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.

MADS-box genes encode transcription factors that affect plant growth and development. Camellia chekiangoleosa is an oil tree species with ornamental value, but there have been few molecular biological studies on the developmental regulation of this species. To explore their possible role in C. chekiangoleosa and lay a foundation for subsequent research, 89 MADS-box genes were identified across the whole genome of C. chekiangoleosa for the first time. These genes were present on all the chromosomes and were found to have expanded by tandem duplication and fragment duplication. Based on the results of a phylogenetic analysis, the 89 MADS-box genes could be divided into either type I (38) or type II (51). Both the number and proportion of the type II genes were significantly greater than those of Camellia sinensis and Arabidopsis thaliana, indicating that C. chekiangoleosa type II genes experienced a higher duplication rate or a lower loss rate. The results of both a sequence alignment and a conserved motif analysis suggest that the type II genes are more conserved, meaning that they may have originated and differentiated earlier than the type I genes did. At the same time, the presence of extra-long amino acid sequences may be an important feature of C. chekiangoleosa. Gene structure analysis revealed the number of introns of MADS-box genes: twenty-one type I genes had no introns, and 13 type I genes contained only 1~2 introns. The type II genes have far more introns and longer introns than the type I genes do. Some MIKC^C genes have super large introns (≥15 kb), which are rare in other species. The super large introns of these MIKC^C genes may indicate richer gene expression. Moreover, the results of a qPCR expression analysis of the roots, flowers, leaves and seeds of C. chekiangoleosa showed that the MADS-box genes were expressed in all those tissues. Overall, compared with that of the type I genes, the expression of the type II genes was significantly higher. The CchMADS31 and CchMADS58 genes (type II) were highly expressed specifically in the flowers, which may in turn regulate the size of the flower meristem and petals. CchMADS55 was expressed specifically in the seeds, which might affect seed development. This study provides additional information for the functional characterization of the MADS-box gene family and lays an important foundation for in-depth study of related genes, such as those involved in the development of the reproductive organs of C. chekiangoleosa.

The Malus crabapple is an important woody ornamental plant. The fading of petals during its development significantly affects their ornamental value. Petal color is related to anthocyanin content and miRNAs play an important role in the post-transcriptional regulation of anthocyanin synthesis. However, the mechanisms underlying miRNA regulation of petal fading have rarely been studied. Transcriptome and small RNA sequencing of petals from the blooming phases of Malus. ‘Indian Summer’ varieties S1 (small bud), S2 (initial-flowering), and S3 (late-flowering) allowed us to identify 230 known miRNAs and 17 novel miRNAs, including 52 differentially expressed miRNAs which targeted 494 genes and formed 823 miRNA–target pairs. Based on the target gene annotation results, miRNA–target pairs were screened that may be involved in the fading process of Malus crabapple petals through three different pathways: anthocyanin synthesis, transport, and degradation, involving mcr-miR858-MYB1\MYB5 and mcr-miR396-McCHI inhibiting anthocyanin synthesis; mcr-miR167, mcr-miR390, mcr-miR535, and mcr-miR858 inhibiting anthocyanin transport from the cytoplasm to the vacuole by targeting ABC transporter genes (ABCB, ABCC, ABCD, and ABCG); and mcr-miR398 targeting the superoxide dismutase genes (CZSOD2 and CCS) to accelerate anthocyanin degradation. These findings offer a novel approach to understanding the mechanism of petal fading and serve as a reference for other plants with floral fading.

The NAC (NAM-ATAF1/2-CUC) transcription factor family is one of the largest plant-specific transcription factor families, playing an important role in plant growth and development and abiotic stress response. As a short-rotation woody plant, Salix integra (S. integra) has high lead (Pb) phytoremediation potential. To understand the role of NAC in S. integra Pb tolerance, 53 SiNAC transcripts were identified using third-generation and next-generation transcriptomic data from S. integra exposed to Pb stress, and a phylogenetic analysis revealed 11 subfamilies. A sequence alignment showed that multiple subfamilies represented by TIP and ATAF had a gene that produced more than one transcript under Pb stress, and different transcripts had different responses to Pb. By analyzing the expression profiles of SiNACs at 9 Pb stress time points, 41 of 53 SiNACs were found to be significantly responsive to Pb. Short time-series expression miner (STEM) analysis revealed that 41 SiNACs had two significant Pb positive response patterns (early and late), both containing 10 SiNACs. The SiNACs with the most significant Pb response were mainly from the ATAF and NAP subfamilies. Therefore, 4 and 3 SiNACs from the ATAF and NAP subfamilies, respectively, were selected as candidate Pb-responsive SiNACs for further structural and functional analysis. The RT-qPCR results of 7 transcripts also confirmed the different Pb response patterns of the ATAF and NAP subfamilies. SiNAC004 and SiNAC120, which were randomly selected from two subfamilies, were confirmed to be nuclear localization proteins by subcellular localization experiments. Functional prediction analysis of the associated transcripts of seven candidate SiNACs showed that the target pathways of ATAF subfamily SiNACs were “sulfur metabolism” and “glutathione metabolism”, and the target pathways of NAP subfamily SiNACs were “ribosome” and “phenylpropanoid biosynthesis”. This study not only identified two NAC subfamilies with different Pb response patterns but also identified Pb-responsive SiNACs that could provide a basis for subsequent gene function verification.

Ginkgo biloba L. has attracted much attention due to its medicinal properties, particularly those of its terpenoid and flavonoid contents. However, the content and utilization efficiency of terpenoids remain low. The enzyme 3-hydroxy-3-methylglutaryl CoA synthase (HMGS) is a major rate-limiting factor, and RNA-seq has revealed that the mRNA expression of this enzyme is differentially expressed during terpenoid biosynthesis. Here, we investigated the function of the GbHMGS1 gene and its overexpression in Populus. We compared the metabolite contents of nontransgenic (CK) Populus with those of transgenic Populus lines through metabolomics analysis. Our results indicate that the GbHMGS1 protein is localized in the cytoplasm. Significant differences in chemical characteristics were found between the transgenic and CK plants, and a total of 31 differentially expressed metabolites were upregulated in the transgenic plants. We also found higher contents of lanosterol (triterpenoid), dehydroabietic acid (diterpenoid), and phytol (diterpenoid) in the transgenic Populus plants than in their CK counterparts. We thus speculate that GbHMGS1 might regulate plant-related product formation and increase metabolite contents. This study revealed the molecular mechanism governing metabolite synthesis and suggested that one triterpenoid and two diterpenoids with significant upregulation can be used as markers for the breeding of plants with specific terpenoid metabolism-related characteristics.