UV-B irradiation promotes anthocyanin biosynthesis in the leaves of Lycium ruthenicum Murray.

Anthocyanins are the most valuable pigments in Lycium ruthenicum Murray (L. ruthenicum). Although ultraviolet-B (UV-B) irradiation is a key environmental factor influencing anthocyanin biosynthesis in L. ruthenicum, the deep molecular mechanism remains unclear. Herein, we examined the changes in the total anthocyanin content and transcriptomic characteristics of L. ruthenicum leaves following UV-B irradiation treatment. The results showed a twofold increase in anthocyanin content in the leaves of L. ruthenicum after the treatment. The transcriptome analysis showed that the expression of 24 structural genes identified in the anthocyanin synthesis pathway was up-regulated. In particular, F3'H (Unigene0009145) and C4H (Unigene0046607) exhibit notable up-regulation, suggesting their potential roles in anthocyanin synthesis. Protein interaction network results revealed that MYB1 (Unigene0047706) had the highest connectivity, followed by bHLH (Unigene0014085). Additionally, UVR8 (Unigene0067978) and COP1 (Unigene0008780) were found to be highly involved in UV-B signal transduction. These findings provide new insights into the genetic and biochemical mechanisms that regulate anthocyanin production, and could guide agricultural practices to reduce environmental impacts and improve crop yield and quality.

Integration of Transcriptome and Metabolome Reveals Wax Serves a Key Role in Preventing Leaf Water Loss in Goji (Lycium barbarum).

Drought stress is one of the main abiotic stresses that limit plant growth and affect fruit quality and yield. Plants primarily lose water through leaf transpiration, and wax effectively reduces the rate of water loss from the leaves. However, the relationship between water loss and the wax formation mechanism in goji (Lycium barbarum) leaves remains unclear. 'Ningqi I' goji and 'Huangguo' goji are two common varieties. In this study, 'Ningqi I' goji and 'Huangguo' goji were used as samples of leaf material to detect the differences in the water loss rate, chlorophyll leaching rate, wax phenotype, wax content, and components of the two materials. The differences in wax-synthesis-related pathways were analyzed using the transcriptome and metabolome methods, and the correlation among the wax components, wax synthesis genes, and transcription factors was analyzed. The results show that the leaf permeability of 'Ningqi I' goji was significantly lower than that of 'Huangguo' goji. The total wax content of the 'Ningqi I' goji leaves was 2.32 times that of the 'Huangguo' goji leaves, and the epidermal wax membrane was dense. The main components of the wax of 'Ningqi I' goji were alkanes, alcohols, esters, and fatty acids, the amounts of which were 191.65%, 153.01%, 6.09%, and 9.56% higher than those of 'Huangguo' goji, respectively. In the transcriptome analysis, twenty-two differentially expressed genes (DEGs) and six transcription factors (TFs) were screened for wax synthesis; during the metabolomics analysis, 11 differential metabolites were screened, which were dominated by lipids, some of which, like D-Glucaro-1, 4-Lactone, phosphatidic acid (PA), and phosphatidylcholine (PE), serve as prerequisites for wax synthesis, and were significantly positively correlated with wax components such as alkanes by the correlation analysis. A combined omics analysis showed that DEGs such as LbaWSD1, LbaKCS1, and LbaFAR2, and transcription factors such as LbaMYB306, LbaMYB60, and LbaMYBS3 were strongly correlated with wax components such as alkanes and alcohols. The high expression of DEGs and transcription factors is an important reason for the high wax content in the leaf epidermis of 'Ningqi I' goji plants. Therefore, by regulating the expression of wax-synthesis-related genes, the accumulation of leaf epidermal wax can be promoted, and the epidermal permeability of goji leaves can be weakened, thereby reducing the water loss rate of goji leaves. The research results can lay a foundation for cultivating drought-tolerant goji varieties.

Development and evaluation of suitable reference genes for qRT-PCR normalization of hybrids derived from Lycium barbarum and Lycium ruthenicum.

BACKGROUND: A correct and stably expressing reference gene is prerequisite for successful quantitative real-time PCR (qRT-PCR). Investigating gene expression profiling during flower development could enhance our understanding of the molecular mechanisms of flower formation and fertility in Lycium. METHODS AND RESULTS: In this study, 11 candidate reference genes in Lycium flower development were selected from transcriptome sequence data and evaluated with five traditional housekeeping genes from previous studies based on qRT-PCR amplification. Comparing the expression stability result of 16 candidate genes using GeNorm, NormFinder, BestKeeper, and Delta Ct algorithms, Lba04g01649 and Lba12g02820 were validated as the optimal reference genes for the flower development of Lycium. CONCLUSIONS: The reference genes identified in this study would improve the accuracy of qRT-PCR quantification of target gene expression in Lycium flower development and facilitate future functional genomics studies on flower development. This research could lay the foundation for the study of the reproduction and development of the Lycium flower.

LbCOPT1 is a copper transporter induced in Lycium barbarum mycorrhizal roots, which allows tobacco with improved growth and nutrient uptake.

KEY MESSAGE: Overexpressing the copper transporter LbCOPT1 leads to a notable increase in the abundance of mycorrhizal arbuscules that suggests the potential application of LbCOPT1 in breeding programs aimed at enhancing symbiotic nutrient uptake in Lycium barbarum L.

Phylogenetic analysis and divergence time estimation of Lycium species in China based on the chloroplast genomes.

BACKGROUND: Lycium is an economically and ecologically important genus of shrubs, consisting of approximately 70 species distributed worldwide, 15 of which are located in China. Despite the economic and ecological importance of Lycium, its phylogeny, interspecific relationships, and evolutionary history remain relatively unknown. In this study, we constructed a phylogeny and estimated divergence time based on the chloroplast genomes (CPGs) of 15 species, including subspecies, of the genus Lycium from China. RESULTS: We sequenced and annotated 15 CPGs in this study. Comparative analysis of these genomes from these Lycium species revealed a typical quadripartite structure, with a total sequence length ranging from 154,890 to 155,677 base pairs (bp). The CPGs was highly conserved and moderately differentiated. Through annotation, we identified a total of 128-132 genes. Analysis of the boundaries of inverted repeat (IR) regions showed consistent positioning: the junctions of the IRb/LSC region were located in rps19 in all Lycium species, IRb/SSC between the ycf1 and ndhF genes, and SSC/IRa within the ycf1 gene. Sequence variation in the SSC region exceeded that in the IR region. We did not detect major expansions or contractions in the IR region or rearrangements or insertions in the CPGs of the 15 Lycium species. Comparative analyses revealed five hotspot regions in the CPG: trnR(UCU), atpF-atpH, ycf3-trnS(GGA), trnS(GGA), and trnL-UAG, which could potentially serve as molecular markers. In addition, phylogenetic tree construction based on the CPG indicated that the 15 Lycium species formed a monophyletic group and were divided into two typical subbranches and three minor branches. Molecular dating suggested that Lycium diverged from its sister genus approximately 17.7 million years ago (Mya) and species diversification within the Lycium species of China primarily occurred during the recent Pliocene epoch. CONCLUSION: The divergence time estimation presented in this study will facilitate future research on Lycium, aid in species differentiation, and facilitate diverse investigations into this economically and ecologically important genus.

Mapping quantitative trait loci associated with self-(in)compatibility in goji berries (Lycium barbarum).

BACKGROUND: Goji (Lycium barbarum L.) is a perennial deciduous shrub widely distributed in arid and semiarid regions of Northwest China. It is highly valued for its medicinal and functional properties. Most goji varieties are naturally self-incompatible, posing challenges in breeding and cultivation. Self-incompatibility is a complex genetic trait, with ongoing debates regarding the number of self-incompatible loci. To date, no genetic mappings has been conducted for S loci or other loci related to self-incompatibility in goji. RESULTS: We used genome resequencing to create a high-resolution map for detecting de novo single-nucleotide polymorphisms (SNP) in goji. We focused on 229 F1 individuals from self-compatible '13-19' and self-incompatible 'new 9' varieties. Subsequently, we conducted a quantitative trait locus (QTL) analysis on traits associated with self-compatibility in goji berries. The genetic map consisted of 249,327 SNPs distributed across 12 linkage groups (LGs), spanning a total distance of 1243.74 cM, with an average interval of 0.002 cM. Phenotypic data related to self-incompatibility, such as average fruit weight, fruit rate, compatibility index, and comparable compatibility index after self-pollination and geitonogamy, were collected for the years 2021-2022, as well as for an extra year representing the mean data from 2021 to 2022 (2021/22). A total of 43 significant QTL, corresponding to multiple traits were identified, accounting for more than 11% of the observed phenotypic variation. Notably, a specific QTL on chromosome 2 consistently appeared across different years, irrespective of the relationship between self-pollination and geitonogamy. Within the localization interval, 1180 genes were annotated, including Lba02g01102 (annotated as an S-RNase gene), which showed pistil-specific expression. Cloning of S-RNase genes revealed that the parents had two different S-RNase alleles, namely S1S11 and S2S8. S-genotype identification of the F1 population indicated segregation of the four S-alleles from the parents in the offspring, with the type of S-RNase gene significantly associated with self-compatibility. CONCLUSIONS: In summary, our study provides valuable insights into the genetic mechanism underlying self-compatibility in goji berries. This highlights the importance of further positional cloning investigations and emphasizes the importance of integration of marker-assisted selection in goji breeding programs.

Carotenoid biosynthesis genes LcLCYB, LcLCYE, and LcBCH from wolfberry confer increased carotenoid content and improved salt tolerance in tobacco.

Carotenoids play essential roles in plant growth and development and provide plants with a tolerance to a series of abiotic stresses. In this study, the function and biological significance of lycopene ß-cyclase, lycopene ε-cyclase, and ß-carotene hydroxylase, which are responsible for the modification of the tetraterpene skeleton procedure, were isolated from Lycium chinense and analyzed. The overexpression of lycopene ß-cyclase, lycopene ε-cyclase, and ß-carotene hydroxylase promoted the accumulation of total carotenoids and photosynthesis enhancement, reactive oxygen species scavenging activity, and proline content of tobacco seedlings after exposure to the salt stress. Furthermore, the expression of the carotenoid biosynthesis genes and stress-related genes (ascorbate peroxidase, catalase, peroxidase, superoxide dismutase, and pyrroline-5-carboxylate reductase) were detected and showed increased gene expression level, which were strongly associated with the carotenoid content and reactive oxygen species scavenging activity. After exposure to salt stress, the endogenous abscisic acid content was significantly increased and much higher than those in control plants. This research contributes to the development of new breeding aimed at obtaining stronger salt tolerance plants with increased total carotenoids and vitamin A content.

Anthocyanin biosynthesis in goji berry is inactivated by deletion in a bHLH transcription factor LrLAN1b promoter.

Black goji berry (Lycium ruthenicum Murray) contains a rich source of health-promoting anthocyanins which are used in herbal medicine and nutraceutical foods in China. A natural variant producing white berries allowed us to identify two key genes involved in the regulation of anthocyanin biosynthesis in goji berries: one encoding a MYB transcription factor (LrAN2-like) and one encoding a basic helix-loop-helix (bHLH) transcription factor (LrAN1b). We previously found that LrAN1b expression was lost in the white berry variant, but the molecular basis for this phenotype was unknown. Here, we identified the molecular mechanism for loss of anthocyanins in white goji berries. In white goji, the LrAN1b promoter region has a 229 bp deletion that removes three MYB-binding elements and one bHLH-binding element, which are key to its expression. Complementation of the white goji berry LrAN1b allele with the LrAN1b promoter restored pigmentation. Virus-induced gene silencing of LrAN1b in black goji berry reduced fruit anthocyanin biosynthesis. Molecular analyses showed that LrAN2-like and another bHLH transcription factor LrJAF13 can activate LrAN1b by binding directly to the MYB-recognizing element and bHLH-recognizing element of its promoter-deletion region. LrAN1b expression is enhanced by the interaction of LrAN2-like with LrJAF13 and the WD40 protein LrAN11. LrAN2-like and LrAN11 interact with either LrJAF13 or LrAN1b to form two MYB-bHLH-WD40 complexes, which hierarchically regulate anthocyanin biosynthesis in black goji berry. This study on a natural variant builds a comprehensive anthocyanin regulatory network that may be manipulated to tailor goji berry traits.

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.

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.

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.

Metabolite-based genome-wide association studies enable the dissection of the genetic bases of flavonoids, betaine and spermidine in wolfberry (Lycium).

Wolfberry is a plant with medicinal and food values. However, its bioactive ingredients and the corresponding genetic bases have not been determined. Here, we de novo generated a chromosome-level genome assembly for wolfberry, yielding a genome sequence of ~1.77 Gb with contig N50 of 50.55 Mb and 39 224 predicted gene models. A variation map, using 307 re-sequenced accessions, was called based on this genome assembly. Furthermore, the fruit metabolome of these accessions was profiled using 563 annotated metabolites, which separated Lycium barbarum L. and non-L. barbarum L. The flavonoids, coumarins, alkaloids and nicotinic acid contents were higher in the former than in the latter. A metabolite-based genome-wide association study mapped 156 164 significant single nucleotide polymorphisms corresponding to 340 metabolites. This included 19 219 unique lead single nucleotide polymorphisms in 1517 significant association loci, of which three metabolites, flavonoids, betaine and spermidine, were highlighted. Two candidate genes, LbUGT (evm.TU.chr07.2692) and LbCHS (evm.TU.chr07.2738), with non-synonymous mutations, were associated with the flavonoids content. LbCHS is a structural gene that interacts with a nearby MYB transcription factor (evm.TU.chr07.2726) both in L. barbarum and L. ruthenicum. Thus, these three genes might be involved in the biosynthesis/metabolism of flavonoids. LbSSADH (evm.TU.chr09.627) was identified as possibly participating in betaine biosynthesis/metabolism. Four lycibarbarspermidines (E-G and O) were identified, and only the lycibarbarspermidines O content was higher in L. barbarum varieties than in non-L. barbarum varieties. The evm.TU.chr07.2680 gene associated with lycibarbarspermidines O was annotated as an acetyl-CoA-benzylalcohol acetyltransferase, suggesting that it is a candidate gene for spermidine biosynthesis. These results provide novel insights into the specific metabolite profile of non-L. barbarum L. and the genetic bases of flavonoids, betaine and spermidine biosynthesis/metabolism.

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.

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.

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.

[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.