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Summary Flavanones and flavones are excellent source of bioactive compounds but the molecular basis of their highly efficient production remains elusive. Chalcone isomerase (CHI) family proteins play essential roles in flavonoid biosynthesis but little are known about the transcription factors controlling their gene expression. Here, we identified a type IV CHI (designated as CitCHIL1) from citrus which enhances the accumulation of citrus flavanones and flavones (CFLs). CitCHIL1 participates in a CFL biosynthetic metabolon and assists the cyclization of naringenin chalcone to (2S)‐naringenin, which leads to the efficient influx of substrates to chalcone synthase (CHS) and improves the catalytic efficiency of CHS. Overexpressing CitCHIL1 in Citrus and Arabidopsis significantly increased flavonoid content and RNA interference‐induced silencing of CitCHIL1 in citrus led to a 43% reduction in CFL content. Three AP2/ERF transcription factors were identified as positive regulators of the CitCHIL1 expression. Of these, two dehydration‐responsive element binding (DREB) proteins, CitERF32 and CitERF33, activated the transcription by directly binding to the CGCCGC motif in the promoter, while CitRAV1 (RAV: related to ABI3/VP1) formed a transcription complex with CitERF33 that strongly enhanced the activation efficiency and flavonoid accumulation. These results not only illustrate the specific function that CitCHIL1 executes in CFL biosynthesis but also reveal a new DREB‐RAV transcriptional complex regulating flavonoid production.

Xanthohumol (XN) and demethylxanthohumol (DMX) are specialized prenylated chalconoids with multiple pharmaceutical applications that accumulate to high levels in the glandular trichomes of hops (Humulus lupulus L.). Although all structural enzymes in the XN pathway have been functionally identified, biochemical mechanisms underlying highly efficient production of XN have not been fully resolved. In this study, we characterized two noncatalytic chalcone isomerase (CHI)-like proteins (designated as HlCHIL1 and HlCHIL2) using engineered yeast harboring all genes required for DMX production. HlCHIL2 increased DMX production by 2.3-fold, whereas HlCHIL1 significantly decreased DMX production by 30%. We show that CHIL2 is part of an active DMX biosynthetic metabolon in hop glandular trichomes that encompasses a chalcone synthase (CHS) and a membrane-bound prenyltransferase, and that type IV CHI-fold proteins of representative land plants contain conserved function to bind with CHS and enhance its activity. Binding assays and structural docking uncover a function of HlCHIL1 to bind DMX and naringenin chalcone to stabilize the ring-open configuration of these chalconoids. This study reveals the role of two HlCHILs in DMX biosynthesis in hops, and provides insight into their evolutionary development from the ancestral fatty acid-binding CHI-fold proteins to specialized auxiliary proteins supporting flavonoid biosynthesis in plants.

The chalcone isomerase gene OsCHI, one of the key genes in the flavonoid biosynthesis pathway, plays an important role in rice (Oryza sativa) resistance to abiotic stresses. This study reveals how the chalcone isomerase gene family member OsCHI3 participates in rice responses to drought stress through the regulation of flavonoid biosynthesis. Overexpression of OsCHI3 increased the tolerance of rice to drought stress. In contrast, CRISPR/Cas9-mediated deletion of OsCHI3 reduced the drought tolerance of rice, an effect that is reversed by exogenous ABA treatment. Transcriptomic and physiological biochemical analyses indicated that flavonoids regulated by OsCHI3 not only scavenge reactive oxygen species (ROS) but also increase drought tolerance in rice by stimulating ABA biosynthesis through the regulation of OsNCED1 and OsABA8ox3 expression. These findings demonstrate that OsCHI3 increases drought stress tolerance in rice by activating the antioxidant defense system and the ABA metabolic pathway, providing new clues for drought-resistant rice breeding research.

In addition to causing oxidative stress, NaCl can act as an inexpensive elicitor to trigger the biosynthesis of secondary metabolites like flavonoids, an important group of therapeutic compounds found in the Haplophyllum genus. Haplophyllum virgatum is an Iranian endemic plant from this genus. So far, the elicitation effect of NaCl on this plant has not been investigated. Cell suspension culture, along with elicitor application, is an appropriate technique to obtain large amounts of plant-produced compounds. Here, the oxidative stress responses were investigated in suspension cells of H. virgatum var. virgatum in the presence of NaCl (0, 100, 150, and 200 mM) at various time courses (0, 8, 12, 24, 48, 72, and 168 h after treatments). Besides, the expression levels of two critical enzymes in the biosynthetic pathway of flavonoids (chalcone synthase and chalcone isomerase) and the R2R3-MYB transcription factor were measured. The accumulation of rutin (the main flavonoid in Rutaceae) in the NaCl-treated cells was also evaluated by high-performance liquid chromatography (HPLC). The obtained results have demonstrated significant enhancements in the oxidative stress indices (malondialdehyde, proline, hydrogen peroxide, and enzymatic and non-enzymatic antioxidants) and the rutin contents in the studied suspension cells. In addition, positive correlations were deduced between the expression levels of mentioned genes and the rutin contents. These findings highlight the possible regulatory role of R2R3-MYB transcription factors in flavonoid biosynthesis in H. virgatum and the role of these secondary metabolites in responses to NaCl. The obtained results also suggest that NaCl is a suitable elicitor for flavonoid production in the cell suspension culture of the plant.Key messageNaCl elicited the accumulation of flavonoids in suspension cells of H. virgatum, probably via upregulation of R2R3-MYB, which then upregulated flavonoid biosynthetic genes, CHS and CHI.

Flavonoids as a class of important secondary metabolites are widely present in land plants, and chalcone isomerase (CHI) is the key rate-limiting enzyme that participates in catalyzing the stereospecific isomerization of chalcones to yield their corresponding flavanones. However, the phylogenetic dynamics and functional divergence of CHI family genes during the evolutionary path of green plants remains poorly understood. Here, a total of 122 CHI genes were identified by performing a genome-wide survey of 15 representative green plants from the most ancestral basal plant chlorophyte algae to higher angiosperm plants. Phylogenetic, orthologous groups (OG) classification, and genome structure analysis showed that the CHI family genes have evolved into four distinct types (types I–IV) containing eight OGs after gene duplication, and further studies indicated type III CHIs consist of three subfamilies (FAP1, FAP2, and FAP3). The phylogeny showed FAP3 CHIs as an ancestral out-group positioned on the outer layers of the main branch, followed by type IV CHIs, which are placed in an evolutionary intermediate between FAP3 CHIs and bona fide CHIs (including type I and type II). The results imply a potential intrinsic evolutionary connection between CHIs existing in the green plants. The amino acid substitutions occurring in several residues have potentially affected the functional divergence between CHI proteins. This is supported by the analysis of transcriptional divergence and cis-acting element analysis. Evolutionary dynamics analyses revealed that the differences in the total number of CHI family genes in each plant are primarily attributed to the lineage-specific expansion by natural selective forces. The current studies provide a deeper understanding of the phylogenetic relationships and functional diversification of CHI family genes in green plants, which will guide further investigation on molecular characteristics and biological functions of CHIs.

Astragalus sinicus is an important winter-growing cover crop. It is widely utilized, not only as a cover crop for its benefits in fertilizing the soil but also as a landscape ground cover plant. Anthocyanins are involved in the pigmentation of plants in leaves and flowers, which is a crucial characteristic trait for A. sinicus. The formation of anthocyanins depends significantly on the enzyme chalcone isomerase (CHI). However, research on the CHI gene of A. sinicus remains unexplored. The rapid amplification of cDNA ends (RACE) approach was used in this research to clone the CHI sequence from A. sinicus (AsiCHI). The expression profiles of the AsiCHI gene in multiple tissues of A. sinicus were subsequently examined by qRT-PCR (Quantitative Real-Time PCR). Furthermore, the function of the AsiCHI was identified by the performance of ectopic expression in Arabidopsis (Arabidopsis thaliana). The outcomes revealed that the full-length cDNA of the AsiCHI gene (GeneBank: OQ870547) measured 972 bp in length and included an open reading frame of 660 bp. The encoded protein contains 219 amino acids with a molecular weight of 24.14 kDa and a theoretical isoelectric point of 5.11. In addition, the remarkable similarity between the AsiCHI protein and the CHI proteins of other Astragalus species was demonstrated by the sequence alignment and phylogenetic analysis. Moreover, the highest expression level of AsiCHI was observed in leaves and showed a positive correlation with anthocyanin content. The functional analysis further revealed that the overexpression of AsiCHI enhanced the anthocyanidin accumulation in the transgenic lines. This study provided a better understanding of AsiCHI and elucidated its role in anthocyanin production.

Anthocyanins are colorful pigments known for contributing antioxidant effects to the human diet which provide health benefits that protect against several forms of cancer and vascular disease. Unfortunately, tomatoes found in nature have very low anthocyanin content. Anthocyanin-rich purple tomatoes by the ectopic co-expression of two transcription factors Delila (Del) and Rosea1 (Ros1) from the snapdragon Antirrhinum majus have been generated. However, the Del/Ros1 (DR)-expressing tomatoes cannot sufficiently upregulate all necessary key endogenous genes, particularly chalcone isomerase (CHI), for full utilization of the anthocyanin production pathway. In this study, CHI from onion Allium cepa was introduced into DR-expressing tomatoes for a further increase of anthocyanin levels in both the peel and the flesh of tomatoes. We achieved up to 400- and 260-fold increases in the levels of anthocyanins in tomato peel and flesh, respectively, in CHI/DR transgenics compared with 100- and 80-fold increases in tomato peel and flesh in the DR-only-expressing lines. Furthermore, CHI/DR-expressing tomatoes increased up to 200-fold more total flavonol content in flesh compared with the wild-type tomatoes. In summary, stacking CHI with DR could significantly increase the levels of anthocyanins and flavonols in tomato fruit.

Flavonoid is one of the widespread groups of plant secondary metabolites that provide several health benefits. However, the explicit mechanism of flavonoid biosynthesis in plants largely remains unclear. Chalcone isomerase an important class of enzyme presents crucial role during flavonoid metabolism in many plants. Here, we isolated the full-length cDNA (1161 bp) of a novel Chalcone Isomerase from safflower encoding 217 amino acid polypeptide using oligos from 5′ and 3′ ends. The result of Sanger sequencing and phylogenetic analysis revealed that CtCHI is highly homologous to other plants, including typical polyadenylation signals AATAA and Poly A tail. The transient expression in tobacco mesophyll cells using Green Fluorescent Protein tagging determined the subcellular localization of CtCHI in cell membrane and nucleus. The CtCHI ectopic expression in different safflower varieties at different flowering stages showed that CtCHI were found in abundance at the bud stage of Jihong No. 1. Further correlation analysis between CtCHI expression and flavonoid accumulation at various flowering phases suggested that CtCHI might play a potential role during flavonoid biosynthesis in safflower. In addition, the overexpression of pBASTA-CtCHI in transgenic Arabidopsis infiltrated with floral dip transformation showed relatively higher expression level and increased flavonoid accumulation than wild type. Moreover, the in vitro enzymatic activity and HPLC analysis of transgenic Arabidopsis confirmed the de novo biosynthesis of Rutin. Taken together, our findings laid the foundation of identifying an important gene that might influence flavonoid metabolism in safflower.

Economically important compounds, such as licorice flavonoids, are present in insufficient amounts in the hairy roots. To overcome this problem, we took the transgenic approach combined with the elicitation technique to increase the flavonoid production. The Glycyrrhiza uralensis Fisch cDNA encoding chalcone isomerase gene (chi) was over-expressed in hairy roots of G. uralensis Fisch mediated by the disarmed Agrobacterium rhizogenes A4. Stable genetic transformation was confirmed by Southern blot analysis. The transgenic and wild cultures were subsequently elicited with PEG8000 (2%) alone, yeast extract (YE) (0.1%) alone, or both of them, and then the total flavonoids were extracted and measured. The results showed that over a culture period of 3 weeks, the wild-type hairy roots, the untreated transgenic hairy roots, and the double-treated transgenic hairy roots accumulated 0.842, 1.394, and 2.838 (g/100 g DW) of total flavonoids, respectively. Moreover, the enhanced accumulation of flavonoids were correlated with the elevated level of chi transcripts and CHI activity, confirming the key role of chi in the flavonoids synthesis. This research demonstrated that the combination of the metabolic engineering and PEG8000-YE elicitation treatment was an effective strategy to increase the flavonoids production in hairy roots of G. uralensis Fisch.

Flavonoids are a diverse group of phenolic secondary metabolites that occur naturally in plants and therefore form an integral component of the human diet. Many of the compounds belonging to this group are potent antioxidants in vitro and epidemiological studies suggest a direct correlation between high flavonoid intake and decreased risk of cardiovascular disease, cancer and other age‐related diseases. Enhancing flavonoid biosynthesis in chosen crops may provide new raw materials that have the potential to be used in foods designed for specific benefits to human health. Using genetic modification, it was possible to generate several tomato lines with significantly altered flavonoid content and to probe the role and importance of several key enzymatic steps in the tomato flavonoid biosynthetic pathway. Most notably an up to 78‐fold increase in total fruit flavonols was achieved through ectopic expression of a single biosynthetic enzyme, chalcone isomerase. In addition, chalcone synthase and flavonol synthase transgenes were found to act synergistically to up‐regulate flavonol biosynthesis significantly in tomato flesh tissues.