Metabolome profiling and transcriptome analysis filling the early crucial missing steps of piperine biosynthesis in Piper nigrum L.

Black pepper (Piper nigrum L.), the world renown as the King of Spices, is not only a flavorsome spice but also a traditional herb. Piperine, a species-specific piper amide, is responsible for the major bioactivity and pungent flavor of black pepper. However, several key steps for the biosynthesis of piperoyl-CoA (acyl-donor) and piperidine (acyl-acceptor), two direct precursors for piperine, remain unknown. In this study, we used guilt-by-association analysis of the combined metabolome and transcriptome, to identify two feruloyldiketide-CoA synthases responsible for the production of the C5 side chain scaffold feruloyldiketide-CoA intermediate, which is considered the first and important step to branch metabolic fluxes from phenylpropanoid pathway to piperine biosynthesis. In addition, we also identified the first two key enzymes for piperidine biosynthesis derived from lysine in P. nigrum, namely a lysine decarboxylase and a copper amine oxidase. These enzymes catalyze the production of cadaverine and 1-piperideine, the precursors of piperidine. In vivo and in vitro experiments verified the catalytic capability of them. In conclusion, our findings revealed enigmatic key steps of piperine biosynthetic pathway and thus provide a powerful reference for dissecting the biosynthetic logic of other piper amides.

Elucidation of the melitidin biosynthesis pathway in pummelo.

Specialized plant metabolism is a rich resource of compounds for drug discovery. The acylated flavonoid glycoside melitidin is being developed as an anti-cholesterol statin drug candidate, but its biosynthetic route in plants has not yet been fully characterized. Here, we describe the gene discovery and functional characterization of a new flavonoid gene cluster (UDP-glucuronosyltransferases (CgUGTs), 1,2 rhamnosyltransferase (Cg1,2RhaT), acyltransferases (CgATs)) that is responsible for melitidin biosynthesis in pummelo (Citrus grandis (L.) Osbeck). Population variation analysis indicated that the tailoring of acyltransferases, specific for bitter substrates, mainly determine the natural abundance of melitidin. Moreover, 3-hydroxy-3-methylglutaryl-CoA reductase enzyme inhibition assays showed that the product from this metabolic gene cluster, melitidin, may be an effective anti-cholesterol statin drug candidate. Co-expression of these clustered genes in Nicotiana benthamiana resulted in the formation of melitidin, demonstrating the potential for metabolic engineering of melitidin in a heterologous plant system. This study establishes a biosynthetic pathway for melitidin, which provides genetic resources for the breeding and genetic improvement of pummelo aimed at fortifying the content of biologically active metabolites.

OsbZIP18, a Positive Regulator of Serotonin Biosynthesis, Negatively Controls the UV-B Tolerance in Rice.

Serotonin (5-hydroxytryptamine) plays an important role in many developmental processes and biotic/abiotic stress responses in plants. Although serotonin biosynthetic pathways in plants have been uncovered, knowledge of the mechanisms of serotonin accumulation is still limited, and no regulators have been identified to date. Here, we identified the basic leucine zipper transcription factor OsbZIP18 as a positive regulator of serotonin biosynthesis in rice. Overexpression of OsbZIP18 strongly induced the levels of serotonin and its early precursors (tryptophan and tryptamine), resulting in stunted growth and dark-brown phenotypes. A function analysis showed that OsbZIP18 activated serotonin biosynthesis genes (including tryptophan decarboxylase 1 (OsTDC1), tryptophan decarboxylase 3 (OsTDC3), and tryptamine 5-hydroxylase (OsT5H)) by directly binding to the ACE-containing or G-box cis-elements in their promoters. Furthermore, we demonstrated that OsbZIP18 is induced by UV-B stress, and experiments using UV-B radiation showed that transgenic plants overexpressing OsbZIP18 exhibited UV-B stress-sensitive phenotypes. Besides, exogenous serotonin significantly exacerbates UV-B stress of OsbZIP18_OE plants, suggesting that the excessive accumulation of serotonin may be responsible for the sensitivity of OsbZIP18_OE plants to UV-B stress. Overall, we identified a positive regulator of serotonin biosynthesis and demonstrated that UV-B-stress induced serotonin accumulation, partly in an OsbZIP18-dependent manner.

Comparative analysis of metabolome of rice seeds at three developmental stages using a recombinant inbred line population.

Plants are considered an important food and nutrition source for humans. Despite advances in plant seed metabolomics, knowledge about the genetic and molecular bases of rice seed metabolomes at different developmental stages is still limited. Here, using Zhenshan 97 (ZS97) and Minghui 63 (MH63), we performed a widely targeted metabolic profiling in seeds during grain filling, mature seeds and germinating seeds. The diversity between MH63 and ZS97 was characterized in terms of the content of metabolites and the metabolic shifting across developmental stages. Taking advantage of the ultra-high-density genetic map of a population of 210 recombinant inbred lines (RILs) derived from a cross between ZS97 and MH63, we identified 4681 putative metabolic quantitative trait loci (mQTLs) in seeds across the three stages. Further analysis of the mQTLs for the codetected metabolites across the three stages revealed that the genetic regulation of metabolite accumulation was closely related to developmental stage. Using in silico analyses, we characterized 35 candidate genes responsible for 30 structurally identified or annotated compounds, among which LOC_Os07g04970 and LOC_Os06g03990 were identified to be responsible for feruloylserotonin and l-asparagine content variation across populations, respectively. Metabolite-agronomic trait association and colocation between mQTLs and phenotypic quantitative trait loci (pQTLs) revealed the complexity of the metabolite-agronomic trait relationship and the corresponding genetic basis.

Natural variation in the OsbZIP18 promoter contributes to branched-chain amino acid levels in rice.

Branched-chain amino acids (BCAAs) are essential amino acids that must be obtained from the diet for humans and animals, and they play important roles in various aspects of plant growth and development. Although BCAA biosynthetic pathways in higher plants have been uncovered, knowledge of their genetic control is still limited, and no positive regulators have been identified to date. Here, we showed that variation in BCAA levels in rice is attributable to differential transcription of OsbZIP18, a basic leucine zipper (bZIP) transcription factor, due to polymorphisms in its promoter. Functional analysis revealed that OsbZIP18 positively regulates BCAA synthesis by binding directly to the ACE and C-box cis-elements in the promoters of the biosynthetic genes branched-chain aminotransferase1 (OsBCAT1) and OsBCAT2. We further demonstrated that OsbZIP18 is strongly induced by nitrogen (N) deficiency and that N starvation results in enhanced BCAA levels in an OsbZIP18-dependent manner. Overall, we identified OsbZIP18, a positive regulator of BCAA biosynthesis, which contributed to natural variation in BCAA levels and mediated BCAA accumulation through de novo synthesis by directly modulating the key biosynthetic genes OsBCAT1 and OsBCAT2.

Metabolome Analysis of Multi-Connected Biparental Chromosome Segment Substitution Line Populations.

Metabolomic analysis coupled with advanced genetic populations represents a powerful tool with which to investigate the plant metabolome. However, genetic analyses of the rice (Oryza sativa) metabolome have been conducted mainly using natural accessions or a single biparental population. Here, the flag leaves from three interconnected chromosome segment substitution line populations with a common recurrent genetic background were used to dissect rice metabolic diversity. We effectively used multiple interconnected biparental populations, constructed by introducing genomic segments into Zhenshan 97 from ACC10 (A/Z), Minghui 63 (M/Z), and Nipponbare (N/Z), to map metabolic quantitative trait loci (mQTL). A total of 1,587 mQTL were generated, of which 684, 479, and 722 were obtained from the A/Z, M/Z, and N/Z chromosome segment substitution line populations, respectively, and we designated 99 candidate genes for 367 mQTL. In addition, 1,001 mQTL were generated specifically from joint linkage analysis with 25 candidate genes assigned. Several of these candidates were validated, such as LOC_Os07g01020 for the in vivo content of pyridoxine and its derivative and LOC_Os04g25980 for cis-zeatin glucosyltransferase activity. We propose a novel biosynthetic pathway for O-methylapigenin C-pentoside and demonstrated that LOC_Os04g11970 encodes a component of this pathway through fine-mapping. We postulate that the methylated apigenin may confer plant disease resistance. This study demonstrates the power of using multiple interconnected populations to generate a large number of veritable mQTL. The combined results are discussed in the context of functional metabolomics and the possible features of assigned candidates underlying respective metabolites.

OsTSD2-mediated cell wall modification affects ion homeostasis and salt tolerance.

Salt stress is a major environmental threat to meeting the food demands of an increasing global population. The identification and exploitation of salt adaption mechanisms in plants are therefore vital for crop breeding. We here define the rice mutant (sstm1) whose salt sensitivity was unambiguously assigned to a single T-DNA insertion through segregational analysis following backcrossing to the wild type line. Insertion was within OsTSD2, which encoded a pectin methyltransferase. The sstm1 and allelic mutants, collectively known as tsd2, displayed higher content of Na+ and lower level of K+ in the shoot, which is likely to lead to reduced salt tolerance. Molecular analysis revealed reduced expression of the genes maintaining K+ /Na+ homeostasis in tsd2, including OsHKT1;5, OsSOS1, and OsKAT1. Furthermore, OsTSD2 influenced ion distribution between the hull and the rice seed, which could improve food safety with heavy metal pollution. Amino acid levels tended to be increased in tsd2 mutants, implicating a role of pectin in the regulation of metabolism. Taken together, we have demonstrated an important facet of salt tolerance, which implicated OsTSD2-mediated cell wall pectin modification as a key component that could be widely applied in crop science.

Evolutionarily Distinct BAHD N-Acyltransferases Are Responsible for Natural Variation of Aromatic Amine Conjugates in Rice.

Phenolamides (PAs) are specialized (secondary) metabolites mainly synthesized by BAHD N-acyltransferases. Here, we report metabolic profiling coupled with association and linkage mapping of 11 PAs in rice (Oryza sativa). We identified 22 loci affecting PAs in leaves and 16 loci affecting PAs in seeds. We identified eight BAHD N-acyltransferases located on five chromosomes with diverse specificities, including four aromatic amine N-acyltransferases. We show that genetic variation in PAs is determined, at least in part, by allelic variation in the tissue specificity of expression of the BAHD genes responsible for their biosynthesis. Tryptamine hydroxycinnamoyl transferase 1/2 (Os-THT1/2) and tryptamine benzoyl transferase 1/2 (Os-TBT1/2) were found to be bifunctional tryptamine/tyramine N-acyltransferases. The specificity of Os-THT1 and Os-TBT1 for agmatine involved four tandem arginine residues, which have not been identified as specificity determinants for other plant BAHD transferases, illustrating the versatility of plant BAHD transferases in acquiring new acyl acceptor specificities. With phylogenetic analysis, we identified both divergent and convergent evolution of N-acyltransferases in plants, and we suggest that the BAHD family of tryptamine/tyramine N-acyltransferases evolved conservatively in monocots, especially in Gramineae. Our work demonstrates that omics-assisted gene-to-metabolite analysis provides a useful tool for bulk gene identification and crop genetic improvement.

Rice putative methyltransferase gene OsTSD2 is required for root development involving pectin modification.

Pectin synthesis and modification are vital for plant development, although the underlying mechanisms are still not well understood. Here, we report the functional characterization of the OsTSD2 gene, which encodes a putative methyltransferase in rice. All three independent T-DNA insertion lines of OsTSD2 displayed dwarf phenotypes and serial alterations in different zones of the root. These alterations included abnormal cellular adhesion and schizogenous aerenchyma formation in the meristematic zone, inhibited root elongation in the elongation zone, and higher lateral root density in the mature zone. Immunofluorescence (with LM19) and Ruthenium Red staining of the roots showed that unesterified homogalacturonan (HG) was increased in Ostsd2 mutants. Biochemical analysis of cell wall pectin polysaccharides revealed that both the monosaccharide composition and the uronic acid content were decreased in Ostsd2 mutants. Increased endogenous ABA content and opposite roles performed by ABA and IAA in regulating cellular adhesion in the Ostsd2 mutants suggested that OsTSD2 is required for root development in rice through a pathway involving pectin synthesis/modification. A hypothesis to explain the relationship among OsTSD2, pectin methylesterification, and root development is proposed, based on pectin's function in regional cell extension/division in a zone-dependent manner.

Genetic analysis of the metabolome exemplified using a rice population.

Plant metabolites are crucial for both plant life and human nutrition. Despite recent advance in metabolomics, the genetic control of plant metabolome remains largely unknown. Here, we performed a genetic analysis of the rice metabolome that provided over 2,800 highly resolved metabolic quantitative trait loci for 900 metabolites. Distinct and overlapping accumulation patterns of metabolites were observed and complex genetic regulation of metabolism was revealed in two different tissues. We associated 24 candidate genes to various metabolic quantitative trait loci by data mining, including ones regulating important morphological traits and biological processes. The corresponding pathways were reconstructed by updating in vivo functions of previously identified and newly assigned genes. This study demonstrated a powerful tool and provided a vast amount of high-quality data for understanding the plasticity of plant metabolome, which may help bridge the gap between the genome and phenome.

Interaction between carbon metabolism and phosphate accumulation is revealed by a mutation of a cellulose synthase-like protein, CSLF6.

Phosphorus is an essential macronutrient for plant growth and development. However, the network that affects phosphate (Pi) accumulation in crops is not well established. It is reported here that OsCSLF6, a member of the cellulose synthase-like family (CSLF), which is found only in grasses, is involved in Pi accumulation. The oscslf6 mutants (oscslf6-1 and oscslf6-2) display Pi toxic symptoms and increased Pi accumulation in both roots and shoots under the Pi-sufficient condition, which correlate with the induced expression of Pi transporters in the knockout mutants. Consistent with the over-accumulation of Pi, a significant decrease in primary root length, adventitious root length, and adventitious root number were observed in the oscslf6 mutants when compared with the wild type (WT) under Pi-sufficient conditions. In addition, the sucrose (Suc) level was increased in the oscslf6 mutants and the expression of sucrose synthases (OsSUS4/5) and sucrose transporters (OsSUT1/2/4/OsSweet14) genes were also induced in the shoots of oscslf6 mutants, suggesting that OsCSLF6 may play a role in affecting Pi accumulation by affecting the level of carbon metabolism.

Comprehensive profiling and natural variation of flavonoids in rice.

Flavonoids constitute a major group of plant phenolic compounds. While extensively studied in Arabidopsis, profiling and naturally occurring variation of these compounds in rice (Oryza sativa), the monocot model plant, are less reported. Using a collection of rice germplasm, comprehensive profiling and natural variation of flavonoids were presented in this report. Application of a widely targeted metabolomics method facilitated the simultaneous identification and quantification of more than 90 flavonoids using liquid chromatography tandem mass spectrometry (LC-MS/MS). Comparing flavonoid contents in various tissues during different developmental stages revealed tissue-specific accumulation of most flavonoids. Further investigation indicated that flavone mono-C-glycosides, malonylated flavonoid O-hexosides, and some flavonoid O-glycosides accumulated at significantly higher levels in indica than in japonica, while the opposite was observed for aromatic acylated flavone C-hexosyl-O-hexosides. In contrast to the highly differential accumulation between the two subspecies, relatively small variations within subspecies were detected for most flavonoids. Besides, an association analysis between flavonoid accumulation and its biosynthetic gene sequence polymorphisms disclosed that natural variation of flavonoids was probably caused by sequence polymorphisms in the coding region of flavonoid biosynthetic genes. Our work paves the way for future dissection of biosynthesis and regulation of flavonoid pathway in rice.

Phase synchronization during the processing of taxonomic and thematic relations.

Semantic relations include "taxonomic" relations based on shared features and "thematic" relations based on co-occurrence in events. The "dual-hub" account proposes that the anterior temporal lobe (ATL) is functionally specialized for taxonomic relations and the inferior parietal lobule (IPL) for thematic relations. This study examined this claim by analyzing the intra- and inter-region phase synchronization of intracranial EEG data from electrodes in the ATL, IPL, and two subregions of the semantic control network: left inferior frontal gyrus (IFG) and posterior middle temporal gyrus (pMTG). Ten participants with epilepsy completed a semantic relatedness judgment task during intracranial EEG recording and had electrodes in at least one hub and at least one semantic control region. Theta band phase synchronization was partially consistent with the dual-hub account: synchronization between the ATL and IFG/pMTG increased when processing taxonomic relations, and synchronization within the IPL and between IPL and pMTG increased when processing thematic relations.

WTV2.0: A high-coverage plant volatilomics method with a comprehensive selective ion monitoring acquisition mode.

Volatilomics is essential for understanding the biological functions and fragrance contributions of plant volatiles. However, the annotation coverage achieved using current untargeted and widely targeted volatomics (WTV) methods has been limited by low sensitivity and/or low acquisition coverage. Here, we introduce WTV 2.0, which enabled the construction of a high-coverage library containing 2111 plant volatiles, and report the development of a comprehensive selective ion monitoring (cSIM) acquisition method, including the selection of characteristic qualitative ions with the minimal ion number for each compound and an optimized segmentation method, that can acquire the smallest but sufficient number of ions for most plant volatiles, as well as the automatic qualitative and semi-quantitative analysis of cSIM data. Importantly, the library and acquisition method we developed can be self-expanded by incorporating compounds not present in the library, utilizing the obtained cSIM data. We showed that WTV 2.0 increases the median signal-to-noise ratio by 7.6-fold compared with the untargeted method, doubled the annotation coverage compared with the untargeted and WTV 1.0 methods in tomato fruit, and led to the discovery of menthofuran as a novel flavor compound in passion fruit. WTV 2.0 is a Python library with a user-friendly interface and is applicable to profiling of volatiles and primary metabolites in any species.

Widely Targeted Volatilomics and Metabolomics Analysis Reveal the Metabolic Composition and Diversity of Zingiberaceae Plants.

Zingiberaceae plants are widely used in the food and pharmaceutical industries; however, research on the chemical composition and interspecific differences in the metabolome and volatilome of Zingiberaceae plants is still limited. In this study, seven species of Zingiberaceae plants were selected, including Curcuma longa L., Zingiber officinale Rosc., Alpinia officinarum Hance, Alpinia tonkinensis Gagnep, Amomum tsaoko Crevost et Lemarie, Alpinia hainanensis K. Schum. and Amomum villosum Lour. Myristica fragrans Houtt. was also selected due to its flavor being similar to that of the Zingiberaceae plant. The metabolome and volatilome of selected plants were profiled by widely targeted approaches; 542 volatiles and 738 non-volatile metabolites were detected, and ß-myrcene, α-phellandrene and α-cadinene were detected in all the selected plants, while chamigren, thymol, perilla, acetocinnamone and cis-α-bisabolene were exclusively detected in certain Zingiberaceae plants. Differential analysis showed that some terpenoids, such as cadalene, cadalene-1,3,5-triene, cadalene-1,3,8-triene and (E)-ß-farnesene, and some lipids, including palmitic acid, linoleic acid and oleic acid were amongst the most varied compounds in Zingiberaceae plants. In conclusion, this study provided comprehensive metabolome and volatilome profiles for Zingiberaceae plants and revealed the metabolic differences between these plants. The results of this study could be used as a guide for the nutrition and flavor improvement of Zingiberaceae plants.

Natural variations of OsAUX5, a target gene of OsWRKY78, control the neutral essential amino acid content in rice grains.

Grain essential amino acid (EAA) levels contribute to rice nutritional quality. However, the molecular mechanisms underlying EAA accumulation and natural variation in rice grains remain unclear. Here we report the identification of a previously unrecognized auxin influx carrier subfamily gene, OsAUX5, which encodes an amino acid transporter that functions in uptake of multiple amino acids. We identified an elite haplotype of Pro::OsAUX5Hap2 that enhances grain EAA accumulation without an apparent negative effect on agronomic traits. Natural variations of OsAUX5 occur in the cis elements of its promoter, which are differentially activated because of the different binding affinity between OsWRKY78 and the W-box, contributing to grain EAA variation among rice varieties. The two distinct haplotypes were shown to have originated from different Oryza rufipogon progenitors, which contributed to the divergence between japonica and indica. Introduction of the indica-type Pro::OsAUX5Hap2 genotype into japonica could significantly increase EAA levels, indicating that indica-type Pro::OsAUX5Hap2 can be utilized to increase grain EAAs of japonica varieties. Collectively, our study uncovers an WRKY78-OsAUX5-based regulatory mechanism controlling grain EAA accumulation and provides a potential target for breeding EAA-rich rice.

Disease resistance conferred by components of essential chrysanthemum oil and the epigenetic regulation of OsTPS1.

The sesquiterpene alpha-bisabolol is the predominant active ingredient in essential oils that are highly valued in the cosmetics industry due to its wound healing, anti-inflammatory, and skin-soothing properties. Alpha-bisabolol was thought to be restricted to Compositae plants. Here we reveal that alpha-bisabolol is also synthesized in rice, a non-Compositae plant, where it acts as a novel sesquiterpene phytoalexin. Overexpressing the gene responsible for the biosynthesis of alpha-bisabolol, OsTPS1, conferred bacterial blight resistance in rice. Phylogenomic analyses revealed that alpha-bisabolol-synthesizing enzymes in rice and Compositae evolved independently. Further experiments demonstrated that the natural variation in the disease resistance level was associated with differential transcription of OsTPS1 due to polymorphisms in its promoter. We demonstrated that OsTPS1 was regulated at the epigenetic level by JMJ705 through the methyl jasmonate pathway. These data reveal the cross-family accumulation and regulatory mechanisms of alpha-bisabolol production.

Metabolomic Analysis Reveals Nutritional Diversity among Three Staple Crops and Three Fruits.

More than 2 billion people worldwide are under threat of nutritional deficiency. Thus, an in-depth comprehension of the nutritional composition of staple crops and popular fruits is essential for health. Herein, we performed LC-MS-based non-targeted and targeted metabolome analyses with crops (including wheat, rice, and corn) and fruits (including grape, banana, and mango). We detected a total of 2631 compounds by using non-targeted strategy and identified more than 260 nutrients. Our work discovered species-dependent accumulation of common present nutrients in crops and fruits. Although rice and wheat lack vitamins and amino acids, sweet corn was rich in most amino acids and vitamins. Among the three fruits, mango had more vitamins and amino acids than grape and banana. Grape and banana provided sufficient 5-methyltetrahydrofolate and vitamin B6, respectively. Moreover, rice and grape had a high content of flavonoids. In addition, the three crops contained more lipids than fruits. Furthermore, we also identified species-specific metabolites. The crops yielded 11 specific metabolites, including flavonoids, lipids, and others. Meanwhile, most fruit-specific nutrients were flavonoids. Our work discovered the complementary pattern of essential nutrients in crops and fruits, which provides metabolomic evidence for a healthy diet.

Research on Hybrid Force Control of Redundant Manipulator with Reverse Task Priority.

This paper presents the reverse priority impedance control of manipulators with reference to redundant robots of a given task. The reverse priority kinematic control of redundant manipulators is first expressed in detail. The motion in the joint space is derived following the opposite order compared with the classical task priority-based solution. Then the Cartesian impedance control is combined with the reverse priority impedance control to solve the reverse hierarchical impedance controlled, so that the Cartesian impedance behavior can be divided into the primary priority impedance control and the secondary priority impedance control. Furthermore, the secondary impedance control task will not disturb the primary impedance control task. The motion in the joint space is affected following the opposite order and working in the corresponding projection operators. The primary impedance control tasks are implemented at the end, so as to avoid the possible deformations caused by the singularities occurring in the secondary impedance control tasks. Hence, the proposed reverse priority impedance control of manipulator can achieve the desired impedance control tasks with proper hierarchy. In this paper, the simulation experiments of the manipulator will verify the proposed reverse priority control algorithm.