KEGG database, the DEGs at three developmental stages were mainly concentrated in metabolic pathways, biosynthesis of secondary metabolites and plant-pathogen interaction. In KOG database, 1 775, 1 751 and 1 541 DEGs were annotated at three developmental stages, respectively. Searching the annotated genes against the PubMed database revealed 18, 26 and 24 DEGs related to the synthesis of active ingredients were mined at the green fruit stage, color turning stage and maturity stage, respectively. These genes are involved in carotenoid, flavonoid, terpenoid, alkaloid, vitamin metabolic pathways, etc. Seven DEGs were verified by RT-qPCR, which showed consistent results with transcriptome sequencing. This study provides preliminary evidences for the differences in the content of active ingredients in different Lycium barbarum L. varieties from the transcriptional level. These evidences may facilitate further exploring the key genes for active ingredients biosynthesis in Lycium barbarum L. and analyzing their expression regulation mechanism.

Integrated Transcriptome and Metabolome Dynamic Analysis of Galls Induced by the Gall Mite Aceria pallida on Lycium barbarum Reveals the Molecular Mechanism Underlying Gall Formation and Development.

Galls have become the best model for exploring plant-gall inducer relationships, with most studies focusing on gall-inducing insects but few on gall mites. The gall mite Aceria pallida is a major pest of wolfberry, usually inducing galls on its leaves. For a better understanding of gall mite growth and development, the dynamics of the morphological and molecular characteristics and phytohormones of galls induced by A. pallida were studied by histological observation, transcriptomics and metabolomics. The galls developed from cell elongation of the epidermis and cell hyperplasia of mesophylls. The galls grew quickly, within 9 days, and the mite population increased rapidly within 18 days. The genes involved in chlorophyll biosynthesis, photosynthesis and phytohormone synthesis were significantly downregulated in galled tissues, but the genes associated with mitochondrial energy metabolism, transmembrane transport, carbohydrates and amino acid synthesis were distinctly upregulated. The levels of carbohydrates, amino acids and their derivatives, and indole-3-acetic acid (IAA) and cytokinins (CKs), were markedly enhanced in galled tissues. Interestingly, much higher contents of IAA and CKs were detected in gall mites than in plant tissues. These results suggest that galls act as nutrient sinks and favor increased accumulation of nutrients for mites, and that gall mites may contribute IAA and CKs during gall formation.

Integrated transcriptome and metabolome provide insight into flavonoid variation in goji berries (Lycium barbarum L.) from different areas in China.

Goji berries (Lycium barbarum L.) were rich in flavonoids, showing high nutritional and medicinal value. However, a thorough evaluation and comparison of the flavonoids in goji berries from various regions and the possible biological regulation pathways with differences are scanty. Here, we investigated the flavonoid metabolites and gene expression levels of goji berries from three major production areas in China using transcriptomics sequencing and metabolomics. The total flavonoid content and total polyphenol content of goji berry in Ningxia (57.87 µg/g and 183.41 µg/g, respectively) were higher than in Qinghai (50.77 µg/g and 156.81 µg/g) and Gansu (47.86 µg/g and 111.17 µg/g). We identified the 105 differentially accumulated flavonoids (DAFs) and 1858 differentially expressed genes (DEGs) from the goji berries in three habitats. Interestingly, gossypetin-3-O-rutinoside and isorhamnetin were significantly expressed between Ningxia and Qinghai berries. The chalcone isomerase (CHI), chalcone synthase (CHS), and flavonol synthase (FLS) genes also played key roles in the regulation of flavonoid synthesis. In addition, MYB1 positively regulated the expression of quercetin-3-O-glucoside, quercetin-7-O-glucoside and isohyperoside. As a result, we speculated that CHI, CHS, FLS genes, and related transcription factors jointly controlled the variation of flavone accumulation in goji berries. These findings may provide a new perspective for understanding the accumulation and molecular mechanisms of goji flavonoids.

Sucrose promotes branch-thorn occurrence of Lycium ruthenicum through dual effects of energy and signal.

Lycium ruthenicum is an important ecoeconomic thorny shrub. In this study, the L. ruthenicum plants of a clone showed two types of 'fewer leaves without thorn' and 'more leaves with thorns' under the same condition after transplanting. Microscopic observation revealed that the apical buds of the thornless (Thless) and thorny (Thorny) branches should be selected as materials for further study. RNA-Seq analysis showed that the KEGG pathway of starch and sucrose metabolism and differentially expressed genes of sugar transport protein 13 (SUT13), sucrose synthase (SUS), trehalose-phosphate phosphatase (TPP) and trehalose-phosphate synthase (TPS) were significantly up-regulated in Thorny. The results of qRT-PCR confirmed the accuracy and credibility of the RNA-Seq. The content of sucrose in Thorny was significantly higher than that in Thless, but the content of trehalose-6-phosphate (T6P) was opposite. Leaf-clipping treatments reduced sucrose content and inhibited the occurrence/development of branch-thorns; exogenous sucrose of 16 g l-1 significantly promoted the occurrence and growth of branch-thorns, and the promotion effects were significantly higher than those treated with non-metabolizable sucrose analogs (isomaltolose and melitose). These findings suggested that sucrose might play a dual role of energy and signal in the occurrence of branch-thorns. Higher sucrose supply in apical buds from more leaves promoted the occurrence of branch-thorns via a lower content of T6P and higher expression levels of SUS, TPP and TPS, whereas fewer leaves inhibited the occurrence. The molecular hypothesis model of the leaf number/sucrose supply regulating the occurrence of branch-thorns in L. ruthenicum was established in the study, which provides foundation for breeding both Thless L. ruthenicum and Thless types of other species.

Analysis of flavonoid metabolism during fruit development of Lycium chinense.

Lycium chinense is an important medicinal plant in the northwest of China. Flavonoids are the major pharmacological components of L. chinense fruits. However, flavonoid metabolism during fruit development of L. chinense remains to be studied. Here, we analyzed the change of flavonoid contents, enzyme activity, and gene expression during fruit development of L. chinense. We found that flavonoids, anthocyanins, and catechins are the most important components of L. chinense fruits. Flavonoid content was increased with fruit development and was high at the late developmental stage. PAL, CHS, and F3H enzymes played a significant role in flavonoid accumulation in fruits. Transcriptomic analysis showed that anthocyanin pathway, flavonol pathway, flavonoid biosynthesis, and phenylpropanoid synthesis pathway were the major pathways involved in flavonoid metabolism in L. chinense. Gene expression analysis indicated that PAL1 and CHS2 genes were critical for flavonoid metabolism in L. chinense fruits. These discoveries help us understand the dynamic changes in flavonoids during fruit development and enhance the use of L. chinense fruits.

Genome-Wide Identification and Analysis of the BBX Gene Family and Its Role in Carotenoid Biosynthesis in Wolfberry (Lycium barbarum L.).

The B-box proteins (BBXs) are a family of zinc-finger transcription factors with one/two B-Box domain(s) and play important roles in plant growth and development as well as stress responses. Wolfberry (Lycium barbarum L.) is an important traditional medicinal and food supplement in China, and its genome has recently been released. However, comprehensive studies of BBX genes in Lycium species are lacking. In this study, 28 LbaBBX genes were identified and classified into five clades by a phylogeny analysis with BBX proteins from Arabidopsis thaliana and the LbaBBXs have similar protein motifs and gene structures. Promoter cis-regulatory element prediction revealed that LbaBBXs might be highly responsive to light, phytohormone, and stress conditions. A synteny analysis indicated that 23, 20, 8, and 5 LbaBBX genes were orthologous to Solanum lycopersicum, Solanum melongena, Capsicum annuum, and Arabidopsis thaliana, respectively. The gene pairs encoding LbaBBX proteins evolved under strong purifying selection. In addition, the carotenoid content and expression patterns of selected LbaBBX genes were analyzed. LbaBBX2 and LbaBBX4 might play key roles in the regulation of zeaxanthin and antheraxanthin biosynthesis. Overall, this study improves our understanding of LbaBBX gene family characteristics and identifies genes involved in the regulation of carotenoid biosynthesis in wolfberry.

The mechanism of bud dehyperhydricity by the method of 'starvation drying combined with AgNO3' in Lycium ruthenicum.

Micropropagation is very important for rapid clonal propagation and scientific research of woody plants. However, the micropropagated materials usually show hyperhydricity, which seriously hinders application of the micropropagation. Lycium ruthenicum is an important species of eco-economic forests. Herein, treatment of 'starvation and drying combined with 30 µM AgNO3' (SDCAg+) removed serious hyperhydricity of L. ruthenicum buds regenerated from its green-inflorescence-explants, and then gene expression, metabolites of various phytohormones, chloroplasts, chlorophyll (Chl) and total soluble proteins of the hyperhydric and dehyperhydric leaves were compared and analyzed. The results suggested that the SDCAg+ treatment might remove hyperhydricity of L. ruthenicum through: reducing water uptake; increasing water loss; up-regulating the expression of chloroplast-ribosomal-protein genes from nuclear genome; down-regulating the expression of cytoplasmic-ribosomal-protein genes; up-regulating the synthesis of the total soluble proteins; restoring the lamellar structure of chloroplast grana and matrix; improving Chl synthesis and reducing Chl metabolism; increasing expression of light-harvesting Chl protein complex genes and content of Chla and b; up-regulating both photosynthesis and starch and sucrose metabolism KEGG pathways; up-regulating abscisic acid, salicylic acid and their signaling; down-regulating cytokinin, jasmonic acid, jasmonoyl-l-isoleucine and their signaling. Also, the above events interact to form a regulatory network of dehyperhydricity by SDCAg+ treatment. Overall, the study indicated key genes/pathways and physiological/subcellular changes involved in dehyperhydricity and then established a dehyperhydric mechanism model of L. ruthenicum. This not only proposed clues for preventing or removing hyperhydricity but also laid foundations for molecular breeding of L. ruthenicum and other species.

[DNA methylation diversity analysis of Lycium barbarum samples from different cultivation areas based on MSAP].

Obvious epigenetic differentiation occurred on Lycium barbarum in different cultivation areas in China. To investigate the difference and change rule of DNA methylation level and pattern of L. barbarum from different cultivation areas in China, the present study employed fluorescence-assisted methylation-sensitive amplified polymorphism(MSAP) to analyze the methylation level and polymorphism of 53 genomic DNA samples from Yinchuan Plain in Ningxia, Bayannur city in Inner Mongolia, Jingyuan county and Yumen city in Gansu, Delingha city in Qinghai, and Jinghe county in Xinjiang. The MSAP technical system suitable for the methylation analysis of L. barbarum genomic DNA was established and ten pairs of selective primers were selected. Among amplified 5'-CCGG-3' methylated sites, there were 35.85% full-methylated sites and 39.88% hemi-methylated sites, showing a high degree of epigenetic differentiation. Stoichiometric analysis showed that the ecological environment was the main factor affecting the epigenetic characteristics of L. barbarum, followed by cultivated varieties. Precipitation, air temperature, and soil pH were the main ecological factors affecting DNA methylation in different areas. This study provided a theoretical basis for the analysis of the epigenetic mechanism of L. barbarum to adapt to the diffe-rent ecological environments and research ideas for the introduction, cultivation, and germplasm traceability of L. barbarum.

Genome-Wide Comparative Analysis of the R2R3-MYB Gene Family in Five Solanaceae Species and Identification of Members Regulating Carotenoid Biosynthesis in Wolfberry.

The R2R3-MYB is a large gene family involved in various plant functions, including carotenoid biosynthesis. However, this gene family lacks a comprehensive analysis in wolfberry (Lycium barbarum L.) and other Solanaceae species. The recent sequencing of the wolfberry genome provides an opportunity for investigating the organization and evolutionary characteristics of R2R3-MYB genes in wolfberry and other Solanaceae species. A total of 610 R2R3-MYB genes were identified in five Solanaceae species, including 137 in wolfberry. The LbaR2R3-MYB genes were grouped into 31 subgroups based on phylogenetic analysis, conserved gene structures, and motif composition. Five groups only of Solanaceae R2R3-MYB genes were functionally divergent during evolution. Dispersed and whole duplication events are critical for expanding the R2R3-MYB gene family. There were 287 orthologous gene pairs between wolfberry and the other four selected Solanaceae species. RNA-seq analysis identified the expression level of LbaR2R3-MYB differential gene expression (DEGs) and carotenoid biosynthesis genes (CBGs) in fruit development stages. The highly expressed LbaR2R3-MYB genes are co-expressed with CBGs during fruit development. A quantitative Real-Time (qRT)-PCR verified seven selected candidate genes. Thus, Lba11g0183 and Lba02g01219 are candidate genes regulating carotenoid biosynthesis in wolfberry. This study elucidates the evolution and function of R2R3-MYB genes in wolfberry and the four Solanaceae species.

Metabolomic and transcriptomic analysis of Lycium chinese and L. ruthenicum under salinity stress.

BACKGROUND: High soil salinity often adversely affects plant physiology and agricultural productivity of almost all crops worldwide, such as the crude drug known as wolfberry. However, the mechanism of this action in wolfberry is not fully understood yet. RESULTS: Here in this study, we studied different mechanisms potentially in Chinese wolfberry (Lycium chinese, LC) and black wolfberry (L. ruthenicum, LR) under salinity stress, by analyzing their transcriptome, metabolome, and hormone changes. The hormone detection analysis revealed that the ABA content was significantly lower in LR than LC under normal condition, and increased sharply under salinity stress in LR but not in LC. The transcriptome analysis showed that the salinity-responsive genes in wolfberry were mainly enriched in MAPK signaling, amino sugar and nucleotide sugar metabolism, carbon metabolism, and plant hormone signal transduction pathways in LC, while mainly related to carbon metabolism and protein processing in endoplasmic reticulum in LR. Metabolome results indicated that LR harbored higher flavone and flavonoid contents than LC under normal condition. However, the flavone and flavonoid contents were hardly changed in LR, but increased substantially in LC when exposed to salinity stress. CONCLUSIONS: Our results adds ABA and flavone to mechanism understanding of salinity tolerance in wolfberry. In addition, flavone plays a positive role in resistance to salinity stress in wolfberry.

The branch-thorn occurrence of Lycium ruthenicum is associated with leaf DNA hypermethylation in response to soil water content.

BACKGROUND: Lycium ruthenicum is an eco-economic shrub which can exist in two forms, thorny and thornless under varying soil moisture conditions. The aim of this study was to determine if the two forms of L. ruthenicum were influenced by soil water content (SWC) and to test the three-way link among SWC, occurrence of branch-thorn and DNA methylation modification. METHODS AND RESULTS: Here, pot experiment was carried out to reveal the influence of SWC on the occurrence of branch-thorn and then paraffin sections, scanning electron microscope and methylation-sensitive amplification polymorphism(MSAP) analysis were used to determine the three-way link among SWC, branch-thorn occurrence and DNA methylation. The results showed that (a) soil drought promoted the development of thorn primordium into branch-thorn and (b) branch-thorn covered axillary bud to protect it against drought and other stresses; (c) the branch-thorn occurrence response to drought was correlated with hypermethylation of CCGG sites and (d) thorny and thornless plants of a clone were distinguished successfully based on the MSAP profiles of their leaves. CONCLUSIONS: Branch-thorns of the L. ruthenicum clone, which occurred in response to drought, covered axillary buds to protect them against drought and other stresses; thorn primordium of the clone did not develop into branch-thorn under the adequate soil moisture condition. The occurrence and absence of the branch-thorns were correlated with the hyper- and hypo-methylation, respectively. We proposed that the branch-thorn plasticity might be an adjustment strategy for the environment, which seems to support the theory of "Use in, waste out".