Molecular regulatory mechanisms of staminate strobilus development and dehiscence in Torreya grandis.

Gymnosperms are mostly dioecious, and their staminate strobili undergo a longer developmental period than those of angiosperms. However, the underlying molecular mechanisms remain unclear. This study aimed to identify key genes and pathways involved in staminate strobilus development and dehiscence in Torreya grandis. Through weighted gene co-expression network analysis (WGCNA), we identified fast elongation-related genes enriched in carbon metabolism and auxin signal transduction, whereas dehiscence-related genes were abundant in alpha-linolenic acid metabolism and the phenylpropanoid pathway. Based on WGCNA, we also identified PHYTOCHROME-INTERACTING FACTOR4 (TgPIF4) as a potential regulator for fast elongation of staminate strobilus and 2 WRKY proteins (TgWRKY3 and TgWRKY31) as potential regulators for staminate strobilus dehiscence. Multiple protein-DNA interaction analyses showed that TgPIF4 directly activates the expression of TRANSPORT INHIBITOR RESPONSE2 (TgTIR2) and NADP-MALIC ENZYME (TgNADP-ME). Overexpression of TgPIF4 significantly promoted staminate strobilus elongation by elevating auxin signal transduction and pyruvate content. TgWRKY3 and TgWRKY31 bind to the promoters of the lignin biosynthesis gene PHENYLALANINE AMMONIA-LYASE (TgPAL) and jasmonic acid metabolism gene JASMONATE O-METHYLTRANSFERASE (TgJMT), respectively, and directly activate their transcription. Overexpression of TgWRKY3 and TgWRKY31 in the staminate strobilus led to early dehiscence, accompanied by increased lignin and methyl jasmonate levels, respectively. Collectively, our findings offer a perspective for understanding the growth of staminate strobili in gymnosperms.

Genome-Wide Identification, Expression Analysis under Abiotic Stress and Co-Expression Analysis of MATE Gene Family in Torreya grandis.

The multidrug and toxin efflux (MATE) family participates in numerous biological processes and plays important roles in abiotic stress responses. However, information about the MATE family genes in Torreya grandis remains unclear. In this study, our genome-wide investigation identified ninety MATE genes in Torreya grandis, which were divided into five evolutionary clades. TgMATE family members are located on eleven chromosomes, and a total of thirty TgMATEs exist in tandem duplication. The promoter analysis showed that most TgMATEs contain the cis-regulatory elements associated with stress and hormonal responses. In addition, we discovered that most TgMATE genes responded to abiotic stresses (aluminum, drought, high temperatures, and low temperatures). Weighted correlation network analysis showed that 147 candidate transcription factor genes regulated the expression of 14 TgMATE genes, and it was verified through a double-luciferase assay. Overall, our findings offer valuable information for the characterization of the TgMATE gene mechanism in responding to abiotic stress and exhibit promising prospects for the stress tolerance breeding of Torreya grandis.

Advances on delta 5-unsaturated-polymethylene-interrupted fatty acids: Resources, biosynthesis, and benefits.

Though the knowledge on delta 5-unsaturated-polymethylene-interrupted fatty acids (Δ5-UPIFAs) is being updated, the issue of their integration still exists within the field. Thus, this review systematically summarizes the sources, biosynthesis and metabolism, analytical methods, preparation, and health-promoting roles of Δ5-UPIFAs. In plants, the content of Δ5-UPIFAs is higher, which is an ideal source. In animals, although the content of Δ5-UPIFAs is not high, there are many species, which is the possible source of some special Δ5-UPIFAs. At present, although the extraction of Δ5-UPIFAs is mainly from plants, the fermentation by organisms, especially for genetically modified microorganisms engineering maybe be a substitue of pepration of Δ5-UPIFAs. Δ5-UPIFAs have been proved to possess multi-beneficial effects, such as lipid lowering, anti-inflammation and so on, so it has a certain potential application value. However, related knowledge of the underlying molecular mechanisms regarding Δ5-UPIFAs limited, and how Δ5-UPIFAs work is not clear. Further clinical and human studies about Δ5-UPIFAs are also needed. Studies on tapping new resources, developing structured lipide rich in Δ5-UPIFA and enhancing delivery were quite deficient. This review emphasizes the further directions on Δ5-UPIFAs with scientific suggestions to pay more attention to the applications of Δ5-UPIFAs in food, pharmaceutical and cosmetic industries.

Integrated Metabolomics, Transcriptome and Functional Analysis Reveal Key Genes Are Involved in Tree Age-Induced Amino Acid Accumulation in Torreya grandis Nuts.

Torreya grandis is native Chinese tree species of economic significance, renowned for its long lifespan and the rich nutritional value of its nuts. In this study, we analyzed the morphological characteristics, metabolites, associated gene expressions, and regulatory mechanism in nuts from young (10 years old) and old (1000 years old) T. grandis trees. We observed that the length, width, and weight of nuts from older trees were considerably greater than those from younger trees. Metabolomic analysis revealed that the concentrations of 18 amino acids and derivatives (including histidine and serine) in nuts from older trees were markedly higher than those in nuts from younger trees. Transcriptome and metabolomic correlation analysis identified 16 genes, including TgPK (pyruvate kinase), TgGAPDH (glyceraldehyde 3-phosphate dehydrogenase), and others, which exhibit higher expression levels in older trees compared to younger trees, as confirmed by qRT-PCR. These genes are associated with the biosynthesis of histidine, glutamic acid, tryptophan, and serine. Transient expression of TgPK in tobacco led to increased pyruvate kinase activity and amino acid content (histidine, tryptophan, and serine). Additionally, dual-luciferase assays and yeast one-hybrid results demonstrated that TgWRKY21 positively regulates TgPK expression by directly binding to the TgPK promoter. These findings not only demonstrate the nutritional differences between nuts from young and old trees but also offer fresh insights into the development of nutritional sources and functional components based on nuts from old trees, enriching our understanding of the potential benefits of utilizing nuts from older trees.

The salt-activated CBF1/CBF2/CBF3-GALS1 module fine-tunes galactan-induced salt hypersensitivity in Arabidopsis.

Plant growth and development are significantly hampered in saline environments, limiting agricultural productivity. Thus, it is crucial to unravel the mechanism underlying plant responses to salt stress. ß-1,4-Galactan (galactan), which forms the side chains of pectic rhamnogalacturonan I, enhances plant sensitivity to high-salt stress. Galactan is synthesized by GALACTAN SYNTHASE1 (GALS1). We previously showed that NaCl relieves the direct suppression of GALS1 transcription by the transcription factors BPC1 and BPC2 to induce the excess accumulation of galactan in Arabidopsis (Arabidopsis thaliana). However, how plants adapt to this unfavorable environment remains unclear. Here, we determined that the transcription factors CBF1, CBF2, and CBF3 directly interact with the GALS1 promoter and repress its expression, leading to reduced galactan accumulation and enhanced salt tolerance. Salt stress enhances the binding of CBF1/CBF2/CBF3 to the GALS1 promoter by inducing CBF1/CBF2/CBF3 transcription and accumulation. Genetic analysis suggested that CBF1/CBF2/CBF3 function upstream of GALS1 to modulate salt-induced galactan biosynthesis and the salt response. CBF1/CBF2/CBF3 and BPC1/BPC2 function in parallel to regulate GALS1 expression, thereby modulating the salt response. Our results reveal a mechanism in which salt-activated CBF1/CBF2/CBF3 inhibit BPC1/BPC2-regulated GALS1 expression to alleviate galactan-induced salt hypersensitivity, providing an activation/deactivation fine-tune mechanism for dynamic regulation of GALS1 expression under salt stress in Arabidopsis.

Comparative proteomic analysis of tomato (Solanum lycopersicum L.) shoots reveals crosstalk between strigolactone and auxin.

As one of the main vegetable crops cultivated in the world, the tomato has advantages of high yield and economic benefits, and plays an important role in promoting farmers' income and social and economic growth. However, lateral branches during the growth process of tomato consume considerable nutrients and reduce the yield of tomato. Phytohormones such as strigolactone and auxin can inhibit the formation of lateral branches. However, the mechanism of their interaction is not particularly clear. To better understand the effects of exogenous strigolactone and auxin on tomato, proteome analyses of tomato shoots treated with exogenous GR24 and indole acetic acid were performed using an integrated approach involving tandem mass tag (TMT) labeling and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). We identified 6685 proteins, of which 5822 contained quantitative information. Many differentially expressed proteins (DEPs) were found in different comparisons, including 415, 148, and 130 DEPs in GR24 vs mock, IAA vs mock, and GR24 + IAA vs mock comparisons, respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that 'photosynthesis - antenna proteins' were significantly enriched in three treatments. Our data can help reveal the interaction between strigolactone and auxin in tomato seedlings.

Physiological and Molecular Analysis of Aluminium-Induced Organic Acid Anion Secretion from Grain Amaranth (Amaranthus hypochondriacus L.) Roots.

Grain amaranth (Amaranthus hypochondriacus L.) is abundant in oxalate and can secrete oxalate under aluminium (Al) stress. However, the features of Al-induced secretion of organic acid anions (OA) and potential genes responsible for OA secretion are poorly understood. Here, Al-induced OA secretion in grain amaranth roots was characterized by ion charomatography and enzymology methods, and suppression subtractive hybridization (SSH) together with quantitative real-time PCR (qRT-PCR) was used to identify up-regulated genes that are potentially involved in OA secretion. The results showed that grain amaranth roots secrete both oxalate and citrate in response to Al stress. The secretion pattern, however, differs between oxalate and citrate. Neither lanthanum chloride (La) nor cadmium chloride (Cd) induced OA secretion. A total of 84 genes were identified as up-regulated by Al, in which six genes were considered as being potentially involved in OA secretion. The expression pattern of a gene belonging to multidrug and toxic compound extrusion (MATE) family, AhMATE1, was in close agreement with that of citrate secretion. The expression of a gene encoding tonoplast dicarboxylate transporter and four genes encoding ATP-binding cassette transporters was differentially regulated by Al stress, but the expression pattern was not correlated well with that of oxalate secretion. Our results not only reveal the secretion pattern of oxalate and citrate from grain amaranth roots under Al stress, but also provide some genetic information that will be useful for further characterization of genes involved in Al toxicity and tolerance mechanisms.

Melatonin alleviates aluminum toxicity by regulating aluminum-responsive and nonresponsive pathways in hickory.

Aluminum (Al) toxicity is a significant constraint on agricultural productivity worldwide. Melatonin (MT) has been shown to alleviate Al toxicity in plants; however, the underlying mechanisms remain largely unknown. Here, we employed a combination of physiological and molecular biology techniques to examine the role of MT in mitigating Al toxicity of hickory. We found that MT decreased the contents of cell wall pectin, hemicellulose, Al, and Al-induced massive reactive oxygen species accumulation in the roots of hickory. Transcriptomic analysis revealed that MT may alleviate root tip Al stress by regulating Al-responsive and nonresponsive pathways. Co-expression regulatory network and dual-luciferase receptor assays revealed that transcription factors, CcC3H12 and CcAZF2, responded to MT and significantly activated the expression of two cell wall pectin-related genes, CcPME61 and CcGAE6, respectively. Further, yeast one-hybrid and electrophoretic mobility shift assay (EMSA) assays verified that CcC3H12 and CcAZF2 regulated CcPME61 and CcGAE6, respectively, by directly binding to their promoters. Overexpression of CcPME61 enhanced the Al sensitivity of Arabidopsis thaliana. Our results indicate that MT can improve Al tolerance of hickory via multiple pathways, which provides a new perspective for the study of the mechanism of MT in alleviating abiotic stress.

Molecular characterization and transcriptional regulation analysis of the Torreya grandis squalene synthase gene involved in sitosterol biosynthesis and drought response.

The kernel of Torreya grandis cv. 'Merrillii' (Cephalotaxaceae) is a rare nut with a variety of bioactive compounds and a high economic value. ß-sitosterol is not only the most abundant plant sterol but also has various biological effects, such as antimicrobial, anticancer, anti-inflammatory, lipid-lowering, antioxidant, and antidiabetic activities. In this study, a squalene synthase gene from T. grandis, TgSQS, was identified and functionally characterized. TgSQS encodes a deduced protein of 410 amino acids. Prokaryotic expression of the TgSQS protein could catalyze farnesyl diphosphate to produce squalene. Transgenic Arabidopsis plants overexpressing TgSQS showed a significant increase in the content of both squalene and ß-sitosterol; moreover, their drought tolerance was also stronger than that of the wild type. Transcriptome data from T. grandis seedlings showed that the expression levels of sterol biosynthesis pathway-related genes, such as HMGS, HMGR, MK, DXS, IPPI, FPPS, SQS, and DWF1, increased significantly after drought treatment. We also demonstrated that TgWRKY3 directly bound to the TgSQS promoter region and regulated its expression through a yeast one-hybrid experiment and a dual luciferase experiment. Together, these findings demonstrate that TgSQS has a positive role in ß-sitosterol biosynthesis and in protecting against drought stress, emphasizing its importance as a metabolic engineering tool for the simultaneous improvement of ß-sitosterol biosynthesis and drought tolerance.

Transcriptome-referenced association study provides insights into the regulation of oil and fatty acid biosynthesis in Torreya grandis kernel.

INTRODUCTION: Torreya grandis is a gymnosperm belonging to Taxodiaceae. As an economically important tree, its kernels are edible and rich in oil with high unsaturated fatty acids, such as sciadonic acid. However, the kernels from different T. grandis landraces exhibit fatty acid and oil content variations. OBJECTIVES: As a gymnosperm, does T. grandis have special regulation mechanisms for oil biosynthesis? The aim of this study was to dissect the genetic architecture of fatty acid and oil content and the underlying mechanism in T. grandis. METHODS: We constructed a high integrity reference sequence of expressed regions of the genome in T. grandis and performed transcriptome-referenced association study (TRAS) for 10 fatty acid and oil traits of kernels in the 170 diverse T. grandis landraces. To confirm the TRAS result, we performed functional validation and molecular biology experiments for oil significantly associated genes. RESULTS: We identified 41 SNPs from 34 transcripts significantly associated with 7 traits by TRAS (-log10 (P) greater than 6.0). Results showed that LOB domain-containing protein 40 (LBD40) and surfeit locus protein 1 (SURF1) may be indirectly involved in the regulation of oil and sciadonic acid biosynthesis, respectively. Moreover, overexpression of TgLBD40 significantly increased seed oil content. The nonsynonymous variant in the TgLBD40 coding region discovered by TRAS could alter the oil content in plants. Pearson's correlation analysis and dual-luciferase assay indicated that TgLBD40 positively enhanced oil accumulation by affecting oil biosynthesis pathway genes, such as TgDGAT1. CONCLUSION: Our study provides new insights into the genetic basis of oil biosynthesis in T. grandis and demonstrates that integrating RNA sequencing and TRAS is a powerful strategy to perform association study independent of a reference genome for dissecting important traits in T. grandis.

The Torreya grandis genome illuminates the origin and evolution of gymnosperm-specific sciadonic acid biosynthesis.

Torreya plants produce dry fruits with assorted functions. Here, we report the 19-Gb chromosome-level genome assembly of T. grandis. The genome is shaped by ancient whole-genome duplications and recurrent LTR retrotransposon bursts. Comparative genomic analyses reveal key genes involved in reproductive organ development, cell wall biosynthesis and seed storage. Two genes encoding a C18 Δ9-elongase and a C20 Δ5-desaturase are identified to be responsible for sciadonic acid biosynthesis and both are present in diverse plant lineages except angiosperms. We demonstrate that the histidine-rich boxes of the Δ5-desaturase are crucial for its catalytic activity. Methylome analysis reveals that methylation valleys of the T. grandis seed genome harbor genes associated with important seed activities, including cell wall and lipid biosynthesis. Moreover, seed development is accompanied by DNA methylation changes that possibly fuel energy production. This study provides important genomic resources and elucidates the evolutionary mechanism of sciadonic acid biosynthesis in land plants.

Ti3C2Tx MXene nanosheets enhance the tolerance of Torreya grandis to Pb stress.

As a 2D nanomaterial, MXene (Ti3C2Tx) has shown enormous potential for use in fields such as biomedical and environmental pollution. However, the utilization of MXene materials in plants has received little attention thus far. The efficient use of MXene materials in agriculture and forestry is first highlighted in this study. Phenotypic and physiological analyses indicated that MXene application significantly enhanced the tolerance of Torreya grandis to Pb stress by reducing Pb accumulation. Furthermore, we illustrated two independent mechanisms of MXene material in reducing Pb accumulation in T. grandis: 1) MXene converted the available form of Pb into stable forms via its strong Pb adsorption ability, resulting in a decrease of the available form of Pb in soils, and 2) MXene application obviously increased the cell wall pectin content to restrict more Pb in the cell wall by regulating the expression of pectin synthesis/metabolism-related genes (TgPLL2, TgPLL11, TgPG5, TgPG30, TgGAUT3 and TgGAUT12) in T. grandis roots. Overall, this finding provides insight into the application of MXene material in modern agriculture and forestry, which will facilitate the rapid development of nanotechnology in sustainable agriculture and forestry.

Multi-omics analysis reveals the molecular responses of Torreya grandis shoots to nanoplastic pollutant.

Micro/nanoplastic has become an emerging pollutant of global concern. At present, ecotoxic researches on micro/nanoplastics mostly focus on marine aquatic organisms and freshwater algae. Research on the ecological impacts of plastics on higher terrestrial plants, especially on forest plants, is relatively limited. Torreya grandis cv. Merrillii, a species of conifer in the family Taxaceae, is a unique and economically valuable tree species in China. The physiological and biochemical responses of T. grandis seedlings to polystyrene nanoplastics (PSNPs) with a diameter of 100 nm were systematically studied inthe present study. The results showed that nanoplastics enhanced the accumulation of the thiobarbituric acid reactive substance and the activities of catalase and peroxidase. The concentrations of iron, sulfur, and zinc were reduced after nanoplastic exposure. PSNP treatment had an important effect on a series of chemical and genetic indicators of T. grandis, includingantioxidants, small RNA, gene transcription, protein expressions, and metabolite accumulation. Multi-omic analysis revealed that PSNPs modulate terpenoid- and flavonoid-biosynthesis pathways by regulating small RNA transcription and protein expression. Our study provided novelty insights into the responses of forest plants to nanoplastic treatment.

Identification of key genes contributing to amino acid biosynthesis in Torreya grandis using transcriptome and metabolome analysis.

Torreya grandis has high economic and nutritional value due to the high nutrients in its kernels. The kernels of different development stages vary enormously in their amino acids content. However, the molecular basis and the regulatory mechanism of amino acid biosynthesis remain unclear. Here, transcriptome and metabolome analysis were performed. Correlation analysis result showed that 4 unigenes were significantly and positively correlated with at least 10 amino acids. The full length CDS of 2 unigenes (TgDAHP2 and TgASA1) were successfully cloned from the 4 unigenes for DAHP, ASA and CITS. Subcelluar localization analysis showed that both TgDAHP2 and TgASA1 were localized to the chloroplast. Overexpression of TgDAHP2 and TgASA1 in Arabidopsis can greatly increase the content of most amino acids. Moreover, 3 transcription factors were found to positively regulate the expression of TgASA1. This research contributes to understand the molecular regulatory mechanisms of amino acid biosynthesis in T. grandis.

Effects of Strigolactone on Torreya grandis Gene Expression and Soil Microbial Community Structure Under Simulated Nitrogen Deposition.

Nitrogen enters the terrestrial ecosystem through deposition. High nitrogen levels can affect physical and chemical properties of soil and inhibit normal growth and reproduction of forest plants. Nitrogen modulates the composition of soil microorganisms. Strigolactones inhibits plant branching, promotes root growth, nutrient absorption, and promotes arbuscular fungal mycelia branching. Plants are subjected to increasing atmospheric nitrogen deposition. Therefore, it is imperative to explore the relationship between strigolactone and nitrogen deposition of plants and abundance of soil microorganisms. In the present study, the effects of strigolactone on genetic responses and soil microorganisms of Torreya grandis, under simulated nitrogen deposition were explored using high-throughput sequencing techniques. T. grandis is a subtropical economic tree species in China. A total of 4,008 differentially expressed genes were identified in additional N deposition and GR24 treatment. These genes were associated with multiple GO terms and metabolic pathways. GO enrichment analysis showed that several DEGs were associated with enrichment of the transporter activity term. Both additional nitrogen deposition and GR24 treatment modulated the content of nutrient elements. The content of K reduced in leaves after additional N deposition treatment. The content of P increased in leaves after GR24 treatment. A total of 20 families and 29 DEGs associated with transporters were identified. These transporters may be regulated by transcription factors. A total of 1,402,819 clean reads and 1,778 amplicon sequence variants (ASVs) were generated through Bacterial 16S rRNA sequencing. Random forest classification revealed that Legionella, Lacunisphaera, Klebsiella, Bryobacter, and Janthinobacterium were significantly enriched in the soil in the additional N deposition group and the GR24 treatment group. Co-occurrence network analysis showed significant differences in composition of soil microbial community under different treatments. These results indicate a relationship between N deposition and strigolactones effect. The results provide new insights on the role of strigolactones in plants and composition of soil microorganisms under nitrogen deposition.

Identification of key genes and enzymes contributing to nutrition conversion of Torreya grandis nuts during post-ripening process.

The seeds of Torreya grandis are necessary to go through a ripening process, which eventually leads to nutrition conversion and the production of edible nuts. However, the molecular basis of nutrition conversion remains unclear. Here, transcriptome sequencing was performed on seeds treated with different temperature and humidity. A total of 881 unigenes related to nutrition conversion were identified. The correlations between nutrient content and gene expression suggested that sucrose phosphate synthase (SPS), dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex (DLST), glycerol-3-phosphate acyltransferase (GPAT) and Pyruvate kinase (PK) may play key roles in nutrition conversion. Transient over-expression of TgDLST, TgPK and TgGPAT in tobacco leaves promoted nutritional conversion. Moreover, enzyme activity analysis indicated that diacylglycerol acyltransferase (DGAT) and pyruvate dehydrogenase (PDH) activities may also accelerate the nutritional conversion. This study uncovers the molecular basis of nutrition conversion in T. grandis seeds, which critical for shortening the time of nutrition conversion.

The effect of ethylene on squalene and ß-sitosterol biosynthesis and its key gene network analysis in Torreya grandis nuts during post-ripening process.

Squalene and ß-sitosterol are health-benefit compounds due to their nutritional and medicinal properties. It has been reported that the content of these bioactive compounds is relatively high in Torreya grandis nuts. However, it is not yet known what changes in squalene and ß-sitosterol accumulation occur during the special post-ripening process of T. grandis nuts and the effect of the well-known ripening hormone ethylene on the regulatory mechanism of their biosynthetic pathways. Thus, we performed transcriptome and metabolite analyses. The results showed that ethylene not only promoted the post-ripening process but also enhanced the accumulation of squalene by inducing gene expression in the mevalonate pathway. At the same time, ethylene treatment also promoted the accumulation of other sterols but inhibited gene expression in the ß-sitosterol biosynthesis pathway. In addition, co-expression and correlation analysis suggested a framework for the transcriptional regulation of squalene and ß-sitosterol biosynthesis genes under ethylene treatment.

New insights into the accumulation of vitamin B3 in Torreya grandis nuts via ethylene induced key gene expression.

Vitamin B3, derived primarily from plant sources, is an essential nutrient for humans. Torreya grandis is rich in vitamin B3, however, the mechanism underlying the biosynthesis and regulation of vitamin B3 in T. grandis remains unclear. A systematic transcriptomic investigation was thus conducted to identify the gene expression pattern of vitamin B3 biosynthesis in 10 T. grandis cultivars. The findings suggest that biosynthesis occurs mainly via the aspartate pathway. Expression and correlation analyses indicate that aspartate oxidase (AOX) and quinolinate synthase (QS) may play important roles in vitamin B3 accumulation. Furthermore, co-expression network and ethephon treatments indicate that the ethylene response factor (ERF) may be involved in the regulation of vitamin B3 biosynthesis in T. grandis nuts. Our findings not only help to elucidate the biosynthesis of vitamin B3, but also provide valuable resource material for future genomic research and molecular-assisted breeding to develop genotypes with higher vitamin B3 levels.

Transcriptome sequencing and metabolomics analyses provide insights into the flavonoid biosynthesis in Torreya grandis kernels.

The kernel of Torreya grandis (T. grandis) is a rare nut with a variety of bioactive compounds. Flavonoids are a very important class of bioactive compounds with high antioxidant activity in T. grandis kernels. However, the flavonoid compositions which mainly contribute to antioxidant capacity and the molecular basis of flavonoid biosynthesis in T. grandis remain unclear. Here, transcriptome sequencing and metabolomics analysis for kernels were performed. In total, 124 flavonoids were identified. Among them, 9 flavonoids were highly correlated with antioxidant activity. Furthermore, unigenes encoding CHS, DFR and ANS showed significant correlation with the 9 flavonoids. Transient overexpression of TgDFR1 in tobacco leaves resulted in increased antioxidant activity. Moreover, several transcription factors from MYB, bHLH and bZIP families were identified by co-expression assay, suggesting that they may regulate flavonoid biosynthesis. Our findings provide a molecular basis and new insights into the flavonoid biosynthesis in T. grandis kernels.

Unraveling the malate biosynthesis during development of Torreya grandis nuts.

Torreya grandis is a characteristic rare economic tree species in subtropical mountainous areas. The kernels of T. grandis have rich content of organic acids, and malate is the predominant organic acid in T. grandis kernels. However, the contents, biosynthesis/metabolism pathway and transcriptional regulation of malate in developing T. grandis kernels remain completely unknown. Here, the organic acid composition in developing T. grandis kernels was first analyzed. The results showed that the content of malate was increased during the maturation T. grandis kernels. A malate synthase (TgMLS) gene might be involved in the accumulation of malate based on transcriptome data, gene expression and enzyme activity analysis. Transient expression of TgMLS in tobacco resulted in the high malate synthase activity and malate content. Furthermore, a basic helix-loop-helix transcription factor (bHLH), TgbHLH87 was identified to positively regulate the TgMLS expression via directly binding the TgMLS promoter. Our finding contributes to mechanism underlying malate accumulation in T. grandis kernels.