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Although the detailed biosynthetic mechanism is still unclear, the unique secondary metabolites of Ginkgo biloba, including ginkgolic acids (GAs) and terpene trilactones, have attracted increasing attention for their potent medicinal, physiological and biochemical properties. In particular, GAs have shown great potential in the fields of antibacterial and insecticidal activities, making it urgent to elucidate their biosynthetic mechanism. In this study, we systematically revealed the landscape of metabolic-transcriptional regulation across continuous growth stages of G. biloba seeds (GBS) based on multi-omics mining and experimental verification, and successfully identified all major types of GAs and terpene trilactones along with more than a thousand kinds of other metabolites. The phenological changes and the essential gene families associated with these unique metabolites were analyzed in detail, and several potential regulatory factors were successfully identified based on co-expression association analysis. In addition, we unexpectedly found the close relationship between large introns and the biosynthesis of these secondary metabolites. These genes with large introns related to the synthesis of secondary metabolites showed higher gene expression and expression stability in different tissues or growth stages. Our results may provide a new perspective for the study of the regulatory mechanism of these unique secondary metabolites in GBS.

Ginkgo biloba L. has a unique evolutionary status. Owing to its high medicinal and ornamental value, ginkgo has also recently become a research hotspot. However, the large genome and long juvenile period, as well as the lack of an effective genetic transformation system, have hindered gaining a full understanding of the comprehensive functions of ginkgo genes. At present, heterologous expression of genes in model plants is the primary method used in ginkgo-related research; however, these distant plant model relatives limit reliable interpretation of the results for direct applications in ginkgo breeding. To overcome these limitations, in this study, an efficient isolation and transient expression system for ginkgo protoplasts was established. A large number of intact and homogeneous ginkgo mesophyll protoplasts were isolated using 2% cellulase and 0.25% pectinase in 0.4 M mannitol. The activity of these protoplasts remained above 90% even after 24 h. Furthermore, when the concentration of the polyethylene glycol 4000 solution was 30%–40% (w/v), the transformation efficiency of the protoplasts reached 40%. Finally, the reliability of the system was verified using subcellular localization, transient overexpression, and protein interaction experiments with ginkgo genes, thereby providing a technical platform for the identification and analysis of ginkgo gene functions. The proposed method partially compensates for the limitations associated with the lack of a genetic transformation system and provides technical support to expand research on elucidating the functions of ginkgo genes.

Flavonoids are important secondary metabolites with extensive pharmacological functions. Ginkgo biloba L. (ginkgo) has attracted extensive attention because of its high flavonoid medicinal value. However, little is understood about ginkgo flavonol biosynthesis. Herein, we cloned the full-length gingko GbFLSa gene (1314 bp), which encodes a 363 amino acid protein that has a typical 2-oxoglutarate (2OG)-Fe(II) oxygenase region. Recombinant GbFLSa protein with a molecular mass of 41 kDa was expressed in Escherichia coli BL21(DE3). The protein was localized to the cytoplasm. Moreover, proanthocyanins, including catechin, epicatechin, epigallocatechin and gallocatechin, were significantly less abundant in transgenic poplar than in nontransgenic (CK) plants. In addition, dihydroflavonol 4-reductase, anthocyanidin synthase and leucoanthocyanidin reductase expression levels were significantly lower than those of their CK counterparts. GbFLSa thus encodes a functional protein that might negatively regulate proanthocyanin biosynthesis. This study helps elucidate the role of GbFLSa in plant metabolism and the potential molecular mechanism of flavonoid biosynthesis.

Light is an important ecological factor that affects plant growth, survival and distribution. Carpinus betulus L. is native to central Europe and is used as an ornamental plant with strong adaptability. It is an important tree species for landscaping and timber use. What’s more, the antioxidant- and anticancer-related properties of C. betulus leaf extracts are remarkable, that make it a possible raw material for medicine. Light intensity is an important environmental factor affecting the growth and physicochemical changes of C. betulus, but the mechanism of its effect on this species still remains unknown. In this study, the growth, photosynthetic characteristics, endogenous hormones and antioxidant activity responses of C. betulus seedlings to four light intensity gradients (T0: normal light; T1: 75%; T2: 50%; T3: 25% of normal light) were studied after 60 days of shading treatments. The results showed a significant effect of low light intensity on the values of the growth and physiological parameters of C. betulus. The low light intensity caused the inhibition of plant biomass accumulation and the degradation of photosynthetic capacity and stomatal behavior and aggravated the cell membrane lipid peroxidation. However, the plant height growth, leaf area, specific leaf area, photosynthetic pigment content, and contents of GA3 and ABA of C. betulus increased with decreasing light intensity. We found that C. betulus can tolerate mild (T1) and moderate (T2) shading stress by developing photoprotective mechanisms and maintaining relatively high concentrations of organic osmolytes and high antioxidant enzyme activities (superoxide dismutase, peroxidase, catalase and ascorbate peroxidase), but the ability of C. betulus to synthesize osmotic substances and enzymatic antioxidants was impaired under severe shading conditions (T3). Our results suggest that C. betulus can make effective use of low light resources by adjusting its morphology, material distribution, photosynthetic rate and antioxidant enzyme system in suitable low-light environments (50%~75% of normal light); however, light intensity levels that are too low (25% of normal light) will have adverse effects on plant growth. The results of this study provide not only new insights into the response mechanisms of C. betulus to light intensity but also a scientific basis for the cultivation and application of C. betulus in China.

Although the effects of girdling on grape berry development have been widely studied, the underlying mechanisms are poorly understood, especially at the molecular level. This study investigated the effect of trunk girdling on grape (Vitis L.) berry maturation. Girdling was performed on 5-year-old ‘Summer Black’ grapevines at early veraison, and transcriptional and physiologic analyses were performed. Trunk girdling promoted sugar accumulation and color development in berries and accelerated berry ripening by 25 days. Genes related to sucrose cleavage and polysaccharide degradation were upregulated at the transcriptional level, which was associated with increased monosaccharide accumulation and berry softening. Anthocyanin biosynthesis and accumulation were also enhanced by trunk girdling through the upregulation of anthocyanin biosynthesis genes including phenylalanine ammonia-lyase and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT). The increased expression of two VvUFGT genes was accompanied by the upregulation of VvMYBA2 under girdling. The upregulation of genes involved in ethylene biosynthesis and hormone (abscisic acid and brassinosteroid) responses and downregulation of genes involved in indoleacetic acid biosynthesis and response may have also promoted berry ripening in the girdling group. A total of 120 differentially expressed transcription factor genes from 29 gene families including MYB, ERF, and MYB-related were identified in the girdling group, which may participate in the regulation of berry development and ripening. These results provide molecular-level insight into the positive effects of trunk girdling on berry development in grapes.

Soybeans are an important economic crop. As the most widely used growth regulator globally, the molecular mechanism of mepiquat chloride (DPC) in soybean remains unknown. In this study, RNA sequencing technology combined with ultra-performance liquid chromatography and tandem mass spectrometry were used to analyze the changes in the leaf transcriptome and metabolomics of soybean leaves at the seedling stage under DPC stress. The results showed that differentially expressed genes related to photosynthesis and cell wall synthesis were significantly downregulated at the transcriptional level. In addition, the syntheses of gibberellin, zeatin, brassinolide, and other plant hormones were inhibited in the signal transduction pathway of plant hormones, thereby inhibiting plant growth. In contrast, at the metabolic level, the expression levels of flavonoid differential metabolites were significantly increased, and the proportions of flavonoids in the two varieties were 61.5 and 66%, respectively. The combined analysis of transcriptome and metabolomics showed that the differential expressed genes and metabolites were mainly enriched in the isoflavonoid biosynthesis and flavonoid biosynthesis pathways. Principally, DPC inhibited plant growth but improved drought resistance. Our study is the first to report the molecular mechanism of DPC regulation in soybean, providing useful insights into the rational application of DPC in soybean.

Stalk strength is one of the most important traits in maize, which affects stalk lodging resistance and, consequently, maize harvestable yield. Rind penetrometer resistance (RPR) as an effective and reliable measurement for evaluating maize stalk strength is positively correlated with stalk lodging resistance. In this study, one F2 and three F2:3 populations derived from the cross of inbred lines 3705I (the low RPR line) and LH277 (the high RPR line) were constructed for mapping quantitative trait loci (QTL), conferring RPR in maize. Fourteen RPR QTLs were identified in four environments and explained the phenotypic variation of RPR from 4.14 to 15.89%. By using a sequential fine-mapping strategy based on the progeny test, two major QTLs, qRPR1-3 and qRPR3-1, were narrowed down to 4-Mb and 550-kb genomic interval, respectively. The quantitative real-time PCR (qRT-PCR) assay was adopted to identify 12 candidate genes responsible for QTL qRPR3-1. These findings should facilitate the identification of the polymorphism loci underlying QTL qRPR3-1 and molecular breeding for RPR in maize.

Plant trichomes are specialized epidermal cells that are widely distributed on plant aerial tissues. The initiation and progression of trichomes are controlled in a coordinated sequence of multiple molecular events. During the past decade, major breakthroughs in the molecular understanding of trichome development were achieved through the characterization of various trichomes defective mutants and trichome-associated genes, which revealed a highly complex molecular regulatory network underlying plant trichome development. This review focuses on the recent millstone in plant trichomes research obtained using genetic and molecular studies, as well as ‘omics’ analyses in model plant Arabidopsis and fiber crop cotton. In particular, we discuss the latest understanding and insights into the underlying molecular mechanisms of trichomes formation at multiple dimensions, including at the chromatin, transcriptional, post-transcriptional, and post-translational levels. We summarize that the integration of multi-dimensional trichome-associated genes will enable us to systematically understand the molecular regulation network that landscapes the development of the plant trichomes. These advances will enable us to address the unresolved questions regarding the molecular crosstalk that coordinate concurrent and ordered the changes in cotton fiber initiation and progression, together with their possible implications for genetic improvement of cotton fiber.

Ultrasonic (US) treatment is an efficient method to induce crop tolerance against heavy metal toxicity; however, US-induced aluminum (Al) tolerance in peanuts was rarely studied. This study was comprised of two treatments, namely, CK, without ultrasonic treatment, and US, an ultrasonic seed treatment, for 15 min. Both treated and non-treated treatments were applied with Al in the form of AlCl3.18H2O at 5 mmol L–1 in Hoagland solution at one leaf stage. Results depicted that plant height, main root length, and number of lateral roots increased significantly under US treatment. Transcriptome analysis revealed that plant hormone signal transduction and transcription factors (TFs) were significantly enriched in the differentially expressed genes (DEGs) in US treatment, and the plant hormones were measured, including salicylic acid (SA) and abscisic acid (ABA) contents, were substantially increased, while indole acetic acid (IAA) and jasmonic acid (JA) contents were decreased significantly in US treatment. The TFs were verified using quantitative real-time (qRT)-PCR, and it was found that multiple TFs genes were significantly upregulated in US treatment, and ALMT9 and FRDL1 genes were also significantly upregulated in US treatment. Overall, the US treatment induced the regulation of hormone content and regulated gene expression by regulating TFs to improve Al tolerance in peanuts. This study provided a theoretical rationale for US treatment to improve Al tolerance in peanuts.

Soybean is an important cash crop in the world, and drought is the main reason for the loss of soybean plants productivity, with drought stress during the most water-sensitive flowering period of soybeans. In this article, drought-tolerant variety Heinong 44 (HN44) and drought-sensitive variety Heinong 65 (HN65) were used as experimental materials. Drought treatment was carried out at the early flowering stage. The method of controlling soil moisture content was used to simulate different degrees of drought, and the physiological changes of these two varieties of soybean under different soil moisture contents were studied. The results showed that with a decrease in soil moisture content, the content of malondialdehyde (MDA) in soybean leaves increased significantly; the activities of peroxidase (POD), catalase (CAT), and ascorbic acid peroxidase (APX) increased first and then decreased; the content of proline, soluble sugar, and soluble protein increased; and the total antioxidant capacity (T-AOC) increased significantly. When the soil moisture content was 15.5%, the degree of membrane lipid peroxidation, osmotic regulatory substances, antioxidant enzyme activity, and T-AOC increased the most, and the decrease in drought-tolerant variety HN44 was significantly less than that of drought-sensitive variety HN65. Our research reveals the response law of soybean crops to physiological characteristics under water deficit and provides theoretical basis and guiding significance for drought-resistant cultivation and breeding of soybean.

Plasmodesmata are channels that link adjacent cells in plant tissues through which molecular exchanges take place. They are involved in multiple processes vital to plant cells, such as responses to hormonal signaling or environmental challenges including osmotic stress, wounding and pathogen attack. Despite the importance of plasmodesmata, their proteome is not well-defined. Here, we have isolated fractions enriched in plasmodesmata from cell suspension cultures of Populus trichocarpa and identified 201 proteins that are enriched in these fractions, thereby providing further insight on the multiple functions of plasmodesmata. Proteomics analysis revealed an enrichment of proteins specifically involved in responses to stress, transport, metabolism and signal transduction. Consistent with the role of callose deposition and turnover in the closure and aperture of the plasmodesmata and our proteomic analysis, we demonstrate the enrichment of callose synthase activity in the plasmodesmata represented by several gene products. A new form of calcium-independent callose synthase activity was detected, in addition to the typical calcium-dependent enzyme activity, suggesting a role of calcium in the regulation of plasmodesmata through two forms of callose synthase activities. Our report provides the first proteomic investigation of the plasmodesmata from a tree species and the direct biochemical evidence for the occurrence of several forms of active callose synthases in these structures. Data are available via ProteomeXchange with identifier PXD010692.

Topping damage can induce the nicotine synthesis in tobacco roots, which involves the activation of JA and auxin signal transduction. It remains unclear how these hormone signals are integrated to regulate nicotine synthesis. Here we isolated a transcription factor NtWRKY-R1 from the group IIe of WRKY family and it had strong negative correlation with the expression of putrescine N-methyltransferase, the key enzyme of nicotine synthesis pathway. NtWRKY-R1 was specifically and highly expressed in tobacco roots, and it contains two transcriptional activity domains in the N- and C-terminal. The promoter region of NtWRKY-R1 contains two cis-elements which are responding to JA and auxin signals, respectively. Deletion of NtWRKY-R1 promoter showed that JA and auxin signals were subdued by NtWRKY-R1, and the expression of NtWRKY-R1 was more sensitive to auxin than JA. Furthermore, Yeast two-hybrid experiment demonstrated that NtWRKY-R1 can interact with the actin-binding protein. Our data showed that the intensity of JA and auxin signals can be translated into the expression of NtWRKY-R1, which regulates the balance of actin polymerization and depolymerization through binding actin-binding protein, and then regulates the expression of genes related to nicotine synthesis. The results will help us better understand the function of the WRKY-IIe family in the signaling crosstalk of JA and auxin under damage stress.

Gene expression analysis is widely used to unravel molecular regulatory mechanisms and identify key genes in plants. Appropriate reference gene is an important prerequisite to ensure the accuracy and reliability of qPCR analysis results. Water dropwort is a plant of the Oenanthe genus in the Apiaceae family, which has high economic benefits. However, the underlying molecular regulatory mechanisms in the growth and development of water dropwort have not been fully understood and the appropriate reference genes in different developmental stages of water dropwort not yet reported. In this study, 10 candidate reference genes (ACTIN, PP2A, SAND, EF-1a, GAPDH, UBQ, MIP, TBP, RPS-18, eIF-4a) were identified and cloned from Oenanthe javanica. The qPCR primers of candidate reference genes were designed and verified. Four statistical algorithms, geNorm, NormFinder, BestKeeper and RefFinder were used to evaluate the expression stability of 10 candidate reference genes in different developmental stages of water dropwort. The results showed that TBP and UBQ were the most stable genes in different developmental stages of water dropwort, while GAPDH was the most unstable gene. The normalization of EXP1 genes at different developmental stages further confirmed the reliability of internal reference genes. The results of this study provide a theoretical basis for selecting appropriate internal reference genes in different developmental stages of water dropwort. This study also provides technical support and reliable basis for the expression analysis of key genes in different developmental stages of water dropwort. [ABSTRACT FROM AUTHOR]

Tea leaf color is not only important from an aesthetics standpoint but is also related to tea quality. To investigate the molecular mechanisms that determine tea leaf color, we examined Camellia sinensis cv. 'Anjin Baicha' (an albino tea cultivar) by tandem mass tag isobaric labeling to generate a high-resolution proteome and acetyl-proteome atlas of three leaf developmental stages. We identified a total of 7,637 proteins and quantified 6,256; of these, 3,232 were classified as differentially accumulated proteins (DAPs). We also identified 3,161 lysine acetylation sites in 1,752 proteins and quantified 2,869 in 1,612 proteins. The acetylation levels at 468 sites were significantly altered across the three developmental stages during periodic albinism; the corresponding proteins were associated with a variety of biological processes. Interestingly, a large number of DAPs and acetylated proteins with increased/decreased acetylation were related to photosynthesis and secondary metabolite biosynthetic pathways, suggesting that the accumulation or acetylation level of these proteins regulates periodic albinism in 'Anjin Baicha.' Additionally, overlap between succinylome and acetylome among three 'Anjin Baicha' developmental stages were found. These data provide important insight into the mechanisms of leaf coloration in the tea plant. The mass spectrometry data have been deposited to Proteome X change via the PRIDE partner repository with the data set identifier PXD008134. [ABSTRACT FROM AUTHOR]