Evolution-guided multiomics provide insights into the strengthening of bioactive flavone biosynthesis in medicinal pummelo.

Pummelo (Citrus maxima or Citrus grandis) is a basic species and an important type for breeding in Citrus. Pummelo is used not only for fresh consumption but also for medicinal purposes. However, the molecular basis of medicinal traits is unclear. Here, compared with wild citrus species/Citrus-related genera, the content of 43 bioactive metabolites and their derivatives increased in the pummelo. Furthermore, we assembled the genome sequence of a variety for medicinal purposes with a long history, Citrus maxima 'Huazhouyou-tomentosa' (HZY-T), at the chromosome level with a genome size of 349.07 Mb. Comparative genomics showed that the expanded gene family in the pummelo genome was enriched in flavonoids-, terpenoid-, and phenylpropanoid biosynthesis. Using the metabolome and transcriptome of six developmental stages of HZY-T and Citrus maxima 'Huazhouyou-smooth' (HZY-S) fruit peel, we generated the regulatory networks of bioactive metabolites and their derivatives. We identified a novel MYB transcription factor, CmtMYB108, as an important regulator of flavone pathways. Both mutations and expression of CmtMYB108, which targets the genes PAL (phenylalanine ammonia-lyase) and FNS (flavone synthase), displayed differential expression between Citrus-related genera, wild citrus species and pummelo species. This study provides insights into the evolution-associated changes in bioactive metabolism during the origin process of pummelo.

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.

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.

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.

Metabolomics-centered mining of plant metabolic diversity and function: Past decade and future perspectives.

Plants are natural experts in organic synthesis, being able to generate large numbers of specific metabolites with widely varying structures that help them adapt to variable survival challenges. Metabolomics is a research discipline that integrates the capabilities of several types of research including analytical chemistry, statistics, and biochemistry. Its ongoing development provides strategies for gaining a systematic understanding of quantitative changes in the levels of metabolites. Metabolomics is usually performed by targeting either a specific cell, a specific tissue, or the entire organism. Considerable advances in science and technology over the last three decades have propelled us into the era of multi-omics, in which metabolomics, despite at an earlier developmental stage than genomics, transcriptomics, and proteomics, offers the distinct advantage of studying the cellular entities that have the greatest influence on end phenotype. Here, we summarize the state of the art of metabolite detection and identification, and illustrate these techniques with four case study applications: (i) comparing metabolite composition within and between species, (ii) assessing spatio-temporal metabolic changes during plant development, (iii) mining characteristic metabolites of plants in different ecological environments and upon exposure to various stresses, and (iv) assessing the performance of metabolomics as a means of functional gene identification , metabolic pathway elucidation, and metabolomics-assisted breeding through analyzing plant populations with diverse genetic variations. In addition, we highlight the prominent contributions of joint analyses of plant metabolomics and other omics datasets, including those from genomics, transcriptomics, proteomics, epigenomics, phenomics, microbiomes, and ion-omics studies. Finally, we discuss future directions and challenges exploiting metabolomics-centered approaches in understanding plant metabolic diversity.

Limitations and advantages of using metabolite-based genome-wide association studies: Focus on fruit quality traits.

In the last decades, linkage mapping has help in the location of metabolite quantitative trait loci (QTL) in many species; however, this approach shows some limitations. Recently, thanks to the most recent advanced in high-throughput genotyping technologies like next-generation sequencing, metabolite genome-wide association study (mGWAS) has been proposed a powerful tool to identify the genetic variants in polygenic agrinomic traits. Fruit flavor is a complex interaction of aroma volatiles and taste being sugar and acid ratio key parameter for flavor acceptance. Here, we review recent progress of mGWAS in pinpoint gene polymorphisms related to flavor-related metabolites in fruits. Despite clear successes in discovering novel genes or regions associated with metabolite accumulation affecting sensory attributes in fruits, GWAS incurs in several limitations summarized in this review. In addition, in our own work, we performed mGWAS on 194 Citrus grandis accessions to investigate the genetic control of individual primary and lipid metabolites in ripe fruit. We have identified a total of 667 associations for 14 primary metabolites including amino acids, sugars, and organic acids, and 768 associations corresponding to 47 lipids. Furthermore, candidate genes related to important metabolites related to fruit quality such as sugars, organic acids and lipids were discovered.

Plant metabolic gene clusters in the multi-omics era.

Secondary metabolism in plants gives rise to a vast array of small-molecule natural products. The discovery of operon-like gene clusters in plants has provided a new perspective on the evolution of specialized metabolism and the opportunity to rapidly advance the metabolic engineering of natural product production. Here, we review historical aspects of the study of plant metabolic gene clusters as well as general strategies for identifying plant metabolic gene clusters in the multi-omics era. We also emphasize the exploration of their natural variation and evolution, as well as new strategies for the prospecting of plant metabolic gene clusters and a deeper understanding of how their structure influences their function.

Function of hydroxycinnamoyl transferases for the biosynthesis of phenolamides in rice resistance to Magnaporthe oryzae.

Phenolamide (PA) metabolites play important roles in the interaction between plants and pathogens. The putrescine hydroxycinnamoyl transferase genes OsPHT3 and OsPHT4 positively regulate rice cell death and resistance to Magnaporthe oryzae. The bZIP transcription factor APIP5, a negative regulator of cell death and rice immunity, directly binds to the OsPHT4 promoter to regulate putrescine-derived PAs. Whether other hydroxycinnamoyl transferase (HT) genes also participate in APIP5-mediated immunity remains unclear. Surprisingly, we find that genes encoding agmatine hydroxycinnamoyl transferases OsAHT1 and OsAHT2, tryptamine hydroxycinnamoyl transferases OsTBT1 and OsTBT2, and tyramine hydroxycinnamoyl transferases OsTHT1 and OsTHT2, responsible for the biosynthesis of polyamine-derived PAs are all up-regulated in APIP5-RNAi transgenic plants compared with segregated wild-type rice. Furthermore, both OsAHT1/2 and OsTBT1/2 are induced during M. oryzae infection, showing expression patterns similar to those previously reported for OsTHT1/2 and OsPHT3/4. Transgenic plants overexpressing either OsAHT2-GFP or OsTBT1-GFP show enhanced resistance against M. oryzae and accumulated more PA metabolites and lignin compared with wild-type plants. Interestingly, as demonstrated for OsPHT4, APIP5 directly binds to the promoters of OsAHT1/2, OsTBT1/2, and OsTHT1/2, repressing their transcription. Together, these results indicate that the HT genes are common targets of APIP5 and that PAs play critical roles in rice immunity.

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.

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.

Rice metabolic regulatory network spanning the entire life cycle.

As one of the most important crops in the world, rice (Oryza sativa) is a model plant for metabolome research. Although many studies have focused on the analysis of specific tissues, the changes in metabolite abundance across the entire life cycle have not yet been determined. In this study, combining both targeted and nontargeted metabolite profiling methods, a total of 825 annotated metabolites were quantified in rice samples from different tissues covering the entire life cycle. The contents of metabolites in different tissues of rice were significantly different, with various metabolites accumulating in the plumule and radicle during seed germination. Combining these data with transcriptome data obtained from the same time period, we constructed the Rice Metabolic Regulation Network. The metabolites and co-expressed genes were further divided into 12 clusters according to their accumulation patterns, with members within each cluster displaying a uniform and clear pattern of abundance across development. Using this dataset, we established a comprehensive metabolic profile of the rice life cycle and used two independent strategies to identify novel transcription factors-namely the use of known regulatory genes as bait to screen for new networks underlying lignin metabolism and the unbiased identification of new glycerophospholipid metabolism regulators on the basis of tissue specificity. This study thus demonstrates how guilt-by-association analysis of metabolome and transcriptome data spanning the entire life cycle in cereal crops provides novel resources and tools to aid in understanding the mechanisms underlying important agronomic traits.

An Oryza-specific hydroxycinnamoyl tyramine gene cluster contributes to enhanced disease resistance.

Genomic clustering of non-homologous genes for the biosynthesis of plant defensive compounds is an emerging theme, but insights into their formation and physiological function remain limited. Here we report the identification of a newly discovered hydroxycinnamoyl tyramine (HT) gene cluster in rice. This cluster contains a pyridoxamine 5'-phosphate oxidase (OsPDX3) producing the cofactor pyridoxal 5'-phosphate (PLP), a PLP-dependent tyrosine decarboxylase (OsTyDC1), and two duplicated hydroxycinnamoyl transferases (OsTHT1 and OsTHT2). These members were combined to represent an enzymological innovation gene cluster. Natural variation analysis showed that the abundance of the toxic tyramine intermediate of the gene cluster among different rice accessions is mainly determined by the coordinated transcription of OsTyDC1 and OsTHT1. Further pathogen incubation assays demonstrated that the end products of the HT gene cluster displayed enhanced resistance to the bacterial pathogen Xanthomonas oryzae pv. Oryzae (Xoo) and fungal pathogen Magnaporthe oryzae (M. oryzae), and the enhanced resistance is associated with the boost of phytoalexins and the activation of defense response. The unique presence of the HT gene cluster in Oryza AA genome, together with the enrichment of transposon elements within this gene cluster region, provides an evolutionary background to accelerate cluster member combinations. Our study not only discovered a gene cluster involved in the phenylpropanoid metabolism but also addressed the key aspects of gene cluster formation. In addition, our results provide a new metabolic pool for plant defense against pathogens.

A monocot-specific hydroxycinnamoylputrescine gene cluster contributes to immunity and cell death in rice.

Phenolamides (PAs), a diverse group of specialized metabolites, including hydroxycinnamoylputrescine (HP), hydroxycinnamoylagmatine, and hydroxycinnamoyltryptamine, are important in plant resistance to biotic stress. However, the genes involved in the biosynthesis and modulation of PAs have not been fully elucidated. This study identified an HP biosynthetic gene cluster in rice (Oryza sativa) comprising one gene (OsODC) encoding a decarboxylase and two tandem-duplicated genes (OsPHT3 and OsPHT4) encoding putrescine hydroxycinnamoyl acyltransferases coexpressed in different tissues. OsODC catalyzes the conversion of ornithine to putrescine, which is used in HP biosynthesis involving OsPHT3 and OsPHT4. OsPHT3 or OsPHT4 overexpression causes HP accumulation and cell death and putrescine hydroxycinnamoyl acyltransferases (PHT) activity-dependent resistance against the fungal pathogen Magnaporthe oryzae. OsODC overexpression plants also confer enhanced resistance to M. oryzae. Notably, the basic leucine zipper transcription factor APIP5, a negative regulator of cell death, directly binds to the OsPHT4 promoter, repressing its transcription. Moreover, APIP5 suppression induces OsPHT4 expression and HP accumulation. Comparative genomic analysis revealed that the HP biosynthetic gene cluster is conserved in monocots. These results characterized a previously unidentified monocot-specific gene cluster that is involved in HP biosynthesis and contributes to defense and cell death in rice.

Selection of a subspecies-specific diterpene gene cluster implicated in rice disease resistance.

Diterpenoids are the major group of antimicrobial phytoalexins in rice1,2. Here, we report the discovery of a rice diterpenoid gene cluster on chromosome 7 (DGC7) encoding the entire biosynthetic pathway to 5,10-diketo-casbene, a member of the monocyclic casbene-derived diterpenoids. We revealed that DGC7 is regulated directly by JMJ705 through methyl jasmonate-mediated epigenetic control3. Functional characterization of pathway genes revealed OsCYP71Z21 to encode a casbene C10 oxidase, sought after for the biosynthesis of an array of medicinally important diterpenoids. We further show that DGC7 arose relatively recently in the Oryza genus, and that it was partly formed in Oryza rufipogon and positively selected for in japonica during domestication. Casbene-synthesizing enzymes that are functionally equivalent to OsTPS28 are present in several species of Euphorbiaceae but gene tree analysis shows that these and other casbene-modifying enzymes have evolved independently. As such, combining casbene-modifying enzymes from these different families of plants may prove effective in producing a diverse array of bioactive diterpenoid natural products.

Differentially evolved glucosyltransferases determine natural variation of rice flavone accumulation and UV-tolerance.

Decoration of phytochemicals contributes to the majority of metabolic diversity in nature, whereas how this process alters the biological functions of their precursor molecules remains to be investigated. Flavones, an important yet overlooked subclass of flavonoids, are most commonly conjugated with sugar moieties by UDP-dependent glycosyltransferases (UGTs). Here, we report that the natural variation of rice flavones is mainly determined by OsUGT706D1 (flavone 7-O-glucosyltransferase) and OsUGT707A2 (flavone 5-O-glucosyltransferase). UV-B exposure and transgenic evaluation demonstrate that their allelic variation contributes to UV-B tolerance in nature. Biochemical characterization of over 40 flavonoid UGTs reveals their differential evolution in angiosperms. These combined data provide biochemical insight and genetic regulation into flavone biosynthesis and additionally suggest that adoption of the positive alleles of these genes into breeding programs will likely represent a potential strategy aimed at producing stress-tolerant plants.