Expression Of Flavonoid Biosynthetic Genes Research Articles

Integrated Metabolome and Transcriptome Analyses Reveal the Mechanisms Regulating Flavonoid Biosynthesis in Blueberry Leaves under Salt Stress

The flavonoids play important roles in plant salt tolerance. Blueberries (Vaccinium spp.) are extremely sensitive to soil salt increases. Therefore, improving the salt resistance of blueberries by increasing the flavonoid content is crucial for the development of the blueberry industry. To explore the underlying molecular mechanism, we performed an integrated analysis of the metabolome and transcriptome of blueberry leaves under salt stress. We identified 525 differentially accumulated metabolites (DAMs) under salt stress vs. control treatment, primarily including members of the flavonoid class. We also identified 20,920 differentially expressed genes (DEGs) based on transcriptome data; of these, 568 differentially expressed transcription factors (TFs) were annotated, and bHLH123, OsHSP20, and HSP20 TFs might be responsible for blueberry leaf salt tolerance. DEGs involved in the flavonoid biosynthesis pathway were significantly enriched at almost all stages of salt stress. Salt treatment upregulated the expression of most flavonoid biosynthetic pathway genes and promoted the accumulation of flavonols, flavonol glycosides, flavans, proanthocyanidins, and anthocyanins. Correlation analysis suggested that 4-coumarate CoA ligases (4CL5 and 4CL1) play important roles in the accumulation of flavonols (quercetin and pinoquercetin) and flavan-3-ol (epicatechin and prodelphinidin C2) under salt stress, respectively. The flavonoid 3′5′-hydroxylases (F3′5′H) regulate anthocyanin (cyanidin 3-O-beta-D-sambubioside and delphinidin-3-O-glucoside chloride) biosynthesis, and leucoanthocyanidin reductases (LAR) are crucial for the biosynthesis of epicatechin and prodelphinidin C2 during salt stress. Taken together, it is one of the future breeding goals to cultivate salt-resistant blueberry varieties by increasing the expression of flavonoid biosynthetic genes, especially 4CL, F3′5′H, and LAR genes, to promote flavonoid content in blueberry leaves.

Unveiling the protective role of anthocyanin in rice: insights into drought-induced oxidative stress and metabolic regulation.

This study investigates the impact of anthocyanin treatment on rice plants under drought stress, focusing on phenotypic, molecular, and biochemical responses. Anthocyanin were treated to one month old plants one week before the droughtexposure. Drought stress was imposed by using 10% polyethylene glycol (PEG 6000). Anthocyanin-treated plants exhibited significant enhancements in various traits, including growth parameters and reproductive characteristics, under normal conditions. When subjected to drought stress, these plants displayed resilience, maintaining or improving essential morphological and physiological features compared to non-treated counterparts. Notably, anthocyanin application mitigated drought-induced oxidative stress, as evidenced by reduced levels of reactive oxygen species (ROS) and lipid membrane peroxidation. The study also elucidates the regulatory role of anthocyanins in the expression of flavonoid biosynthetic genes, leading to increased levels of key secondary metabolites. Furthermore, anthocyanin treatment influenced the levels of stress-related signaling molecules, including melatonin, proline, abscisic acid (ABA), and salicylic acid (SA), contributing to enhanced stress tolerance. The enzymatic activity of antioxidants and the expression of drought-responsive genes were modulated by anthocyanins, emphasizing their role in antioxidant defense and stress response. Additionally, anthocyanin treatment positively influenced macronutrient concentrations, particularly calcium ion (Ca+), potassium ion (K+), and sodium ion (Na+), essential for cell wall and membrane stability. The findings collectively highlight the multifaceted protective effects of anthocyanins, positioning them as potential key players in conferring resilience to drought stress in rice plants. The study provides valuable insights into the molecular and physiological mechanisms underlying anthocyanin-mediated enhancement of drought stress tolerance, suggesting promising applications in agricultural practices for sustainable crop production.

Arabidopsis transcription factor TCP13 promotes shade avoidance syndrome-like responses by directly targeting a subset of shade-responsive gene promoters.

TCP13 belongs to a subgroup of TCP transcription factors implicated in the shade avoidance syndrome (SAS), but its exact role remains unclear. Here, we show that TCP13 promotes the SAS-like response by enhancing hypocotyl elongation and suppressing flavonoid biosynthesis as a part of the incoherent feed-forward loop in light signaling. Shade is known to promote the SAS by activating PHYTOCHROME-INTERACTING FACTOR (PIF)-auxin signaling in plants, but we found no evidence in a transcriptome analysis that TCP13 activates PIF-auxin signaling. Instead, TCP13 mimics shade by activating the expression of a subset of shade-inducible and cell elongation-promoting SAUR genes including SAUR19, by direct targeting of their promoters. We also found that TCP13 and PIF4, a molecular proxy for shade, repress the expression of flavonoid biosynthetic genes by directly targeting both shared and distinct sets of biosynthetic gene promoters. Together, our results indicate that TCP13 promotes the SAS-like response by directly targeting a subset of shade-responsive genes without activating the PIF-auxin signaling pathway.

Roles and Preliminary Mechanism of Tobacco cis-Abienol in Inducing Tomato Resistance against Bacterial Wilt.

Bacterial wilt negatively impacts the yield and quality of tomatoes. cis-Abienol, a labdane diterpenoid abundantly produced in the trichome secretion of Nicotiana spp., can induce bacterial wilt resistance in plants; however, study on its practical application and acting mechanism is very limited. This study established the application conditions of cis-abienol for inducing tomato bacterial wilt resistance by pot-inoculation experiments and investigated the underlying mechanism by determining the physio-biochemical indexes and transcriptomic changes. The results showed that applying cis-abienol to the roots was the most effective approach for inducing tomato bacterial wilt resistance. The optimal concentration was 60 μg/mL, and 2-3 consecutive applications with 3-6 days intervals were sufficient to induce the bacterial wilt resistance of tomato plants. cis-Abienol could enhance the antioxidant enzyme activity and stimulate the defensive signal transduction in tomato roots, leading to the upregulation of genes involved in the mitogen-activated protein kinase cascade. It also upregulated the expression of JAZ genes and increased the content of jasmonic acid (JA) and salicylic acid (SA), which control the expression of flavonoid biosynthetic genes and the content of phytoalexins in tomato roots. cis-Abienol-induced resistance mainly depends on the JA signalling pathway, and the SA signalling pathway is also involved in this process. This study established the feasibility of applying the plant-derived terpenoid cis-abienol to induce plant bacterial wilt resistance, which is of great value for developing eco-friendly bactericides.

UV-B induces the expression of flavonoid biosynthetic pathways in blueberry (Vaccinium corymbosum) calli.

Ultraviolet-B (UV-B) radiation is an environmental signal that affects the accumulation of secondary metabolites in plants. In particular, UV-B promotes flavonoid biosynthesis, leading to improved fruit quality. To explore the underlying molecular mechanism, we exposed blueberry (Vaccinium corymbosum) calli to UV-B radiation and performed a transcriptome deep sequencing (RNA-seq) analysis to identify differentially expressed genes (DEGs). We detected 16,899 DEGs among different treatments, with the largest number seen after 24 h of UV-B exposure relative to controls. Functional annotation and enrichment analysis showed a significant enrichment for DEGs in pathways related to plant hormone signal transduction and phenylpropanoid and flavonoid biosynthesis. In agreement with the transcriptome data, flavonol, anthocyanin and proanthocyanidin accumulated upon UV-B radiation, and most DEGs mapping to the phenylpropanoid and flavonoid biosynthetic pathways using the KEGG mapper tool were upregulated under UV-B radiation. We also performed a weighted gene co-expression network analysis (WGCNA) to explore the relationship among genes involved in plant hormone signal transduction, encoding transcription factors or participating in flavonoid biosynthesis. The transcription factors VcMYBPA1, MYBPA2.1, MYB114, MYBA2, MYBF, and MYB102 are likely activators, whereas MYB20, VcMYB14, MYB44, and VcMYB4a are inhibitors of the flavonoid biosynthetic pathway, as evidenced by the direction of correlation between the expression of these MYBs and flavonoid biosynthesis-related genes. The transcription factors bHLH74 and bHLH25 might interact with MYB repressors or directly inhibited the expression of flavonoid biosynthetic genes to control flavonoid accumulation. We also observed the downregulation of several genes belonging to the auxin, gibberellin and brassinosteroid biosynthetic pathways, suggesting that MYB inhibitors or activators are directly or indirectly regulated to promote flavonoid biosynthesis under UV-B radiation.

Overexpression of dihydroflavonol 4-reductase (CoDFR) boosts flavonoid production involved in the anthracnose resistance.

The outbreak of anthracnose caused by Colletotrichum spp. represents a devastating epidemic that severely affects oil tea (Camellia oleifera) production in China. However, the unknown resistance mechanism to anthracnose in C. oleifera has impeded the progress of breeding disease-resistant varieties. In this study, we investigated the physiological responses of resistant and susceptible lines during C. gloeosporioides infection. Our results showed that the accumulation of malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) in both disease-resistant and susceptible lines increased by C. gloeosporioides infection. Also, disease-resistant lines exhibited lower MDA, but higher POD, SOD, and CAT activities compared to susceptible lines. The accumulation of flavonoids in both resistant and susceptible C. oleifera leaves increased following C. gloeosporioides infection, and the increase was greater in resistant lines. Further, we identified and functionally characterized the dihydroflavonol 4-reductase (CoDFR) from the resistant C. oleifera line. We showed that the full-length coding sequence (CDS) of CoDFR is 1044 bp encoding 347 amino acids. The overexpression of CoDFR in tobacco altered the expression of flavonoid biosynthetic genes, resulting in an increased flavonoid content in leaves. CoDFR transgenic tobacco plants exhibited increased anthracnose resistance. Furthermore, the transgenic plants had higher salicylic acid content. These findings offer potential insights into the pivotal role of CoDFR involved in flavonoid-mediated defense mechanisms during anthracnose invasion in resistant C. oleifera.

Photoperiod and water-deficient conditions differentially regulate structural flavonoid biosynthetic genes in peanuts

ABSTRACT Peanut (Arachis hypogaea L.) harbors a plethora of flavonoids that impart its health-promoting properties. To understand its biosynthesis and accumulation, a comparative expression analysis of flavonoid biosynthetic genes encoding enzymes responsible for synthesizing the main structure of flavonoids has been conducted in two differentially performing peanut varieties (PG 1247 and Bari 2011) in response to photoperiod and water availability. Transcript profiling of flavonoid biosynthetic genes indicated time-dependent expression of genes except for AhFLS in selected varieties in response to photoperiod variation. Many genes depicted a low degree of variation within varieties and water-deficient treatments. However, AhANS showed the highest variation for stress duration and varieties. In conclusion, this study has characterized the role of flavonoid biosynthesis in peanuts in response to photoperiod and water availability. Moreover, correlation analysis unraveled the coordination among the expression of flavonoid biosynthetic genes.

OsSPLs Regulate Male Fertility in Response to Different Temperatures by Flavonoid Biosynthesis and Tapetum PCD in PTGMS Rice.

Photoperiod and thermo-sensitive genic male sterile (PTGMS) rice is an important resource for two line hybrid rice production. The SQUAMOSA–promoter binding, such as the (SPL) gene family, encode the plant specific transcription factors that regulate development and defense responses in plants. However, the reports about SPLs participating in male fertility regulation are limited. Here, we identified 19 OsSPL family members and investigated their involvement in the fertility regulation of the PTGMS rice lines, PA2364S and PA2864S, with different fertility transition temperatures. The results demonstrated that OsSPL2, OsSPL4, OsSPL16 and OsSPL17 affect male fertility in response to temperature changes through the MiR156-SPL module. WGCNA (weighted gene co-expression network analysis) revealed that CHI and APX1 were co-expressed with OsSPL17. Targeted metabolite and flavonoid biosynthetic gene expression analysis revealed that OsSPL17 regulates the expression of flavonoid biosynthesis genes CHI, and the up regulation of flavanones (eriodictvol and naringenin) and flavones (apigenin and luteolin) content contributed to plant fertility. Meanwhile, OsSPL17 negatively regulates APX1 to affect APX (ascorbate peroxidase) activity, thereby regulating ROS (reactive oxygen species) content in the tapetum, controlling the PCD (programmed cell death) process and regulating male fertility in rice. Overall, this report highlights the potential role of OsSPL for the regulation of male fertility in rice and provides a new insight for the further understanding of fertility molecular mechanisms in PTGMS rice.

Transcriptome Analysis of MYB Genes and Patterns of Anthocyanin Accumulation During Seed Development in Wheat.

Plants accumulate key metabolites as a response of biotic/abiotic stress conditions. In seed coats, anthocyanins, carotenoids, and chlorophylls can be found. They have been associated as important antioxidants that affect germination. In wheat, anthocyanins can impart the seed coat color which have been recognized as health-promoting nutrients. Transcription factors act as master regulators of cellular processes. Transcription complexes such as MYB-bHLH-WD40 (MBW) regulate the expression of multiple target genes in various plant species. In this study, the spatiotemporal accumulation of seed coat pigments in different developmental stages (10, 20, 30, and 40 days after pollination) was analyzed using cryo-cuts. Moreover, the accumulation of phenolic, anthocyanin, and chlorophyll contents was quantified, and the expression of flavonoid biosynthetic genes was evaluated. Finally, transcriptome analysis was performed to analyze putative MYB genes related to seed coat color, followed by further characterization of putative genes. TaTCL2, an MYB gene, was cloned and sequenced. It was determined that TaTCL2 contains a SANT domain, which is often present in proteins participating in the response to anthocyanin accumulation. Moreover, TaTCL2 transcript levels were shown to be influenced by anthocyanin accumulation during grain development. Interaction network analysis showed interactions with GL2 (HD-ZIP IV), EGL3 (bHLH), and TTG1 (WD40). The findings of this study elucidate the mechanisms underlying color formation in Triticum aestivum L. seed coats.

Meta-Analysis of the Effect of Overexpression of MYB Transcription Factors on the Regulatory Mechanisms of Anthocyanin Biosynthesis.

MYBs (v-myb avian myeloblastosis viral oncogene homologs) are important transcriptional regulators that play critical roles in the regulation of anthocyanin biosynthesis. The overexpression of MYB genes has been reported in different plant species. However, the inconsistent strategies to assess transgenic plants have made it difficult to explain the complex mechanisms of regulation of anthocyanin biosynthesis by MYBs. We report here a meta-analysis of 608 studies from 206 publications assessing the effects of MYB overexpression on anthocyanins and evaluate the experimental variables that have an influence on transgenic plant performance. We found that MYB expression enhanced the magnitude of 20 out of 26 examined plant parameters by at least of 21% and reduced the magnitude of 1 indicator by at least 37%. We explored the variety of moderating variables causing these variations. A deeper color induced by MYBs caused higher plant attributes as compared to normal color changes. MYB genes from dicots stimulated the accumulation of anthocyanins, flavonols and impacted the expressions of PAL, CHS, CHI, FLS, F3′5′H, ANS, UFGT, and ANR as compared to monocots. Heterologous expression and homologous expression showed a great difference in anthocyanin biosynthesis. Transient gene transformation had a significant effect on the expression of flavonoid biosynthetic genes, and stable transformation had a significant effect on flavonoid accumulation. Stress could result in a significantly increased accumulation of flavonoids, especially anthocyanin, flavonol, and proanthocyanidin. Our study, thus, provides new insights into the function of MYBs in the regulatory mechanisms of flavonoid biosynthesis and the use of genetic engineering for improving anthocyanins contents.

Transcriptional regulation of flavonoid biosynthesis in Artemisia annua by AaYABBY5

Artemisia annua is a medicinal plant rich in terpenes and flavonoids with useful biological activities such as antioxidant, anticancer, and antimalarial activities. The transcriptional regulation of flavonoid biosynthesis in A. annua has not been well-studied. In this study, we identified a YABBY family transcription factor, AaYABBY5, as a positive regulator of anthocyanin and total flavonoid contents in A. annua. AaYABBY5 was selected based on its similar expression pattern to the phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and flavonol synthase (FLS) genes. A transient dual-luciferase assay in Nicotiana bethamiana with the AaYABBY5 effector showed a significant increase in the activity of the downstream LUC gene, with reporters AaPAL, AaCHS, AaCHI, and AaUFGT. The yeast one-hybrid system further confirmed the direct activation of these promoters by AaYABBY5. Gene expression analysis of stably transformed AaYABBY5 overexpression, AaYABBY5 antisense, and control plants revealed a significant increase in the expression of AaPAL, AaCHS, AaCHI, AaFLS, AaFSII, AaLDOX, and AaUFGT in AaYABBY5 overexpression plants. Moreover, their total flavonoid content and anthocyanin content were also found to increase. AaYABBY5 antisense plants showed a significant decrease in the expression of flavonoid biosynthetic genes, as well as a decrease in anthocyanin and total flavonoid contents. In addition, phenotypic analysis revealed deep purple-pigmented stems, an increase in the leaf lamina size, and higher trichome densities in AaYABBY5 overexpression plants. Together, these data proved that AaYABBY5 is a positive regulator of flavonoid biosynthesis in A. annua. Our study provides candidate transcription factors for the improvement of flavonoid concentrations in A. annua and can be further extended to elucidate its mechanism of regulating trichome development.

Exogenous ethanol treatment alleviates oxidative damage of Arabidopsis thaliana under conditions of high-light stress.

Abiotic stresses, such as high light and salinity, are major factors that limit crop productivity and sustainability worldwide. Chemical priming is a promising strategy for improving the abiotic stress tolerance of plants. Recently, we discovered that ethanol enhances high-salinity stress tolerance in Arabidopsis thaliana and rice by detoxifying reactive oxygen species (ROS). However, the effect of ethanol on other abiotic stress responses is unclear. Therefore, we investigated the effect of ethanol on the high-light stress response. Measurement of chlorophyll fluorescence showed that ethanol mitigates photoinhibition under high-light stress. Staining with 3,3'-diaminobenzidine (DAB) showed that the accumulation of hydrogen peroxide (H2O2) was inhibited by ethanol under high-light stress conditions in A. thaliana. We found that ethanol increased the gene expressions and enzymatic activities of antioxidative enzymes, including ASCORBATE PEROXIDASE1 (AtAPX1), Catalase (AtCAT1 and AtCAT2). Moreover, the expression of flavonoid biosynthetic genes and anthocyanin contents were upregulated by ethanol treatment during exposure to high-light stress. These results imply that ethanol alleviates oxidative damage from high-light stress in A. thaliana by suppressing ROS accumulation. Our findings support the hypothesis that ethanol improves tolerance to multiple stresses in field-grown crops.

Metabolome and transcriptome profiling of Theobroma cacao provides insights into the molecular basis of pod color variation.

The Theobroma cacao presents a wide diversity in pod color among different cultivars. Although flavonoid biosynthesis has been studied in many plants, molecular mechanisms governing the diversity of coloration in cacao pods are largely unknown. The flavonoid metabolite profiles and flavonoid biosynthetic gene expression in the pod exocarps of light green pod 'TAS 410' (GW), green pod 'TAS 166' (GF), and mauve pod 'TAS 168' (PF) were determined. Changes in flavonoid metabolites, particularly the anthocyanins (cyanidin 3-O-galactoside, cyanidin 3-O-glucoside, and cyanidin O-syringic acid) were significantly up-accumulated in the mauve phenotype (PF) compared to the light green or green phenotypes, endowing the pod color change from light green or green to mauve. Consistently, the PF phenotype showed different expression patterns of flavonoid biosynthetic structural genes in comparison with GW/GF phenotypes. The expression level of LAR and ANR in GW/GF was significantly higher than PF, while the expression level of UFGT in GW/GF was lower than PF. These genes likely generated more anthocyanins in the exocarps samples of PF than that of GW/GF. Simultaneously, colorless flavan-3-ols (catechin, epicatechin and proanthocyanidin) content in the exocarp samples of PF was lower than GW/GF. Additionally, MYB (gene18079) and bHLH (gene5045 and gene21575) may participate in the regulation of the pod color. This study sheds light on the molecular basis of cacao pod color variation, which will contribute to breeding cacao varieties with enhanced flavonoid profiles for nutritional applications.

Expression Characterization of Flavonoid Biosynthetic Pathway Genes and Transcription Factors in Peanut Under Water Deficit Conditions

Drought is one of the hostile environmental stresses that limit the yield production of crop plants by modulating their growth and development. Peanut (Arachis hypogaea) has a wide range of adaptations to arid and semi-arid climates, but its yield is prone to loss due to drought. Other than beneficial fatty acids and micronutrients, peanut harbors various bioactive compounds including flavonoids that hold a prominent position as antioxidants in plants and protect them from oxidative stress. In this study, understanding of the biosynthesis of flavonoids in peanut under water deficit conditions was developed through expression analysis and correlational analysis and determining the accumulation pattern of phenols, flavonols, and anthocyanins. Six peanut varieties (BARD479, BARI2011, BARI2000, GOLDEN, PG1102, and PG1265) having variable responses against drought stress have been selected. Higher water retention and flavonoid accumulation have been observed in BARI2011 but downregulation has been observed in the expression of genes and transcription factors (TFs) which indicated the maintenance of normal homeostasis. ANOVA revealed that the expression of flavonoid genes and TFs is highly dependent upon the genotype of peanut in a spatiotemporal manner. Correlation analysis between expression of flavonoid biosynthetic genes and TFs indicated the role of AhMYB111 and AhMYB7 as an inhibitor for AhF3H and AhFLS, respectively, and AhMYB7, AhTTG1, and AhCSU2 as a positive regulator for the expression of Ah4CL, AhCHS, and AhF3H, respectively. However, AhbHLH and AhGL3 revealed nil-to-little relation with the expression of flavonoid biosynthetic pathway genes. Correlational analysis between the expression of TFs related to the biosynthesis of flavonoids and the accumulation of phenolics, flavonols, and anthocyanins indicated coregulation of flavonoid synthesis by TFs under water deficit conditions in peanut. This study would provide insight into the role of flavonoid biosynthetic pathway in drought response in peanut and would aid to develop drought-tolerant varieties of peanut.

The pigeon pea CcCIPK14-CcCBL1 pair positively modulates drought tolerance by enhancing flavonoid biosynthesis.

Calcineurin B-like (CBL)-interacting protein kinases (CIPKs) play a central role in Ca2+ signalling and promote drought tolerance in plants. The CIPK gene family in pigeon pea (Cajanus cajan L.), a major food crop affected by drought, has not previously been characterised. Here, we identified 28 CIPK genes in the pigeon pea genome. Five CcCIPK genes were strongly upregulated in roots upon drought treatment and were selected for further characterisation. Overexpression of CcCIPK13 and CcCIPK14 increased survival rates by two- to three-fold relative to controls after 14days of drought. Furthermore, the three major flavonoids, genistin, genistein and apigenin, were significantly upregulated in the same transgenic plants. Using CcCIPK14 as bait, we performed a yeast two-hybrid screen and identified six interactors, including CcCBL1. CcCIPK14 exhibited autophosphorylation and phosphorylation of CcCBL1 in vitro. CcCBL1-overexpressed plants displayed higher survival rates upon drought stress as well as higher expression of flavonoid biosynthetic genes and flavonoid content. CcCIPK14-overexpressed plants in which CcCBL1 transcript levels were reduced by RNA interference had lower survival rates, which indicated CcCBL1 in the same pathway as CcCIPK14. Together, our results demonstrate a role for the CcCIPK14-CcCBL1 complex in drought stress tolerance through the regulation of flavonoid biosynthesis in pigeon pea.

Histone variant H2A.Z and transcriptional activators may antagonistically regulate flavonoid biosynthesis

Flavonoid is an important group of plant secondary metabolites. The biosynthesis of this flavonoid group can be precisely regulated by many environmental factors. Currently, histone variants have been documented as important factors in the regulation of eukaryotic gene expression. H2A.Z histone variant has been found to function in numerous plant physiological programs including flavonoid biosynthesis. Moreover, the environmental changes have been shown to significantly influence the replacement between histone proteins and their variants leading to alterations in gene expression. Based on the recent studies, this mini-review is to provide an updated view on the functions of histone variant H2A.Z in the regulation of flavonoid biosynthetic gene expression. In addition, this also suggests a model in which the H2A.Z-containing nucleosomes can be evicted upon environmental stress conditions to facilitate the targeting of transcriptional activators to these flavonoid biosynthetic genes resulting in gene activation and flavonoid accumulation in Arabidopsis plants.

Application of melatonin promotes anthocyanin accumulation in crabapple leaves

Anthocyanins are a class of compounds that are widespread in plants, where they provide protection against stresses, and are also beneficial to human health as dietary components. Melatonin application is known to affect anthocyanin production, but the relationship between anthocyanin and melatonin is still unclear. In this study, we analyzed anthocyanin contents and the expression levels of anthocyanin biosynthetic and regulatory genes in tissue cultured plantlets of two Malus crabapple cultivars following various exogenous melatonin treatments under light and dark conditions. The application of exogenous melatonin not only promoted anthocyanin accumulation in leaves, but also increased the contents of flavonols and proanthocyanins (PAs), via a process that was not dependent on light. Quantitative real time PCR (qRT-PCR) analyses indicated that the expression of flavonoid biosynthetic genes, flavonoid related transcription factors and melatonin biosynthetic genes was induced by melatonin. We propose that anthocyanin biosynthesis is regulated by melatonin in crabapple leaves via the expression of flavonoid related transcription factors. This study provides insight into the mechanism of melatonin induction of anthocyanin biosynthesis in woody plants, and suggests that pretreatment with melatonin may represent a cultivation strategy to increase the flavonoid contents of plants.

R2R3-MYB transcription factor MYB6 promotes anthocyanin and proanthocyanidin biosynthesis but inhibits secondary cell wall formation in Populus tomentosa.

The secondary cell wall is an important carbon sink in higher plants and its biosynthesis requires coordination of metabolic fluxes in the phenylpropanoid pathway. In Arabidopsis (Arabidopsis thaliana), MYB75 and the KNOX transcription factor KNAT7 form functional complexes to regulate secondary cell wall formation in the inflorescence stem. However, the molecular mechanism by which these transcription factors control different branches of the phenylpropanoid pathway remains poorly understood in woody species. We isolated an R2R3-MYB transcription factor MYB6 from Populus tomentosa and determined that it was expressed predominately in young leaves. Overexpression of MYB6 in transgenic poplar upregulated flavonoid biosynthetic gene expression, resulting in significantly increased accumulation of anthocyanin and proanthocyanidins. MYB6-overexpression plants showed reduced secondary cell wall deposition, accompanied by repressed expression of secondary cell wall biosynthetic genes. We further showed that MYB6 interacted physically with KNAT7 and formed functional complexes that acted to repress secondary cell wall development in poplar and Arabidopsis. The results provide an insight into the transcriptional mechanisms involved in the regulation of the metabolic fluxes between the flavonoid and lignin biosynthetic pathways in poplar.

γ-Aminobutyric acid is closely associated with accumulation of flavonoids

ABSTRACTγ-Aminobutyric acid (GABA) is an important neurotransmitter in mammals whose receptor is reported to be regulated by flavonoids. In plants, it is considered to be at the intersection of carbon and nitrogen metabolism, but its relationship with flavonoid metabolism remains unclear. Our recent RNA-seq analysis showed that expression of flavonoid biosynthetic genes was influenced in poplar by the blockage of α-ketoglutarate dehydrogenase (α-KGDH) activity and the application of GABA under NaCl stress, accompanied by the changes in GABA shunt activity. Here, we further found that the flavonoid accumulation was significantly affected by blocking the activities of α-KGDH and GABA transaminase as well as applying exogenous GABA, coupled with the changes of endogenous GABA contents. Key genes involved in the flavonoid biosynthetic pathway were also significantly influenced, including two PALs, 4CL, and two CHSs. Our results suggest that the GABA shunt is closely associated with the metabolism of flavonoids, which would benefit future understanding of GABA’s roles in carbon allocation by regulating the pathway of flavonoid biosynthesis under normal or stress conditions.

Tricin levels and expression of flavonoid biosynthetic genes in developing grains of purple and brown pericarp rice.

The methylated flavone tricin has been associated with numerous health benefits, including reductions in intestinal and colon cancers in animal models. Tricin is found in a wide range of plant species and in many different tissues. However, whole cereal grains, such as rice, barley, oats, and wheat, are the only food sources of tricin, which is located in the bran portion of the grain. Variation in tricin levels was found in bran from rice genotypes with light brown, brown, red, and purple pericarp color, with the purple pericarp genotypes having the highest levels of tricin. Here, we analyzed tricin and tricin derivative levels in developing pericarp and embryo samples of a purple pericarp genotype, IAC600, that had high tricin and tricin derivative levels in the bran, and a light brown pericarp genotype, Cocodrie, that had no detectable tricin or tricin derivatives in the bran. Tricin and tricin derivatives were detected in both the pericarp and embryo of IAC600 but only in the embryo of Cocodrie. The purple pericarp rice had higher total levels of free tricin plus tricin derivatives than the light brown pericarp rice. When expressed on a per grain basis, most of the tricin component of IAC600 was in the pericarp. In contrast, Cocodrie had no detectable tricin in the pericarp samples but did have detectable chrysoeriol, a precursor of tricin, in the pericarp samples. We also used RNA-Seq analysis of developing pericarp and embryo samples of the two cultivars to compare the expression of genes involved in the flavonoid biosynthetic pathway. The results presented here suggest that understanding the basis of tricin accumulation in rice pericarp may lead to an approach to increasing tricin levels in whole grain rice. From analysis of gene expression levels in the pericarp samples it appears that regulation of the flavone specific genes is independent of regulation of the anthocyanin biosynthetic genes. It therefore may be feasible to develop brown pericarp rice cultivars that accumulate tricin in the pericarp.