Cellulose synthase-like OsCSLD4: a key regulator of agronomic traits, disease resistance, and metabolic indices in rice.

KEY MESSAGE: Cellulose synthase-like OsCSLD4 plays a pivotal role in regulating diverse agronomic traits, enhancing resistance against bacterial leaf blight, and modulating metabolite indices based on the multi-omics analysis in rice. To delve deeper into this complex network between agronomic traits and metabolites in rice, we have compiled a dataset encompassing genome, phenome, and metabolome, including 524 diverse accessions, 11 agronomic traits, and 841 metabolites, enabling us to pinpoint eight hotspots through GWAS. We later discovered four distinct metabolite categories, encompassing 15 metabolites that are concurrently present on the QTL qC12.1, associated with leaf angle of flag and spikelet length, and finally focused the cellulose synthase-like OsCSLD4, which was pinpointed through a rigorous process encompassing sequence variation, haplotype, ATAC, and differential expression across diverse tissues. Compared to the wild type, csld4 exhibited significant reductions in the plant height, flag leaf length, leaf width, spikelet length, 1000-grain weight, grain width, grain thickness, fertility, yield per plant, and bacterial blight resistance. However, there were significant increase in tiller numbers, degree of leaf rolling, flowering period, growth period, grain length, and empty kernel rate. Furthermore, the content of four polyphenol metabolites, excluding metabolite N-feruloyltyramine (mr1268), notably rose, whereas the levels of the other three polyphenol metabolites, smiglaside C (mr1498), 4-coumaric acid (mr1622), and smiglaside A (mr1925) decreased significantly in mutant csld4. The content of amino acid L-tyramine (mr1446) exhibited a notable increase, whereas the alkaloid trigonelline (mr1188) displayed a substantial decrease among the mutants. This study offered a comprehensive multi-omics perspective to analyze the genetic mechanism of OsCSLD4, and breeders can potentially enhance rice's yield, bacterial leaf blight resistance, and metabolite content, leading to more sustainable and profitable rice production.

Insights into Tissue-Specific Specialized Metabolism in Wampee (Clausena lansium (Lour.) Skeels) Varieties.

Wampee (Clausena lansium (Lour.) Skeels) has natural bioactive components with diverse health benefits, but its detailed metabolism and tissue distribution are not fully understood. Here, widely targeted metabolomics analysis methods were employed to analyze the wampee fruit (peel, pulp, and seed) of 17 different varieties. A total of 1286 metabolites were annotated, including lipids, flavonoids, polyphenols, carbazole alkaloids, coumarins, and organic acids, among others. The quantitative analysis and matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) analysis indicated remarkable variations in metabolite categories and content in the peel, pulp, and seed of wampee fruit. Additionally, the difference analysis found that the metabolic components of peel contributed dominantly to the differences among varieties, and 7 potential biomarkers were identified. In this study, a comprehensive metabolome landscape of wampee fruit was established, which provided important information for the isolation and identification of functional components, food industry application, and nutritional improvement breeding.

Dissecting the genetic basis of UV-B responsive metabolites in rice.

BACKGROUND: UV-B, an important environmental factor, has been shown to affect the yield and quality of rice (Oryza sativa) worldwide. However, the molecular mechanisms underlying the response to UV-B stress remain elusive in rice. RESULTS: We perform comprehensive metabolic profiling of leaves from 160 diverse rice accessions under UV-B and normal light conditions using a widely targeted metabolomics approach. Our results reveal substantial differences in metabolite accumulation between the two major rice subspecies indica and japonica, especially after UV-B treatment, implying the possible role and mechanism of metabolome changes in subspecies differentiation and the stress response. We next conduct a transcriptome analysis from four representative rice varieties under UV-B stress, revealing genes from amino acid and flavonoid pathways involved in the UV-B response. We further perform a metabolite-based genome-wide association study (mGWAS), which reveals 3307 distinct loci under UV-B stress. Identification and functional validation of candidate genes show that OsMYB44 regulates tryptamine accumulation to mediate UV-B tolerance, while OsUVR8 interacts with OsMYB110 to promote flavonoid accumulation and UV-B tolerance in a coordinated manner. Additionally, haplotype analysis suggests that natural variation of OsUVR8groupA contributes to UV-B resistance in rice. CONCLUSIONS: Our study reveals the complex biochemical and genetic foundations that govern the metabolite dynamics underlying the response, tolerance, and adaptive strategies of rice to UV-B stress. These findings provide new insights into the biochemical and genetic basis of the metabolome underlying the crop response, tolerance, and adaptation to UV-B stress.

Variation in a Poaceae-conserved fatty acid metabolic gene cluster controls rice yield by regulating male fertility.

A wide variety of metabolic gene clusters exist in eukaryotic genomes, but fatty acid metabolic gene clusters have not been discovered. Here, combining with metabolic and phenotypic genome-wide association studies, we identify a major locus containing a six-gene fatty acid metabolic gene cluster on chromosome 3 (FGC3) that controls the cutin monomer hydroxymonoacylglycerols (HMGs) contents and rice yield, possibly through variation in the transcription of FGC3 members. We show that HMGs are sequentially synthesized in the endoplasmic reticulum by OsFAR2, OsKCS11, OsGPAT6, OsCYP704B2 and subsequently transported to the apoplast by OsABCG22 and OsLTPL82. Mutation of FGC3 members reduces HMGs, leading to defective male reproductive development and a significant decrease in yield. OsMADS6 and OsMADS17 directly regulate FGC3 and thus influence male reproduction and yield. FGC3 is conserved in Poaceae and likely formed prior to the divergence of Pharus latifolius. The eukaryotic fatty acid and plant primary metabolic gene cluster we identified show a significant impact on the origin and evolution of Poaceae and has potential for application in hybrid crop breeding.

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.

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.

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.

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.

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.

Integrated Metabolomics and Transcriptomics Analyses Reveal the Metabolic Differences and Molecular Basis of Nutritional Quality in Landraces and Cultivated Rice.

Rice (Oryza sativa L.) is one of the most globally important crops, nutritionally and economically. Therefore, analyzing the genetic basis of its nutritional quality is a paramount prerequisite for cultivating new varieties with increased nutritional health. To systematically compare the nutritional quality differences between landraces and cultivated rice, and to mine key genes that determine the specific nutritional traits of landraces, a seed metabolome database of 985 nutritional metabolites covering amino acids, flavonoids, anthocyanins, and vitamins by a widely targeted metabolomic approach with 114 rice varieties (35 landraces and 79 cultivars) was established. To further reveal the molecular mechanism of the metabolic differences in landrace and cultivated rice seeds, four cultivars and six landrace seeds were selected for transcriptome and metabolome analysis during germination, respectively. The integrated analysis compared the metabolic profiles and transcriptomes of different types of rice, identifying 358 differentially accumulated metabolites (DAMs) and 1982 differentially expressed genes (DEGs), establishing a metabolite-gene correlation network. A PCA revealed anthocyanins, flavonoids, and lipids as the central differential nutritional metabolites between landraces and cultivated rice. The metabolite-gene correlation network was used to screen out 20 candidate genes postulated to be involved in the structural modification of anthocyanins. Five glycosyltransferases were verified to catalyze the glycosylation of anthocyanins by in vitro enzyme activity experiments. At the same time, the different mechanisms of the anthocyanin synthesis pathway and structural diversity in landrace and cultivated rice were systematically analyzed, providing new insights for the improvement and utilization of the nutritional quality of rice landrace varieties.

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.

Integration of rhythmic metabolome and transcriptome provides insights into the transmission of rhythmic fluctuations and temporal diversity of metabolism in rice.

Various aspects of the organisms adapt to cyclically changing environmental conditions via transcriptional regulation. However, the role of rhythmicity in altering the global aspects of metabolism is poorly characterized. Here, we subjected four rice (Oryza sativa) varieties to a range of metabolic profiles and RNA-seq to investigate the temporal relationships of rhythm between transcription and metabolism. More than 40% of the rhythmic genes and a quarter of metabolites conservatively oscillated across four rice accessions. Compared with the metabolome, the transcriptome was more strongly regulated by rhythm; however, the rhythm of metabolites had an obvious opposite trend between day and night. Through association analysis, the time delay of rhythmic transmission from the transcript to the metabolite level was ∼4 h under long-day conditions, although the transmission was nearly synchronous for carbohydrate and nucleotide metabolism. The rhythmic accumulation of metabolites maintained highly coordinated temporal relationships in the metabolic network, whereas the correlation of some rhythmic metabolites, such as branched-chain amino acids (BCAAs), was significantly different intervariety. We further demonstrated that the cumulative diversity of BCAAs was due to the differential expression of branched-chain aminotransferase 2 at dawn. Our research reveals the flexible pattern of rice metabolic rhythm existing with conservation and diversity.

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.

OsRLCK160 contributes to flavonoid accumulation and UV-B tolerance by regulating OsbZIP48 in rice.

Plants produce specialized metabolites to adapt to the ever-changing environments. Flavonoids are antioxidants essential for growth, development, and breeding with increased stress resistance in crops. However, the mechanism of the involvement of flavonoids in ultraviolet-B (UV-B) stress in rice (Oryza sativa) is largely unknown. In this study, we cloned and functionally identified a receptor-like kinase (OsRLCK160) and a bZIP transcription factor (OsbZIP48) positively regulating flavonoid accumulation through metabolite-based genome-wide association study of the flavonoid content in rice. Meanwhile, OsRLCK160 interacted with and phosphorylated OsbZIP48 to regulate the flavonoid accumulation and participate in UV-B tolerance in rice. Our study indicates the importance of applying OsRLCK160 and OsbZIP48 to advance the fundamental understanding of stable rice production and breed UV-B-tolerant rice varieties, which may contribute to breeding high-yield rice varieties.