CsPHRs-CsJAZ3 incorporates phosphate signaling and jasmonate pathway to regulate catechin biosynthesis in Camellia sinensis.

Catechins constitute abundant metabolites in tea and have potential health benefits and high economic value. Intensive study has shown that the biosynthesis of tea catechins is regulated by environmental factors and hormonal signals. However, little is known about the coordination of phosphate (Pi) signaling and the jasmonic acid (JA) pathway on biosynthesis of tea catechins. We found that Pi deficiency caused changes in the content of catechins and modulated the expression levels of genes involved in catechin biosynthesis. Herein, we identified two transcription factors of phosphate signaling in tea, named CsPHR1 and CsPHR2, respectively. Both regulated catechin biosynthesis by activating the transcription of CsANR1 and CsMYB5c. We further demonstrated CsSPX1, a Pi pathway repressor, suppressing the activation by CsPHR1/2 of CsANR1 and CsMYB5c. JA, one of the endogenous plant hormones, has been reported to be involved in the regulation of secondary metabolism. Our work demonstrated that the JA signaling repressor CsJAZ3 negatively regulated catechin biosynthesis via physical interaction with CsPHR1 and CsPHR2. Thus, the CsPHRs-CsJAZ3 module bridges the nutrition and hormone signals, contributing to targeted cultivation of high-quality tea cultivars with high fertilizer efficiency.

Biofortified Rice Provides Rich Sakuranetin in Endosperm.

Sakuranetin plays a key role as a phytoalexin in plant resistance to biotic and abiotic stresses, and possesses diverse health-promoting benefits. However, mature rice seeds do not contain detectable levels of sakuranetin. In the present study, a transgenic rice plant was developed in which the promoter of an endosperm-specific glutelin gene OsGluD-1 drives the expression of a specific enzyme naringenin 7-O-methyltransferase (NOMT) for sakuranetin biosynthesis. The presence of naringenin, which serves as the biosynthetic precursor of sakuranetin made this modification feasible in theory. Liquid chromatography tandem mass spectrometry (LC-MS/MS) validated that the seeds of transgenic rice accumulated remarkable sakuranetin at the mature stage, and higher at the filling stage. In addition, the panicle blast resistance of transgenic rice was significantly higher than that of the wild type. Specially, the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging was performed to detect the content and spatial distribution of sakuranetin and other nutritional metabolites in transgenic rice seeds. Notably, this genetic modification also did not change the nutritional and quality indicators such as soluble sugars, total amino acids, total flavonoids, amylose, total protein, and free amino acid content in rice. Meanwhile, the phenotypes of the transgenic plant during the whole growth and developmental periods and agricultural traits such as grain width, grain length, and 1000-grain weight exhibited no significant differences from the wild type. Collectively, the study provides a conceptual advance on cultivating sakuranetin-rich biofortified rice by metabolic engineering. This new breeding idea may not only enhance the disease resistance of cereal crop seeds but also improve the nutritional value of grains for human health benefits.

Comparison of Nutritional Diversity in Five Fresh Legumes Using Flavonoids Metabolomics and Postharvest Botrytis cinerea Defense Analysis of Peas Mediated by Sakuranetin.

Legumes possess several bioactive nutrients, including flavonoids, and the study of the flavonoid profile of legumes is of great significance to human health. Using widely targeted metabolomics, we revealed the flavonoid profiles of five popular fresh legumes: cowpea, soybean, pea, fava bean, and kidney bean. A total of 259 flavonoids were identified, and the flavonoid accumulation patterns of the five legumes were remarkably different. In addition to analyzing common and species-specific flavonoids in the five legumes, we also generalized representative flavonoids of various subclasses. We related these to the health-promoting effects of legumes. Furthermore, legumes' total flavonoid content and antioxidant system activity were also detected. Intriguingly, sakuranetin, the sole flavonoid phytoalexin that can be induced by UV radiation, was detected only in the peas by metabolomics. Meanwhile, we found that UV treatment could significantly increase the sakuranetin content and the postharvest Botrytis cinerea resistance of pea pods. This study provides clues for the target diet, industrial development of legumes, and a new idea for the postharvest preservation of peas.

Searching for the Virulence-contributing Genes of the Magnaporthe oryzae by Transcriptome Analysis.

Magnaporthe oryzae is a fungal pathogen that causes rice blast. Plant metabolites such as plant hormones and phytoalexin can promote or inhibit the rice blast infection. To study the effect of plant metabolites on M. oryzae, we selected salicylic acid (SA), abscisic acid (ABA), and a phytoalexin sakuranetin to treat M. oryzae grown on the medium. Through the analysis of transcriptome data, 185 and 38 genes, 803 and 156 genes, and 1525 and 428 genes were up- or down-regulated after SA, ABA, or sakuranetin treatment. Among these differentially expressed genes (DEGs), most of them were annotated to the cellular process and metabolic process in the biological process category and binding and catalytic activity in the molecular function category by GO analysis. According to KEGG pathway analysis, metabolism is the pathway with the highest number of DEGs, and the main enriched pathway is carbohydrate, lipid, and amino acid metabolism. In addition, we also found two ABA-induced up-regulated genes that may contribute to M. oryzae infection from the transcriptome data. We verified their expressions in M. oryzae that infected rice.

WRKY33 negatively regulates anthocyanin biosynthesis and cooperates with PHR1 to mediate acclimation to phosphate starvation.

Anthocyanin accumulation is acknowledged as a phenotypic indicator of phosphate (Pi) starvation. However, negative regulators of this process and their molecular mechanisms remain largely unexplored. In this study, we demonstrate that WRKY33 acts as a negative regulator of phosphorus-status-dependent anthocyanin biosynthesis. WRKY33 regulates the expression of the gene encoding dihydroflavonol 4-reductase (DFR), a rate-limiting enzyme in anthocyanin production, both directly and indirectly. WRKY33 binds directly to the DFR promoter to repress its expression and also interferes with the MBW complex through interacting with PAP1 to indirectly influence DFR transcriptional activation. Under -Pi conditions, PHR1 interacts with WRKY33, and the protein level of WRKY33 decreases; the repression of DFR expression by WRKY33 is thus attenuated, leading to anthocyanin accumulation in Arabidopsis. Further genetic and biochemical assays suggest that PHR1 is also involved in regulating factors that affect WRKY33 protein turnover. Taken together, our findings reveal that Pi starvation represses WRKY33, a repressor of anthocyanin biosynthesis, to finely tune anthocyanin biosynthesis. This "double-negative logic" regulation of phosphorus-status-dependent anthocyanin biosynthesis is required for the maintenance of plant metabolic homeostasis during acclimation to Pi starvation.

The OsBZR1-OsSPX1/2 module fine-tunes the growth-immunity trade-off in adaptation to phosphate availability in rice.

The growth-promoting hormones brassinosteroids (BRs) and their key signaling component BZR1 play a vital role in balancing normal growth and defense reactions. Here, we discovered that BRs and OsBZR1 upregulated sakuranetin accumulation and conferred basal defense against Magnaporthe oryzae infection under normal conditions. Resource shortages, including phosphate (Pi) deficiency, potentially disrupt this growth-defense balance. OsSPX1 and OsSPX2 have been reported to sense Pi concentration and interact with the Pi signal mediator OsPHR2, thus regulating Pi starvation responses. In this study, we discovered that OsSPX1/2 interacts with OsBZR1 in both Pi-sufficient and Pi-deficient conditions, inhibiting BR-responsive genes. When Pi is sufficient, OsSPX1/2 is captured by OsPHR2, enabling most of OsBZR1 to promote plant growth and maintain basal resistance. In response to Pi starvation, more OsSPX1/2 is released from OsPHR2 to inhibit OsBZR1 activity, resulting in slower growth. Collectively, our study reveals that the OsBZR1-SPX1/2 module balances the plant growth-immunity trade-off in response to Pi availability.

Unveiling targeted spatial metabolome of rice seed at the dough stage using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry imaging.

Rice (Oryza sativa) seeds contain a variety of metabolites, which not only provide energy for their own growth and development, but also are an important source of nutrition for humans. It is crucial to study the distribution of metabolites in rice seeds, but the spatial metabolome of rice seeds is rarely investigated. In this study, Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) imaging was used to reveal the spatial distribution of free soluble sugars (glucose, fructose, sucrose, and maltose), amino acids (9 essential amino acids and 2 amino acids affecting rice eating quality: L-aspartic acid and L-glutamic acid), and 4 metabolites in the flavonoids synthesis pathway (cinnamic acid, naringenin chalcone, naringenin, and dihydrokaempferol) in rice seed at the dough stage. It was found that the 4 free soluble sugars present similar spatial distribution, mainly distributed in the seed cortex and embryo with high abundance. The majority of amino acids are also concentrated in the rice cortex and embryo, while the others are abundant in the whole seed. Besides cinnamic acid distributed in the seed cortex and embryo, the naringenin chalcone, naringenin, and dihydrokaempferol were also found in the endosperm and had lower content. Furthermore, a colocalization phylogenetic tree according to the spatial distribution imaging of each metabolite was constructed. This study revealed the distribution diversity of metabolites in different segmentations of rice seed at the dough stage, providing clues for the nutritional differences between brown rice and white rice, and serving as a reference for people to target a healthy diet.

Corrigendum: Involvement of PtPHR1 in phosphates starvation-induced alkaloid biosynthesis in Pinellia ternata (Thunb.) Breit.

[This corrects the article DOI: 10.3389/fpls.2022.914648.].

Corrigendum: Genome-wide characterization of NAC transcription factors in Camellia sinensis and the involvement of CsNAC28 in drought tolerance.

[This corrects the article DOI: 10.3389/fpls.2022.1065261.].

Analysis of the Flavonoidome Reveals the Different Health-Promoting Flavonoid Characteristics in Fruit.

Flavonoids are one of the important metabolites of plants, and many flavonoids have functions of antioxidant or antimicrobial, which can help plants resist environmental stress. On the other hand, flavonoids also have a health-promoting effect for humans, such as antioxidant and anti-aging, and some flavonoids can assist in disease treatment. Fruit is one of the main sources of plant food and flavonoids intake for humans. Understanding the flavonoidome of various fruits is helpful to choose fruit combinations according to different demands. In this study, we explored the composition and relative content of flavonoids in 22 fruits and analyzed some health-promoting flavonoids in fruits. In addition, we selected several fruits and measured their antioxidant capacity through experiments. Our study initially established a database of fruit flavonoidome, and can provide reference for nutrition research, fruit breeding and industrial development.

The CsHSFA-CsJAZ6 module-mediated high temperature regulates flavonoid metabolism in Camellia sinensis.

High temperatures (HTs) seriously affect the yield and quality of tea. Catechins, derived from the flavonoid pathway, are characteristic compounds that contribute to the flavour of tea leaves. In this study, we first showed that the flavonoid content of tea leaves was significantly reduced under HT conditions via metabolic profiles; and then demonstrated that two transcription factors, CsHSFA1b and CsHSFA2 were activated by HT and negatively regulate flavonoid biosynthesis during HT treatment. Jasmonate (JA), a defensive hormone, plays a key role in plant adaption to environmental stress. However, little has been reported on its involvement in HT response in tea. Herein, we demonstrated that CsHSFA1b and CsHSFA2 activate CsJAZ6 expression through directly binding to heat shock elements in its promoter, and thereby repress the JA pathway. Most secondary metabolites are regulated by JA, including catechin in tea. Our study reported that CsJAZ6 directly interacts with CsEGL3 and CsTTG1 and thereby reduces catechin accumulation. From this, we proposed a CsHSFA-CsJAZ6-mediated HT regulation model of catechin biosynthesis. We also determined that negative regulation of the JA pathway by CsHSFAs and its homologues is conserved in Arabidopsis. These findings broaden the applicability of the regulation of JAZ by HSF transcription factors and further suggest the JA pathway as a valuable candidate for HT-resistant breeding and cultivation.

Sakuranetin protects rice from brown planthopper attack by depleting its beneficial endosymbionts.

Plants produce chemical defenses that poison insect herbivores or deter their feeding, but herbivores are also accompanied by microbial endosymbionts crucial for their nutrition, reproduction, and fitness. Hence, plant defenses could target a herbivore's beneficial endosymbionts, but this has not yet been demonstrated. Here, we studied flavonoids that are induced when rice is attacked by a phloem-feeding pest, the brown planthopper (BPH), which harbors beneficial yeast-like symbionts (YLS) essential for insect nutrition, such as by remedying deficiencies in sterols. BPH attack dramatically increased sakuranetin accumulations in leaf sheaths and phloem exudates. Sakuranetin is an antifungal phytoalexin derived from the antibacterial precursor, naringenin, via catalysis of naringenin-O-methyltransferase (NOMT). When added to artificial diets, sakuranetin decreased BPH survivorship, suggesting that it functions as an induced defense. Mutation of NOMT abolished sakuranetin accumulation and increased BPH oviposition and hatching rates. High-throughput amplicon sequencing revealed that BPH fed on sakuranetin-deficient nomt lines were enriched in YLS with only minor changes in the bacterial endosymbionts, compared to those feeding on sakuranetin-rich wild-type (WT) plants. In-vitro feeding of sakuranetin suggested that this flavonoid directly inhibited the growth of YLS. BPH feeding on nomt lines accumulated higher cholesterol levels, which might be attributed to increases in the supply of sterol precursors from the YLS, while nomt lines suffered more damage than WT plants did from BPH herbivory. BPH-elicited accumulation of sakuranetin requires intact jasmonate (JA) signaling. This study reveals that rice uses a JA-induced antifungal flavonoid phytoalexin in defense against BPH by inhibiting its beneficial endosymbionts.

Comparative metabolomics of flavonoids in twenty vegetables reveal their nutritional diversity and potential health benefits.

Vegetables are rich in flavonoids and are widely consumed in our daily life. However, comprehensive information on flavonoids components in vegetable varieties and the distribution of flavonoids with health-promoting effects in different vegetables are rarely investigated. Here, we analyzed the constitution of flavonoids among 20 vegetables by widely-targeted metabolome analysis. A total of 403 flavonoids were detected and classified as flavonoid, flavonols, anthocyanins, isoflavones, flavonoid carbonoside, dihydroflavone, chalcones, flavanols, dihydroflavonol, tannin, proanthocyanidins, and other flavonoids. Interestingly, we found that the content and types of flavonoids in bean sprouts and hot pepper were relatively abundant, whereas those were lower in carrot, lettuce, and Zizania latifolia. Then, we characterized the representative flavonoids including flavonoid, flavonols, chalcones, and isoflavones, and related them to the health-promoting effects of vegetables. Finally, we examined the relevance of the flavonoids to antioxidant capacity. Both bean sprouts and hot pepper possessed higher antioxidant enzyme activity, which were responsible for their great antioxidant capacity. Our study established a database of major flavonoids components in vegetables and further provides a new hint for the selection and breeding of vegetables based on their health-promoting effects.

Biotinylated Tn5 transposase-mediated CUT&Tag efficiently profiles transcription factor-DNA interactions in plants.

In contrast to CUT&Tag approaches for profiling bulk histone modifications, current CUT&Tag methods for analysing specific transcription factor (TF)-DNA interactions remain technically challenging due to TFs having relatively low abundance. Moreover, an efficient CUT&Tag strategy for plant TFs is not yet available. Here, we first applied biotinylated Tn5 transposase-mediated CUT&Tag (B-CUT&Tag) to produce high-quality libraries for interrogating TF-DNA interactions. B-CUT&Tag combines streptavidin-biotin-based DNA purification with routine CUT&Tag, optimizing the removal of large amounts of intact chromatin not targeted by specific TFs. The biotinylated chromatin fragments are then purified for construction of deep sequencing libraries or qPCR analysis. We applied B-CUT&Tag to probe genome-wide DNA targets of Squamosa promoter-binding-like protein 9 (SPL9), a well-established TF in Arabidopsis; the resulting profiles were efficient and consistent in demonstrating its well-established target genes in juvenile-adult transition/flowering, trichome development, flavonoid biosynthesis, wax synthesis and branching. Interestingly, our results indicate functions of AtSPL9 in modulating growth-defence trade-offs. In addition, we established a method for applying qPCR after CUT&Tag (B-CUT&Tag-qPCR) and successfully validated the binding of SPL9 in Arabidopsis and PHR2 in rice. Our study thus provides a convenient and highly efficient CUT&Tag strategy for profiling TF-chromatin interactions that is widely applicable to the annotation of cis-regulatory elements for crop improvement.

Comprehensive Genomic Analysis of Trihelix Family in Tea Plant (Camellia sinensis) and Their Putative Roles in Osmotic Stress.

In plants, Trihelix transcription factors are responsible for regulating growth, development, and reaction to various abiotic stresses. However, their functions in tea plants are not yet fully understood. This study identified a total of 40 complete Trihelix genes in the tea plant genome, which are classified into five clades: GT-1 (5 genes), GT-2 (8 genes), GTγ (2 genes), SH4 (7 genes), and SIP1 (18 genes). The same subfamily exhibits similar gene structures and functional domains. Chromosomal mapping analysis revealed that chromosome 2 has the most significant number of trihelix family members. Promoter analysis identified cis-acting elements in C. sinensis trihelix (CsTH), indicating their potential to respond to various phytohormones and stresses. The expression analysis of eight representative CsTH genes from four subfamilies showed that all CsTHs were expressed in more tissues, and three CsTHs were significantly induced under ABA, NaCl, and drought stress. This suggests that CsTHs plays an essential role in tea plant growth, development, and response to osmotic stress. Furthermore, yeast strains have preliminarily proven that CsTH28, CsTH36, and CsTH39 can confer salt and drought tolerance. Our study provides insights into the phylogenetic relationships and functions of the trihelix transcription factors in tea plants. It also presents new candidate genes for stress-tolerance breeding.

Genome-wide characterization of NAC transcription factors in Camellia sinensis and the involvement of CsNAC28 in drought tolerance.

The NAM, ATAF1/2, and CUC2 (NAC) transcription factors, which are members of a plant-specific gene family, play critical roles during the growth and development of plants and in their adaption to environmental stress. Few NAC transcription factors have been functionally characterized in tea plants (Camellia sinensis). Based on the analysis of the gene structure, motif pattern, and evolutionary relationship, we identified 104 NAC genes in C. sinensis. Among them, CsNAC28 is constitutively expressed in all organs, and most significantly, exhibiting remarkable responsiveness to abscisic acid (ABA) treatment and drought stress. ABA is a primary stress-related hormone. Recently, ABA-responsive element binding factor 2 (CsABF2) was identified in the ABA pathway of C. sinensis. However, the involvement of the CsABF2-mediated ABA pathway in regulating CsNACs was not known. Herein, a series of biochemical and genetic approaches supported the fact that CsNAC28 could potentially act as a transcription factor in the downstream of CsABF2. Furthermore, we investigated the function of CsNAC28 in the adapting of a plant to drought stress. The results showed that overexpression of CsNAC28 in Arabidopsis conferred hypersensitivity to ABA treatment and decreased the accumulation of reactive oxygen species (ROS), resulting in improved dehydration tolerance. Under conditions of drought, the expression levels of ABA pathway-related genes and drought stress‒inducible genes were greater in CsNAC28 overexpression lines than in the wild type. Our study's comprehensive characterization of NAC genes in C. sinensis could serve as a foundation for exploring the molecular mechanism of CsNAC-mediated drought responsiveness.

Flavonoids in vegetables: improvement of dietary flavonoids by metabolic engineering to promote health.

Flavonoids are the most abundant polyphenols in plants, and have antioxidant effects as well as other bioactivities (e.g., anti-inflammatory, anti-cancer, anti-allergic, and neuroprotective effects). Vegetables are rich in flavonoids and are indispensable in our daily diet. Moreover, the vegetables as chassis for producing natural products would emerge as a promising means for cost-effective and sustainable production of flavonoids. Understanding the metabolic engineering of flavonoids in vegetables allows us to improve their nutrient composition. In this review, a comprehensive overview of flavonoids in vegetables, including the characterized types and distribution, health-promoting effects, associated metabolic pathways, and applied metabolic engineering are provided. We also introduce breakthroughs in multi-omics approaches that pertain to the elucidation of flavonoids metabolism in vegetables, as well as prospective and potential genome-editing technologies. Based on the varied composition and content of flavonoids among vegetables, dietary suggestions are further provided for human health.

Metabolomics Reveals Nutritional Diversity among Six Coarse Cereals and Antioxidant Activity Analysis of Grain Sorghum and Sweet Sorghum.

Coarse cereals are rich in dietary fiber, B vitamins, minerals, secondary metabolites, and other bioactive components, which exert numerous health benefits. To better understand the diversity of metabolites in different coarse cereals, we performed widely targeted metabolic profiling analyses of six popular coarse cereals, millet, coix, buckwheat, quinoa, oat, and grain sorghum, of which 768 metabolites are identified. Moreover, quinoa and buckwheat showed significantly different metabolomic profiles compared with other coarse cereals. Analysis of the accumulation patterns of common nutritional metabolites among six coarse cereals, we found that the accumulation of carbohydrates follows a conserved pattern in the six coarse cereals, while those of amino acids, vitamins, flavonoids, and lipids were complementary. Furthermore, the species-specific metabolites in each coarse cereal were identified, and the neighbor-joining tree for the six coarse cereals was constructed based on the metabolome data. Since sorghum contains more species-specific metabolites and occupies a unique position on the neighbor-joining tree, the metabolite differences between grain sorghum 654 and sweet sorghum LTR108 were finally compared specifically, revealing that LTR108 contained more flavonoids and had higher antioxidant activity than 654. Our work supports an overview understanding of nutrient value in different coarse cereals, which provides the metabolomic evidence for the healthy diet. Additionally, the superior antioxidant activity of sweet sorghum provides clues for its targeted uses.

Involvement of PtPHR1 in phosphates starvation-induced alkaloid biosynthesis in Pinellia ternata (Thunb.) Breit.

Nowadays, because of the great benefit to human health, more and more efforts have been made to increase the production of alkaloids in Pinellia ternata (Thunb.) Breit. Phosphate (Pi) plays a critical role in plant growth and development, as well as secondary metabolism. However, its effect and regulation mechanism of Pi signaling on alkaloid biosynthesis call for further exploration. Here, we reported that Pi starvation could induce alkaloid accumulation in P. ternata. We cloned a cDNA sequence encoding PtPHR1 from P. ternata, which was further identified by nuclear localization, transcription activity, and binding ability to the PHR1-binding sequence. We found that the transformation of PtPHR1 into the Arabidopsis phr1 mutant (designated as PtPHR1OE/phr1) led to the rescue of the phenotype of the phr1 mutant to that of the wild-type, including the expression level of Pi starvation-induced genes and anthocyanin accumulation. The combination of these biochemical and genetic experiments indicated that PtPHR1 was intended to have a role similar to that of AtPHR1 in Pi signaling and metabolic responses. Interestingly, we found that Pi starvation also induced the production of benzoic acid, an intermediate in the biosynthetic pathway of phenylpropylamino alkaloids. Furthermore, this induction effect was impaired in the phr1 mutant but partly recovered in PtPHR1OE/phr1 plants. Together, our data suggest that Pi starvation promoted benzoic acid-derived alkaloid biosynthesis in P. ternata under the control of PtPHR1. Our finding that PtPHR1 is involved in the regulation of Pi signaling on alkaloid biosynthesis shows a direct link between the Pi nutrient supply and secondary metabolism.