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B-box (BBX) genes play important roles in plant growth, light morphogenesis, and environmental stress responses. Ginkgo (Ginkgo biloba L.) is known as a living fossil species that has a strong ability to adapt to environmental changes and tolerate harsh conditions. In this study, we chose this species to investigate the function of the GbBBX25 gene. We isolated the BBX gene from ginkgo and named it GbBBX25; this gene consists of an 819 bp open reading frame (ORF) that encodes 273 amino acids with two B-box domains but no CCT domain. GbBBX25 was localized in only the nucleus. The expression of GbBBX25 transcripts was observed in the leaves and was significantly enhanced under salt stress conditions. To further verify its function, we overexpressed the GbBBX25 gene in Populus davidiana × Populus bolleana and found that the transgenic Populus had greater soluble sugar levels and higher peroxidase (POD) activity in response to salt stress than nontransgenic (NT) Populus. Five genes related to salt stress were induced in transgenic plants with significantly higher expression levels than those in NT plants. This finding suggests that GbBBX25 improves the salt adaptation abilities of transgenic Populus and provides a scientific basis for related research.

Ginkgo biloba L. leaves are a flavonoid resource for the pharmaceutical industry. The flavonoid 3'-hydroxylase (F3'H) is a key enzyme in the flavonoid biosynthesis pathway. However, the role of F3'H in flavonoid biosynthesis and metabolism is unclear. In this study, we characterized and functionally analyzed the ginkgo F3'H gene GbF3'H1 that encodes a protein of 520 amino acids. Expression profiling showed that GbF3'H1 was highly expressed in the leaves of ginkgo in September. Subcellular localization showed that GbF3'H1 occurred predominately in the cytoplasm. Transgenic poplars overexpressing GbF3'H1 had more red pigmentation in leaves than did wild-type (WT) plants. Furthermore, the concentrations of epigallocatechin, gallocatechin, and catechin in the downstream products synthesized by flavonoids were significantly higher in the transgenic plants than in the WT plants. These results indicate that the overexpression of GbF3'H1 enhances flavonoid production in transgenic plants and provides new insights into flavonoid biosynthesis and metabolism.

Ginkgo (Ginkgo biloba L.) is a tree valued for the high medicinal and nutritional value of its leaves and seeds. However, the metabolite profiles of ginkgo leaves and seeds and their changes during development have not been comprehensively analyzed, which hinders improvements in the utilization of ginkgo. A comprehensive and systematic untargeted LC-MS metabolomics analysis of the metabolites in ginkgo leaves (male and female) and seeds at two developmental stages identified 8146 known metabolites, which mainly included lipids and lipid-like molecules, phenylpropanoids and polyketides, organoheterocyclic compounds, organic acids and derivatives, organic oxygen compounds, and benzenoids. Some of the identified metabolites have known healthcare and food value, and some of the others are newly discovered metabolites with potential for new drug development. The small number of differential Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways between leaves of male and female gingko trees indicated that the developmental stage affected the metabolic pathways more significantly than sex. Among the flavonoid constituents of ginkgo, 653 flavonoid metabolites were identified, and these included some new flavonoid components, which confirmed that the developmental stage is a critical factor in secondary metabolite variations. This study illuminated the metabolites and medicinal and edible values of ginkgo leaves and seeds at different developmental stages and thus supports further effective utilization of ginkgo leaves and seeds.

The flavonoids in Ginkgo biloba L. (ginkgo) have important medicinal uses due to their antioxidant, antitumor, and blood circulation-promoting effects. However, the genetic mechanisms underlying flavonoid biosynthesis in ginkgo remain elusive. Flavonoid 3&prime, 5&prime, hydroxylase (F3&prime, H) is an important enzyme in flavonoid synthesis. We detected a novel differentially expressed GbF3&prime, H1 gene homologous to the F3&prime, H enzyme involved in the flavonoid synthesis pathway through transcriptome sequencing. In this study, we characterized this gene, performed an expression analysis, and heterologously overexpressed GbF3&prime, H1 in Populus. Our results showed that GbF3&prime, H1 is abundant in the leaf and highly expressed during April. We also found four metabolites closely related to flavonoid biosynthesis. Importantly, the contents of 4&prime, 5-dihydroxy-7-glucosyloxyflavanone, epicatechin, and gallocatechin were significantly higher in transgenic plants than in nontransgenic plants. Our findings revealed that the GbF3&prime, H1 gene functions in the biosynthesis of flavonoid-related metabolites, suggesting that GbF3&prime, H1 represents a prime candidate for future studies (e.g., gene-editing) aiming to optimize ginkgo flavonoid production, especially that of flavan-3-ols.

The flavonoids in Ginkgo biloba L. (ginkgo) have important medicinal uses due to their antioxidant, antitumor, and blood circulation-promoting effects. However, the genetic mechanisms underlying flavonoid biosynthesis in ginkgo remain elusive. Flavonoid 3′, 5′-hydroxylase (F3′5′H) is an important enzyme in flavonoid synthesis. We detected a novel differentially expressed GbF3′5′H1 gene homologous to the F3′5′H enzyme involved in the flavonoid synthesis pathway through transcriptome sequencing. In this study, we characterized this gene, performed an expression analysis, and heterologously overexpressed GbF3′5′H1 in Populus. Our results showed that GbF3′5′H1 is abundant in the leaf and highly expressed during April. We also found four metabolites closely related to flavonoid biosynthesis. Importantly, the contents of 4′,5-dihydroxy-7-glucosyloxyflavanone, epicatechin, and gallocatechin were significantly higher in transgenic plants than in nontransgenic plants. Our findings revealed that the GbF3′5′H1 gene functions in the biosynthesis of flavonoid-related metabolites, suggesting that GbF3′5′H1 represents a prime candidate for future studies (e.g., gene-editing) aiming to optimize ginkgo flavonoid production, especially that of flavan-3-ols.

Ginkgo biloba breeding commonly concentrates on the selection of superior trees that have high flavonoid contents. The flavonoids present in Ginkgo leaves have strong medicinal implications, including anti-dengue, anti-HIV, anticancer, antioxidant and anti-inflammatory properties. Flavonoids play important roles in plant immune responses; however, the molecular mechanisms underlying these interesting responses remain unclear. To obtain a comprehensive understanding, we performed the transcriptome sequencing of Ginkgo with different flavonoid contents. Using an Illumina sequencing platform, we obtained approximately 533,952,528 clean reads. After the sequences were filtered and assembled, the transcriptome data generated 37,625 unigenes, of which 21,472 (57.07%) were successfully annotated in five public databases. Among those genes, many candidates were involved in flavonoid biosynthesis, transport and regulation. Expression profiles were generated, and 457 genes were found to be significantly differentially expressed between the Sample_GB_FH1/2/3 (FH) and Sample_GB_FL1/2/3 (FL) libraries; 246 (53.83%) genes were up-regulated, and 211 (46.17%) were down-regulated. These genes included 14 genes that were enriched in flavonoid transport, 1 MYB gene that encoded a putative transcription factor (TF), and 1 dihydroflavonol reductase (DFR) gene that was involved in the flavonoid pathway. Our results provide comprehensive gene expression information about the Ginkgo transcriptome and can facilitate our understanding of the molecular mechanisms of flavonoid development in Ginkgo. Furthermore, our results markedly expand both the available Ginkgo genetic library and analyses of the species and provide valuable information to the Ginkgo-related pharmaceutical industry.

Agroforestry practices aim to achieve environmentally friendly land use. Fungi play a primarily role in soil organic carbon and nutrient maintenance, while the response of the soil fungi community to land use changes is little explored. Here, a high-throughput sequencing method was applied to understand the fungal community structure distinction in ginkgo agroforestry systems and adjacent croplands and nurseries. Our results showed that the agroforestry systems achieved better soil fertility and carbon contents. The agroforestry practices significantly altered the composition of soil fungal communities comparing with pure gingko plantation, adjacent cropland, and nursery. The dominant fungal phyla were always Ascomycota and Basidiomycota. The relative abundance of Ascomycota was correlated with the TN and AP, while the abundance of Basidiomycota was negatively correlated with the TN and NN. The soil organic carbon, total nitrogen, and nitrate nitrogen explained 59.80% and 63.36% of the total variance in the fungal community composition in the topsoil and subsoil, and the available phosphorus also played a key role in the topsoil. Considering soil fertility maintenance and fungal community survival and stability, the agroforestry systems achieved better results, and the ginkgo and wheat system was the best among the five planting systems we studied. In the ginkgo and wheat system, applying readily available mineral nitrogen fertilizer either alone or in combination with organic amendments will improve the soil quality and fertility.

Background Long noncoding RNAs (lncRNAs) play an important role in diverse biological processes and have been widely studied in recent years. However, the roles of lncRNAs in leaf pigment formation in ginkgo (Ginkgo biloba L.) remain poorly understood. Results In this study, lncRNA libraries for mutant yellow-leaf and normal green-leaf ginkgo trees were constructed via high-throughput sequencing. A total of 2044 lncRNAs were obtained with an average length of 702 nt and typically harbored 2 exons. We identified 238 differentially expressed lncRNAs (DELs), 32 DELs and 49 differentially expressed mRNAs (DEGs) that constituted coexpression networks. We also found that 48 cis-acting DELs regulated 72 target genes, and 31 trans-acting DELs regulated 31 different target genes, which provides a new perspective for the regulation of the leaf-color mutation. Due to the crucial regulatory roles of lncRNAs in a wide range of biological processes, we conducted in-depth studies on the DELs and their targets and found that the chloroplast thylakoid membrane subcategory and the photosynthesis pathways (ko00195) were most enriched, suggesting their potential roles in leaf coloration mechanisms. In addition, our correlation analysis indicates that eight DELs and 68 transcription factors (TFs) might be involved in interaction networks. Conclusions This study has enriched the knowledge concerning lncRNAs and provides new insights into the function of lncRNAs in leaf-color mutations, which will benefit future selective breeding of ginkgo.

Ginkgo biloba (ginkgo) is a well-known ornamental tree. The yellow-leaf ginkgo mutant is an ideal material for studying leaf pigment synthesis and mutation. In this study, we used the Illumina sequencing of ginkgo small RNAs to gain insight into the function of microRNAs (miRNAs) in leaf color mutation. We constructed six small RNA libraries, and identified a total of 426 known and 265 novel miRNAs. Nineteen differentially expressed miRNAs (DEMs) were detected between the yellow-leaf and the green-leaf groups, including 8 upregulated miRNAs. The target genes of DEMs were predicted, resulting in the identification of 205 target mRNAs and 230 target gene binding sites. We found that the novel 158_mature and gma-miR2118a-3p target genes were involved in pigment biosynthesis. Three miRNAs, the novel 151_mature, ptc-miR396e-3p and aly-miR156a-5p appeared to be the potential key regulators of leaf color mutation. These DEMs and their corresponding target genes likely play key roles in the regulation of the yellow-leaf mutant. This study constructed miRNA profiles and provides new insight and a reference for related studies on miRNA regulation of leaf color pigment synthesis.

Ginkgo (Ginkgo biloba L.) is a unique medicinal tree species that mainly depends on the flavonoids and terpenoids found in its leaves. However, the biosynthesis of these metabolites is controlled and regulated by gene actions which remain poorly understood. To improve our understanding of this process, we produced a transcriptome library from young and mature leaves of ginkgo trees using the Illumina HiSeq X Ten sequencing platformin this study. We obtained a total of 57.5 million clean reads, compared with seven databases and revealed 39,404 annotated unigenes. The key differentially expressed genes (DEGs) in young and mature leaves of ginkgo were explored through GO and KEGG metabolic pathway analysis. A total of 1,256 DEGs, including 561 upregulated and 695 downregulated genes were obtained in the ginkgo transcriptome in the mature leaves compared with that of the young leaves. These genes included 23 bHLH, 9 MYB, 5 WRKY, and 4 bZIP genes that act as regulators in flavonoid and terpenoid biosynthesis. One key gene involved in the diterpenoid pathway was downregulated. A gene in the phenylpropanoid biosynthesis pathway was significantly upregulated and significantly negatively correlated with serine in metabolites. Additionally, a metabolomics analysis detected 283 metabolites, and 38 significantly different metabolites were identified. The combined metabolome and transcriptome approach employed in this study constitutes an effective method for assessing the relationships between the expression of critical genes and metabolites related to biosynthetic pathways. Our study provides new ideas for analyzing biosynthesis at the molecular level and will encourage further development of the ginkgo-related pharmaceutical industry.

Ginkgo (Ginkgo biloba L.) has attracted much attention due to its medicinal properties, such as its high flavonoid and lactone contents. To improve its content of active substances through phenotypic selection, variation in crown type and its association with the main active substance contents were investigated at four test sites. The results showed significant differences in the tree height (HT), diameter at breast height (DBH), branching angle (BA), leaf area (LA), and leaf density among the crown types. The results also indicated that the isorhamnetin contents in Ginkgo leaves differed significantly among the crown types and tended to increase with increasing crown size, suggesting the effectiveness of the classification in crown types and its potential use in selective breeding. Trees with the wide-crown medium type had the highest growth rate in HT and DBH, and the greatest leaf yield. Selection of wide-crown medium types will greatly increase active substance contents, but selection of individuals whose crown type has a high content is also necessary to fully exploit the variation among Ginkgo individuals and enhance breeding efficiency. This study will provide a basis and effective method for selection, breeding, and industrial production.