Multi-omics explores the potential regulatory role of acetylation modification in flavonoid biosynthesis of Ginkgo biloba.

Flavonoids are crucial medicinal active ingredients in Ginkgo biloba. However, the effect of protein post-translational modifications (PTMs) on flavonoid biosynthesis remains poorly explored. Lysine acetylation, a reversible PTM, plays a crucial role in metabolic regulation. This study aims to investigate the potential role of acetylation in G. biloba flavonoid biosynthesis. Through comprehensive analysis of transcriptomes, metabolomes, proteomes, and acetylated proteins in different tissues, a total of 11,788 lysine acetylation sites were identified on 4324 acetylated proteins, including 89 acetylation sites on 23 proteins. Additionally, 128 types of differentially accumulated flavonoids were identified among tissues, and a dataset of differentially expressed genes related to the flavonoid biosynthesis pathway was constructed. Twelve (CHI, C3H1, ANR, DFR, CCoAOMT1, F3H1, F3H2, CCoAOMT2, C3H2, HCT, F3'5'H, and FG2) acetylated proteins that might be involved in flavonoid biosynthesis were identified. Specifically, we found that the modification levels of CCoAOMT1 and F3'5'H sites correlated with the catalytic production of homoeriodictyol and dihydromyricetin, respectively. Inhibitors of lysine deacetylase (trichostatin A, TSA) impacted total flavonoid content in different tissues and increased flavonoid levels in G. biloba roots. Treatment with TSA revealed that expression levels of GbF3'5'H and GbCCoAOMT1 in stems and leaves aligned with total flavonoid content variations, while in roots, expression levels of GbC3H2 and GbFG2 corresponded to total flavonoid content changes. Collectively, these findings reveal for the first time the important role of acetylation in flavonoid biosynthesis.

The Ginkgo biloba microRNA160-ERF4 module participates in terpene trilactone biosynthesis.

Terpene trilactones (TTLs) are important secondary metabolites in ginkgo (Ginkgo biloba); however, their biosynthesis gene regulatory network remains unclear. Here, we isolated a G. biloba ethylene response factors 4 (GbERF4) involved in TTL synthesis. Overexpression of GbERF4 in tobacco (Nicotiana tabacum) significantly increased terpenoid content and upregulated the expression of key enzyme genes (3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), 1-deoxy-D-xylulose-5-phosphate synthase (DXS), acetyl-CoA C-acetyltransferase (AACT), and geranylgeranyl diphosphate synthase (GGPPS)) in the terpenoid pathway in tobacco, suggesting that GbERF4 functions in regulating the synthesis of terpenoids. The expression pattern analysis and previous microRNA (miRNA) sequencing showed that gb-miR160 negatively regulates the biosynthesis of TTLs. Transgenic experiments showed that overexpression of gb-miR160 could significantly inhibit the accumulation of terpenoids in tobacco. Targeted inhibition and dual-luciferase reporter assays confirmed that gb-miR160 targets and negatively regulates GbERF4. Transient overexpression of GbERF4 increased TTL content in G. biloba, and further transcriptome analysis revealed that DXS, HMGS, CYPs, and transcription factor genes were upregulated. In addition, yeast one-hybrid and dual-luciferase reporter assays showed that GbERF4 could bind to the promoters of the HMGS1, AACT1, DXS1, levopimaradiene synthase (LPS2), and GGPPS2 genes in the TTL biosynthesis pathway and activate their expression. In summary, this study investigated the molecular mechanism of the gb-miR160-GbERF4 regulatory module in regulating the synthesis of TTLs. It provides information for enriching the understanding of the regulatory network of TTL biosynthesis and offers important gene resources for the genetic improvement of G. biloba with high contents of TTLs.

Cloning and functional analysis of Gb4CL1 and Gb4CL2 from Ginkgo biloba.

4-Coumarate-CoA ligase (4CL) gene plays vital roles in plant growth and development, especially the regulation of lignin metabolism and flavonoid synthesis. To investigate the potential function of 4CL in the lignin biosynthesis of Ginkgo biloba, this study identified two 4CL genes, Gb4CL1 and Gb4CL2, from G. biloba genome. Based on the phylogenetic tree analysis, Gb4CL1 and Gb4CL2 protein were classified into Class I, which has been confirmed to be involved in lignin biosynthesis. Therefore, it can be inferred that these two genes may also participate in lignin metabolism. The tissue-specific expression patterns of these two genes revealed that Gb4CL1 was highly expressed in microstrobilus, whereas Gb4CL2 was abundant in immature leaves. The onion transient expression assay indicated that Gb4CL1 was predominantly localized in the nucleus, indicating its potential involvement in nuclear functions, while Gb4CL2 was observed in the cell wall, suggesting its role in cell wall-related processes. Phytohormone response analysis revealed that the expression of both genes was upregulated in response to indole acetic acid, while methyl jasmonate suppressed it, gibberellin exhibited opposite effects on these genes. Furthermore, Gb4CL1 and Gb4CL2 expressed in all tissues containing lignin that showed a positive correlation with lignin content. Thus, these findings suggest that Gb4CL1 and Gb4CL2 are likely involved in lignin biosynthesis. Gb4CL1 and Gb4CL2 target proteins were successfully induced in Escherichia coli BL21 with molecular weights of 85.5 and 89.2 kDa, proving the integrity of target proteins. Our findings provided a basis for revealing that Gb4CL participated in lignin synthesis in G. biloba.

Genomic-wide identification and expression analysis of AP2/ERF transcription factors in Zanthoxylum armatum reveals the candidate genes for the biosynthesis of terpenoids.

Terpenoids are the main active components in the Zanthoxylum armatum leaves, which have extensive medicinal value. The Z. armatum leaf is the main by-product in the Z. armatum industry. However, the transcription factors involved in the biosynthesis of terpenoids are rarely reported. This study was performed to identify and classify the APETALA2/ethylene-responsive factor (AP2/ERF) gene family of Z. armatum. The chromosome distribution, gene structure, conserved motifs, and cis-acting elements of the promoter of the species were also comprehensively analyzed. A total of 214 ZaAP2/ERFs were identified. From the obtained transcriptome and terpenoid content data, four candidate ZaAP2/ERFs involved in the biosynthesis of terpenoids were selected via correlation and weighted gene co-expression network analysis. A phylogenetic tree was constructed using 13 AP2/ERFs related to the biosynthesis of terpenoids in other plants. ZaERF063 and ZaERF166 showed close evolutionary relationships with the ERFs in other plant species and shared a high AP2-domain sequence similarity with the two closest AP2/ERF proteins, namelySmERF8 from Salvia miltiorrhiza and AaERF4 from Artemisia annua. Further investigation into the effects of methyl jasmonate (MeJA) treatment on the content of terpenoids in Z. armatum leaves revealed that MeJA significantly induced the upregulation of ZaERF166 and led to a significant increase in the terpenoids content in Z. armatum leaves, indicating that ZaERF166 might be involved in the accumulation of terpenoids of Z. armatum. Results will be beneficial for the functional characterization of AP2/ERFs in Z. armatum and establishment of the theoretical foundation to increase the production of terpenoids via the manipulation of the regulatory elements and strengthen the development and utilization of Z. armatum leaves.

The long noncoding RNAs lnc10 and lnc11 regulating flavonoid biosynthesis in Ginkgo biloba.

Although long non-coding RNAs have been recognized to play important roles in plant, their possible functions and potential mechanism in Ginkgo biloba flavonoid biosynthesis are poorly understood. Flavonoids are important secondary metabolites and healthy components of Ginkgo biloba. They have been widely used in food, medicine, and natural health products. Most previous studies have focused on the molecular mechanisms of structural genes and transcription factors that regulate flavonoid biosynthesis. Few reports have examined the biological functions of flavonoid biosynthesis by long non-coding RNAs in G. biloba. Long noncoding RNAs associated with flavonoid biosynthesis in G. biloba have been identified through RNA sequencing, but the function of lncRNAs has not been reported. In this study, the expression levels of lnc10 and lnc11 were identified. Quantitative real-time polymerase chain reaction analysis revealed that lnc10 and lnc11 were expressed in all detected organs, and they showed significantly higher levels in immature and mature leaves than in other organs. In addition, to fully identify the function of lnc10 and lnc11 in flavonoid biosynthesis in G. biloba, lnc10 and lnc11 were cloned from G. biloba, and were transformed into Arabidopsis and overexpressed. Compared with the wild type, the flavonoid content was increased in transgenic plants. Moreover, the RNA-sequencing analysis of wild-type, lnc10-overexpression, and lnc11-overexpression plants screened out 2019 and 2552 differentially expressed genes, and the transcript levels of structural genes and transcription factors associated with flavonoid biosynthesis were higher in transgenic Arabidopsis than in the wild type, indicating that lnc10 and lnc11 activated flavonoid biosynthesis in the transgenic lines. Overall, these results suggest that lnc10 and lnc11 positively regulate flavonoid biosynthesis in G. biloba.

Multiomics analysis provides new insights into the regulatory mechanism of carotenoid biosynthesis in yellow peach peel.

Carotenoids, as natural tetraterpenes, play a pivotal role in the yellow coloration of peaches and contribute to human dietary health. Despite a relatively clear understanding of the carotenoid biosynthesis pathway, the regulatory mechanism of miRNAs involved in carotenoid synthesis in yellow peaches remain poorly elucidated. This study investigated a total of 14 carotenoids and 40 xanthophyll lipids, including six differentially accumulated carotenoids: violaxanthin, neoxanthin, lutein, zeaxanthin, cryptoxanthin, and (E/Z)-phytoene. An integrated analysis of RNA-seq, miRNA-seq and degradome sequencing revealed that miRNAs could modulate structural genes such as PSY2, CRTISO, ZDS1, CHYB, VDE, ZEP, NCED1, NCED3 and the transcription factors NAC, ARF, WRKY, MYB, and bZIP, thereby participating in carotenoid biosynthesis and metabolism. The authenticity of miRNAs and target gene was corroborated through quantitative real-time PCR. Moreover, through weighted gene coexpression network analysis and a phylogenetic evolutionary study, coexpressed genes and MYB transcription factors potentially implicated in carotenoid synthesis were identified. The results of transient expression experiments indicated that mdm-miR858 inhibited the expression of PpMYB9 through targeted cleavage. Building upon these findings, a regulatory network governing miRNA-mediated carotenoid synthesis was proposed. In summary, this study comprehensively identified miRNAs engaged in carotenoid biosynthesis and their putative target genes, thus enhancing the understanding of carotenoid accumulation and regulatory mechanism in yellow peach peel and expanding the gene regulatory network of carotenoid synthesis.

Contemporary understanding of transcription factor regulation of terpenoid biosynthesis in plants.

KEY MESSAGE: This review summarized how TFs function independently or in response to environmental factors to regulate terpenoid biosynthesis via fine-tuning the expression of rate-limiting enzymes. Terpenoids are derived from various species and sources. They are essential for interacting with the environment and defense mechanisms, such as antimicrobial, antifungal, antiviral, and antiparasitic properties. Almost all terpenoids have high medicinal value and economic performance. Recently, the control of enzyme genes on terpenoid biosynthesis has received a great deal of attention, but transcriptional factors regulatory network on terpenoid biosynthesis and accumulation has yet to get a thorough review. Transcription factors function as activators or suppressors independently or in response to environmental stimuli, fine-tuning terpenoid accumulation through regulating rate-limiting enzyme expression. This study investigates the advancements in transcription factors related to terpenoid biosynthesis and systematically summarizes previous works on the specific mechanisms of transcription factors that regulate terpenoid biosynthesis via hormone signal-transcription regulatory networks in plants. This will help us to better comprehend the regulatory network of terpenoid biosynthesis and build the groundwork for terpenoid development and effective utilization.

1,25(OH)2D3 ameliorates doxorubicin­induced cardiomyopathy by inhibiting the NLRP3 inflammasome and oxidative stress.

Doxorubicin (DOX), as a chemotherapy agent with marked therapeutic effect, can be used to treat certain types of cancer such as leukemia, lymphoma and breast cancer. However, the toxic effects of DOX on cardiomyocytes limit its clinical application. Oxidative stress has been documented to serve a pivotal role in DOX-induced cardiomyopathy. Previous studies have reported that 1,25(OH)2D3 has antioxidant and anti-inflammatory effects and can inhibit the renin-angiotensin system. However, the effects of 1,25(OH)2D3 on the pathophysiological processes of DOX-induced cardiomyopathy and its mechanisms remain poorly understood. To investigate these potential effects, C57BL/6J mice were used to construct a DOX-induced cardiomyopathy model and treated with 1,25(OH)2D3. At 4 weeks after the first injection of DOX, cardiac function and myocardial injury were evaluated by echocardiograph and ELISA. Masson's trichrome staining and RT-qPCR were used to assess myocardial fibrosis, and immunohistochemistry and western blotting were performed to analyze expression levels of inflammation and oxidative stress, and the NLRP3 inflammasome pathway. ChIP assay was used to assess the effects of 1,25(OH)2D3 on histone modification in the NLRP3 and Nrf2 promoters. The results showed that 1,25(OH)2D3 treatment increased LVEF and LVFS, reduced serum levels of BNP and cTnT, inhibited the collagen deposition and profibrotic molecular expression, and downregulated the levels of inflammatory cytokines in DOX-induced cardiomyopathy. ROS and antioxidant indices were also ameliorated after 1,25(OH)2D3 treatment. In addition, 1,25(OH)2D3 was found to inhibit the NLRP3 inflammasome and KEAP-Nrf2 pathways through regulation of the levels of H3K4me3, H3K27me3 and H2AK119Ub in the NLRP3 and Nrf2 promoters. In conclusion, the present study demonstrated that 1,25(OH)2D3 regulated histone modification in the NLRP3 and Nrf2 promoters, which in turn inhibits the activation of NLRP3 inflammasome and oxidative stress in cardiomyocytes, alleviating DOX-induced cardiomyopathy. Therefore, 1,25(OH)2D3 may be a potential drug candidate for the treatment of DOX-induced cardiomyopathy.

Ginkgo biloba GbbZIP08 transcription factor is involved in the regulation of flavonoid biosynthesis.

Ginkgo biloba is the oldest relict plant on Earth and an economic plant resource derived from China. Flavonoids extracted from G. biloba are beneficial to the prevention and treatment of cardiovascular and cerebrovascular diseases. Basic leucine zipper (bZIP) transcription factors (TFs) have been recognized to play important roles in plant secondary metabolism. In this study, GbbZIP08 was isolated and characterized. It encodes a protein containing 154 amino acids, which belongs to hypocotyl 5 in group H of the bZIP family. Tobacco transient expression assay indicated that GbbZIP08 was localized in the plant nucleus. GbbZIP08 overexpression showed that the contents of total flavonoids, kaempferol, and anthocyanin in transgenic tobacco were significantly higher than those in the wild type. Transcriptome sequencing analysis revealed significant upregulation of structural genes in the flavonoid biosynthesis pathway. In addition, phytohormone signal transduction pathways, such as the abscisic acid, salicylic acid, auxin, and jasmonic acid pathways, were enriched with a large number of differentially expressed genes. TFs such as MYB, AP2, WRKY, NAC, bZIP, and bHLH, were also differentially expressed. The above results indicated that GbbZIP08 overexpression promoted flavonoid accumulation and increased the transcription levels of flavonoid-synthesis-related genes in plants.

Role of bZIP transcription factors in the regulation of plant secondary metabolism.

MAIN CONCLUSION: This study provides an overview of the structure, classification, regulatory mechanisms, and biological functions of the basic (region) leucine zipper transcription factors and their molecular mechanisms in flavonoid, terpenoid, alkaloid, phenolic acid, and lignin biosynthesis. Basic (region) leucine zippers (bZIPs) are evolutionarily conserved transcription factors (TFs) in eukaryotic organisms. The bZIP TFs are widely distributed in plants and play important roles in plant growth and development, photomorphogenesis, signal transduction, resistance to pathogenic microbes, biotic and abiotic stress, and secondary metabolism. Moreover, the expression of bZIP TFs not only promotes or inhibits the accumulation of secondary metabolites in medicinal plants, but also affects the stress response of plants to the external adverse environment. This paper describes the structure, classification, biological function, and regulatory mechanisms of bZIP TFs. In addition, the molecular mechanism of bZIP TFs regulating the biosynthesis of flavonoids, terpenoids, alkaloids, phenolic acids, and lignin are also elaborated. This review provides a summary for in-depth study of the molecular mechanism of bZIP TFs regulating the synthesis pathway of secondary metabolites and plant molecular breeding, which is of significance for the generation of beneficial secondary metabolites and the improvement of plant varieties.

Genome-wide identification of HD-ZIP gene family and screening of genes related to prickle development in Zanthoxylum armatum.

Zanthoxylum armatum is an important cash crop for medicinal and food purposes in Asia. However, its stems and leaves are covered with a large number of prickles, which cause many problems in the production process. The homeodomain leucine zipper (HD-ZIP) gene family is a class of transcription factors unique to plants that play an important role in biological processes such as morphogenesis, signal transduction, and secondary metabolite synthesis. However, little is known about HD-ZIP gene information that may be involved in prickle development of Z. armatum. Here, we identified 76 ZaHDZ genes from the Z. armatum genome and classified them into four subfamilies (I-IV) based on phylogenetic analysis, a classification further supported by gene structure and conserved motif analysis. Seventy-six ZaHDZ genes were unevenly distributed on chromosomes. Evolutionary analysis revealed that the expansion of ZaHDZ genes mainly were due to whole-genome duplication (WGD) or segmental duplication, and they experienced strong purifying selection pressure in the process of evolution. A total of 47 cis-elements were identified in the promoter region of ZaHDZ genes. Quantitative real-time polymerase chain reaction analysis was performed on subfamily IV ZaHDZ gene expression levels in five tissues and under four hormone treatments. Finally, ZaHDZ16 was predicted to be the candidate gene most likely to be involved in prickle development of Z. armatum. These results contribute to a better understanding of the characteristics of HD-ZIP gene family and lay a foundation for further study on the function of genes related to prickle development of Z. armatum.

Advances in Research on the Involvement of Selenium in Regulating Plant Ecosystems.

Selenium is an essential trace element which plays an important role in human immune regulation and disease prevention. Plants absorb inorganic selenium (selenite or selenate) from the soil and convert it into various organic selenides (such as seleno amino acids, selenoproteins, and volatile selenides) via the sulfur metabolic pathway. These organic selenides are important sources of dietary selenium supplementation for humans. Organoselenides can promote plant growth, improve nutritional quality, and play an important regulatory function in plant ecosystems. The release of selenium-containing compounds into the soil by Se hyperaccumulators can promote the growth of Se accumulators but inhibit the growth and distribution of non-Se accumulators. Volatile selenides with specific odors have a deterrent effect on herbivores, reducing their feeding on plants. Soil microorganisms can effectively promote the uptake and transformation of selenium in plants, and organic selenides in plants can improve the tolerance of plants to pathogenic bacteria. Although selenium is not an essential trace element for plants, the right amount of selenium has important physiological and ecological benefits for them. This review summarizes recent research related to the functions of selenium in plant ecosystems to provide a deeper understanding of the significance of this element in plant physiology and ecosystems and to serve as a theoretical basis and technical support for the full exploitation and rational application of the ecological functions of selenium-accumulating plants.

Comparative physiological and transcriptome analysis reveals the potential mechanism of selenium accumulation and tolerance to selenate toxicity of Broussonetia papyrifera.

Broussonetia papyrifera is an important fodder tree that is widely distributed in China. Enhancing the selenium (Se) content in B. papyrifera may help to improve the nutritional value of the feed. In this study, sodium selenite and selenate were foliar applied to investigate the mechanisms of Se tolerance and accumulation in B. papyrifera. The results showed that both Se forms significantly increased the total Se content, and the proportion of organic Se was significantly higher in the sodium selenite treatment than in the control. In addition, the soluble sugar, phenolic acid and flavonoid contents and antioxidant enzyme activities were increased by exogenous Se. The de novo RNA sequencing results showed that 644 and 1804 differentially expressed genes were identified in the selenite and selenate comparison groups, respectively. Pathway enrichment analysis demonstrated that 24 of the 108 pathways were significantly enriched, of which sulfur assimilation genes in the sodium selenite-treated groups were upregulated, whereas Se conjugation and transporter genes, such as SBP1, PCS, GSTs, ABCs and GPX, were significantly induced under selenate treatment. The hub genes identified by weighted-gene co-expression network analysis further confirmed that sulfur assimilation, conjugation and transporter genes might play a vital role in Se assimilation and tolerance. From this, a model of Se metabolism in B. papyrifera was proposed based on the above physiological and RNA sequencing data. This study is the first study to report that B. papyrifera has a strong ability to accumulate and tolerate exogenous Se, thereby providing a foundation for further characterization of the accumulation and tolerance mechanism of B. papyrifera. Our findings can provide technical support for producing Se-enriched fodder.

Combined metabolome and transcriptome analysis reveal the mechanism of selenate influence on the growth and quality of cabbage (Brassica oleracea var. capitata L.).

Selenium is an essential trace element for human and animal health, and an appropriate amount of Se can promote the growth and development of plants. Cabbage is a popular cruciferous vegetable with a good ability to accumulate Se, and Se-enriched cabbage can be used as an important Se source for humans. However, the effects of Se-enriched cultivation and the Se accumulation mechanism in cabbage are still unclear. In this study, the effects of different concentrations (0, 0.1, 0.2, 0.4, 0.8, and 1.6 mmol/L) of selenate on cabbage growth and quality were explored. A low concentration of selenate (0.1 mmol/L) promoted growth and nutritional quality. The contents of total Se, S, selenocystine, and selenomethionine significantly increased following selenate application. Important secondary metabolites, namely glucosinolates, phenolic acids, and flavonoids, participate in the response to selenate in cabbage. Comparative transcriptome and metabolomics analysis revealed that SULTR2.2, SULTR3.1, APS, APK2, HMT, MMT, and NTR2 played important roles in Se absorption and conversion. Additionally, the SUR1, UGT74B1, and ST5b genes and cytochrome P450 family genes CYP83A1, CYP79A2, and CYP79F1 may be the crucial genes in the glucosinolates biosynthesis and regulation pathway. The PAL, 4CL, CAD, CHS3, FLS, and CYP73A5 genes were involved in flavonoid and phenolic acid accumulation under selenate treatment. These results reveal the internal relationships in the regulatory network of Se metabolism and secondary metabolite biosynthesis in cabbage and help further the understanding of the physiological and molecular mechanism of how Se biofortification affects cabbage quality, thereby providing genetic resources and a technical basis for the breeding and cultivation of Se-enriched cabbage with excellent nutritional quality.

Genome-wide transcriptome analysis reveals the regulatory network governing terpene trilactones biosynthesis in Ginkgo biloba.

Ginkgo biloba L. is currently the only remaining gymnosperm of the Ginkgoaceae Ginkgo genus, and its history can be traced back to the Carboniferous 200 million years ago. Terpene trilactones (TTLs) are one of the main active ingredients in G. biloba, including ginkgolides and bilobalide. They have a good curative effect on cardiovascular and cerebrovascular diseases because of their special antagonistic effect on platelet-activating factors. Therefore, it is necessary to deeply mine genes related to TTLs and to analyze their transcriptional regulation mechanism, which will hold vitally important scientific and practical significance for quality improvement and regulation of G. biloba. In this study, we performed RNA-Seq on the root, stem, immature leaf, mature leaf, microstrobilus, ovulate strobilus, immature fruit and mature fruit of G. biloba. The TTL regulatory network of G. biloba in different organs was revealed by different transcriptomic analysis strategies. Weighted gene co-expression network analysis (WGCNA) revealed that the five modules were closely correlated with organs. The 12 transcription factors, 5 structural genes and 24 Cytochrome P450 (CYP450) were identified as candidate regulators for TTL accumulation by WGCNA and cytoscape visualization. Finally, 6 APETALA2/ethylene response factors, 2 CYP450s and bHLH were inferred to regulate the metabolism of TTLs by correlation analysis. This study is the comprehensive in authenticating transcription factors, structural genes and CYP450 involved in TTL biosynthesis, thereby providing molecular evidence for revealing the comprehensive regulatory network involved in TTL metabolism in G. biloba.

Transcriptome and miRNA sequencing analyses reveal the regulatory mechanism of α-linolenic acid biosynthesis in Paeonia rockii.

Paeonia rockii is a promising woody oil crop because its seeds are rich in polyunsaturated fatty acids especially α-linolenic acid (ALA). ALA is an essential fatty acid that the human body cannot synthesize and is the direct synthetic precursor of eicosapentaenoic and docosahexaenoic acids, which play crucial roles in the development of the blood vessels, brain and nervous system of humans. However, the mechanisms underlying the dynamic changes in ALA during seed development are unknown. In this study, we found that the fatty acid content gradually increased with P. rockii seed development, with ALA being the main unsaturated acid component (37-44%). The content of ALA reached the peak value of 306.26 mg/g DW 20 days before the seeds had fully maturated. Seeds from three different developmental stages were selected for transcriptome and miRNA sequencing analyses to explore the molecular mechanism of ALA accumulation in P. rockii seeds. A total of 39 differentially expressed genes were screened for their involvement in ALA biosynthesis, among which FAD2/8, GPAT, PDAT, LACS, LPAAT, and KAS II might be the key structural genes of ALA accumulation. The differential expression of these genes was dependent on the regulation of five miRNAs (mdm_miR156b, novel miR_91, novel miR_133, novel miR_291, and novel miR_405) and four transcription factors (AP2, SNL2, TGA-like, and SPL). This study reveals the mechanism behind the dynamic changes of ALA contents in P. rockii during seed development, and also provides an important theoretical basis for the breeding of excellent varieties of P. rockii.

Genome-wide identification and expression pattern analysis of the TCP transcription factor family in Ginkgo biloba.

Plant-specific TCP transcription factors play an essential role in plant growth and development. They can regulate leaf curvature, flower symmetry and the synthesis of secondary metabolites. The flavonoids in Ginkgo biloba leaf are one of the main medicinally bioactivate compounds, which have pharmacological and beneficial health effects for humans. In this study, a total of 13 TCP genes were identified in G. biloba, and 5 of them belonged to PCF subclades (GbTCP03, GbTCP07, GbTCP05, GbTCP13, GbTCP02) while others belonged to CIN (GbTCP01, GbTCP04, GbTCP06, GbTCP08, GbTCP09, GbTCP10, GbTCP11, GbTCP12) subclades according to phylogenetic analysis. Numerous cis-acting elements related to various biotic and abiotic signals were predicted on the promoters by cis-element analysis, suggesting that the expression of GbTCPs might be co-regulated by multiple signals. Transcript abundance analysis exhibited that most of GbTCPs responded to multiple phytohormones. Among them, the relative expression levels of GbTCP06, GbTCP11, and GbTCP13 were found to be significantly influenced by exogenous ABA, SA and MeJA application. In addition, a total of 126 miRNAs were predicted to target 9 TCPs (including GbTCP01, GbTCP02, GbTCP04, GbTCP05, GbTCP06, GbTCP08, GbTCP11, GbTCP12, GbTCP13). The correlation analysis between the expression level of GbTCPs and the flavonoid contents showed that GbTCP03, GbTCP04, GbTCP07 might involve in flavonoid biosynthesis in G. biloba. In short, this study mainly provided a theoretical foundation for better understanding the potential function of TCPs in G. biloba.

SMRT and Illumina RNA sequencing reveal the complexity of terpenoid biosynthesis in Zanthoxylum armatum.

Sichuan pepper (Zanthoxylum armatum DC) is a popular spice and is often prescribed in traditional Chinese medicine to treat vomiting, diarrhea, ascariasis and eczema, among other conditions. Volatile oils from Z. armatum leaves contain active ingredients, with terpenoids being one of the main components. In the present study, the combination of sequencing data of Z. armatum from PacBio single molecule real time (SMRT) and Illumina RNA sequencing (RNA-Seq) platforms facilitated an understanding of the gene regulatory network of terpenoid biosynthesis in pepper leaves. The leaves of three developmental stages from two Z. armatum cultivars, 'Rongchangwuci' (WC) and 'Zhuye' (ZY), were selected as test materials to construct sequencing libraries. A total of 143,122 predictions of unique coding sequences, 105,465 simple sequence repeats, 20,145 transcription factors and 4719 long non-coding RNAs (lncRNAs) were identified, and 142,829 transcripts were successfully annotated. The occurrence of alternative splicing events was verified by reverse transcription PCR, and quantitative real-time PCR was used to confirm the expression pattern of six randomly selected lncRNAs. A total of 96,931 differentially expressed genes were filtered from different samples. According to functional annotation, a total of 560 candidate genes were involved in terpenoid synthesis, of which 526 were differentially expressed genes (DEGs). To identify the key genes involved in terpenoid biosynthesis, the module genes in different samples, including structural and transcription factors genes, were analyzed using the weighted gene co-expression network method, and the co-expression network of genes was constructed. Thirty-one terpenoids were identified by gas chromatography-mass spectrometry. The correlation between 18 compounds with significantly different contents and genes with high connectivity in the module was jointly analyzed in both cultivars, yielding 12 candidate DEGs presumably involved in the regulation of terpenoid biosynthesis. Our findings showed that full-length transcriptome SMRT and Illumina RNA-Seq can play an important role in studying organisms without reference genomes and elucidating the gene regulation of a biosynthetic pathway.

Genome-wide characterization of bZIP gene family identifies potential members involved in flavonoids biosynthesis in Ginkgo biloba L.

Ginkgo biloba L. is an ancient relict plant with rich pharmacological activity and nutritional value, and its main physiologically active components are flavonoids and terpene lactones. The bZIP gene family is one of the largest gene families in plants and regulates many processes including pathogen defense, secondary metabolism, stress response, seed maturation, and flower development. In this study, genome-wide distribution of the bZIP transcription factors was screened from G. biloba database in silico analysis. A total of 40 bZIP genes were identified in G. biloba and were divided into 10 subclasses. GbbZIP members in the same group share a similar gene structure, number of introns and exons, and motif distribution. Analysis of tissue expression pattern based on transcriptome indicated that GbbZIP08 and GbbZIP15 were most highly expressed in mature leaf. And the expression level of GbbZIP13 was high in all eight tissues. Correlation analysis and phylogenetic tree analysis suggested that GbbZIP08 and GbbZIP15 might be involved in the flavonoid biosynthesis. The transcriptional levels of 20 GbbZIP genes after SA, MeJA, and low temperature treatment were analyzed by qRT-PCR. The expression level of GbbZIP08 was significantly upregulated under 4°C. Protein-protein interaction network analysis indicated that GbbZIP09 might participate in seed germination by interacting with GbbZIP32. Based on transcriptome and degradome data, we found that 32 out of 117 miRNAs were annotated to 17 miRNA families. The results of this study may provide a theoretical foundation for the functional validation of GbbZIP genes in the future.