Anthocyanins are secondary metabolites derived from the general phenylpropanoid pathway and are widespread throughout the plant kingdom. Anthocyanin accumulation is regulated by transcription factors. However, little is known about the role of CaMYC from the bHLH family in pepper anthocyanin synthesis. In this study, a purple-leaved pepper (Capsicum annuum L.) was selected and subjected to light and cold stress at the seedling stage. The result showed that anthocyanin content in purple leaves was higher than in green leaves, which was related to anthocyanins synthesis. CaMYC is an important factor and may be responsible for anthocyanin synthesis. Light and cold stress can result in strong anthocyanin accumulation. CaMYC plays a key role in increasing structural gene expression, which regulates anthocyanin synthesis by the combination of CaMYB and CaWD40. Further analysis showed that CaMYC-silencing resulted in low expression of structural genes in the anthocyanin synthesis pathway, suggesting that CaMYC has a positive role in pepper anthocyanin metabolism. Our study will provide an insight for anthocyanin synthesis in pepper.
... Previously, virus-induced gene silencing (VIGS) of the CaMYBA gene induced the downregulation of most anthocyanins that synthesize structural genes (Aguilar- Barragań and Ochoa-Alejo, 2014;Zhang et al., 2015) and bHLH gene CaMYC (Zhang et al., 2015). However, VIGS of CaMYC also reduced the expression levels of most anthocyanins that synthesize structural genes, but not CaMYBA (Lu et al., 2019). VIGS of CaTTG1 reduced the expression levels of some anthocyanins that synthesize structural genes (Aguilar-Barragań and Ochoa-Alejo, 2014). ...
... Although the transcription factors CaMYBA, CaMYC, and CaTTG1 are expressed in the leaf of C. annuum L (Zhang et al., 2015;Lu et al., 2019), whether or not they regulate the anthocyanin pathway in pepper leaf as a complex remains to be confirmed. We compared CaMYBA and CaMYC with MYB and bHLH proteins that have been reported to regulate anthocyanin synthesis in other species. ...
... In Arabidopsis and Petunia, MYB, bHLH, and WD40 transcription factors form an MBW complex to regulate anthocyanin biosynthesis (Gonzalez et al., 2008;Albert et al., 2011). According to previous reports, CaMYBA, CaMYC, and CaTTG1 have been shown to be highly correlated with anthocyanin accumulation in pepper, and they may regulate anthocyanin biosynthesis in pepper (Borovsky et al., 2004;Aguilar-Barragań and Ochoa-Alejo, 2014;Zhang et al., 2015;Lu et al., 2019;Jung et al., 2019;Wang et al., 2025;Liu et al., 2024). However, whether they can form an MBW complex has not been reported. ...
Anthocyanins are flavonoid-derived metabolites that contribute to plant and human health. At present, few studies have studied the biosynthesis and accumulation mechanism of anthocyanins in pepper leaves. The role of CaMYBA–CaMYC–CaTTG1 complex in anthocyanin biosynthesis in pepper leaves was studied. Yeast two-hybrid and dual-luciferase experiments showed that CaMYBA, CaMYC, and CaTTG1 could form an MYB–bHLH–WD40 (MBW) complex. They also have transcriptional activation on the anthocyanin synthesis structural genes CaCHS, CaCHI, CaF3H, CaF3′5′H, CaANS, CaDFR, and CaUFGT. Silencing CaMYBA or CaMYC could decrease the content of anthocyanin in pepper leaves. Transient overexpression of CaMYBA in tobacco indicated that CaMYBA determines the function of an MBW complex. Further analysis showed that CaMYBA could activate the expression of CaMYC by binding to its promoter. Overall, our study expands the understanding of the regulatory mechanism of anthocyanin synthesis in pepper leaves and has important significance for creating more pepper plants with different color patterns by gene editing engineering.
... The flavonoid content measurements were performed according to the method of [42]. The collected, frozen samples were ground to a powder and extracted with an extracting solution (3 M 1HCl: 3H2O:16methanol, volume ratio) overnight at 4 °C in the dark. ...
The lengthy maturation period is the key cause of the reduced hot pepper market value. An experiment was performed to assess the effects of R/B/W light qualities on reducing the maturation period. The maturation period was shortened by 16 days under 4R:1B:5W compared to the control. The appearance of the first flower is a limiting factor affecting the maturation period. It exhibited a dark purple color and an abnormal petal under 4R:1B:5W compared to the control, which was associated with increased contents of flavonoids, total organic acids, and phenolics and decreased total amino acid and carbohydrate contents. Plant hormone, flavonoid, ABC transporters, key metabolites (such as amino acid, carbohydrate, organic acid) was associated with first flower development. Ethylene response factors (ERFs) mediated by light, especially ERF021, were identified as potential hub regulators during the development of the first flower due to their involvement in all the associated metabolic pathways.
... 75 A bHLH TF, CaMYC, in association with CaMYB and CaWD40 proteins positively regulates anthocyanin biosynthesis in purple leaved C. annuum under high light intensity and cold treatments. 76,77 A GTB TF, namely, Ca3GT, was highly expressed in the mature purple C. annuum fruits and showed sequence variations in the promoter region between the green and purple genotype, signifying its possible role in anthocyanin biosynthesis. 78 ...
Capsicum (chili pepper) is a widely popular and highly consumed fruit crop with beneficial secondary metabolites such as capsaicinoids, carotenoids, flavonoids, and polyphenols, among others. Interestingly, the secondary metabolite profile is a dynamic function of biosynthetic enzymes, regulatory transcription factors, developmental stage, abiotic and biotic environment, and extraction methods. We propose active manipulable genetic, environmental, and extraction controls for the modulation of quality and quantity of desired secondary metabolites in Capsicum species. Specific biosynthetic genes such as Pun (AT3) and AMT in the capsaicinoids pathway and PSY, LCY, and CCS in the carotenoid pathway can be genetically engineered for enhanced production of capsaicinoids and carotenoids, respectively. Generally, secondary metabolites increase with the ripening of the fruit; however, transcriptional regulators such as MYB, bHLH, and ERF control the extent of accumulation in specific tissues. The precise tuning of biotic and abiotic factors such as light, temperature, and chemical elicitors can maximize the accumulation and retention of secondary metabolites in pre- and postharvest settings. Finally, optimized extraction methods such as ultrasonication and supercritical fluid method can lead to a higher yield of secondary metabolites. Together, the integrated understanding of the genetic regulation of biosynthesis, elicitation treatments, and optimization of extraction methods can maximize the industrial production of secondary metabolites in Capsicum.
... Furthermore, bHLH transcription factors help plants in resisting diverse environmental stresses, such as cold, salt, and drought [27][28][29][30][31][32]. The biosynthesis of secondary metabolites has also been shown to be regulated by bHLH TFs, such as flavonoids and anthocyanins [33][34][35][36][37]. Terpenoids are essential members of secondary metabolites, and extensive research has shown that the biosynthesis of terpenoids is closely correlated with bHLH transcription factors in many species. ...
Basic helix-loop-helix (bHLH) transcription factors (TFs) play irreplaceable roles in plant growth and development, especially in plant secondary metabolism. However, the functions of most bHLH TFs in Lilium ‘Siberia’ are still unknown, especially their roles in regulating floral fragrance. In this study, two bHLH TFs in lily, i.e., LibHLH22 and LibHLH63, were identified and functionally characterized. A bioinformatics analysis revealed that LibHLH22 and LibHLH63 were unstable proteins. Subcellular localization demonstrated that LibHLH22 and LibHLH63 proteins were in the cell nucleus. Quantitative real-time PCR showed that the highest expression level of LibHLH22 was at the initial flowering stage and in the stigma, and the highest expression level of LibHLH63 was at the budding stage and in the filaments. The results of transient overexpression and virus-induced gene silencing (VIGS) of LibHLH22 and LibHLH63 in lily petals showed that these two transcription factors significantly promoted the expression of LiDXR and LiTPS2, and thus, markedly enhanced the release of floral fragrance. Our results indicated that LibHLH22 and LibHLH63 could effectively regulate the fragrance of Lilium ‘Siberia’, laying the foundation for fragrance breeding and improving the terpenoid transcriptional regulatory pathway.
Purple paprika (Capsicum annuum) is a vegetable with potential economic value; however, enhancing and maintaining the purple fruit color is challenging. We investigated the effects of night blue-light supplementation on fruit growth, anthocyanin content, and gene expression in purple paprika. During two duplicated experiments conducted in spring and autumn, purple paprika plants were subjected to blue-light supplementation at night. Ten days after fruits were pollinated, night blue light with photosynthetic photon flux density (PPFD) of 80 to 100 mmol·s21·m22 was supplied from 1800 HR to 0500 HR (11 hours). Fruit samples were harvested 15, 20, and 40 days after pollination from control (no-light treatment) and blue light–treated fruits. The fruit size, fresh weight, anthocyanin content, and expression levels of anthocyanin biosynthesis-related genes (HY5, bHLH, WDR, MYB, PAL, CHS, F3H, DFR, ANS, andUFGT) were determined. Blue-light supplementation increased the anthocyanin-related gene expressions in the fruit peel, enhancing anthocyanin synthesis and accumulation. However, there were no
significant differences between the growth of control and blue light–treated fruits. These findings highlight the potential of night blue-light supplementation to enhance anthocyanin content in purple paprika fruits without affecting overall fruit growth.
Capsicum (chili pepper) is a highly valued crop of the Solanaceae family desired for its characteristic sensory and culinary properties around the world. Capsicum fruits exhibit huge variations in the phenotypic traits, such as shape, size, color, texture, shelf life, and so on, that appeal directly to the consumer preference. The fruits of the Capsicum genus are also diversely rich in several health-benefitting bioactive compounds such as capsaicinoids (mainly capsaicin and dihydrocapsaicin), vitamins (C and E), carotenoids, antioxidants, flavonoids, and so on. Often, highly pungent and stress-resistant Capsicum varieties suit the breeders’ choice. In the past few years, pepper genetics, breeding, and improvement have made an unscalable amount of progress in terms of the characterization of genes and quantitative trait loci controlling plant- and fruit-related character traits, metabolites, and biotic and abiotic stress resistance complemented with marker-assisted selection and genetic manipulation. High-throughput methods like whole genome sequencing and transcriptome analyses have bridged the gap between genetic resources and molecular biology to develop highly competent pepper varieties. Furthermore, biotechnological advances have facilitated rapid transgene interrogation, target-based gene silencing, and overexpression for the functional characterization of candidate genes for the genetic improvement of the pepper crop.
Main conclusion
This review summarizes the anti-stress effects of flavonoids in plants and highlights its role in the regulation of polar auxin transport and free radical scavenging mechanism.
Abstract
As secondary metabolites widely present in plants, flavonoids play a vital function in plant growth, but also in resistance to stresses. This review introduces the classification, structure and synthetic pathways of flavonoids. The effects of flavonoids in plant stress resistance were enumerated, and the mechanism of flavonoids in plant stress resistance was discussed in detail. It is clarified that plants under stress accumulate flavonoids by regulating the expression of flavonoid synthase genes. It was also determined that the synthesized flavonoids are transported in plants through three pathways: membrane transport proteins, vesicles, and bound to glutathione S-transferase (GST). At the same time, the paper explores that flavonoids regulate polar auxin transport (PAT) by acting on the auxin export carrier PIN-FORMED (PIN) in the form of ATP-binding cassette subfamily B/P-glycoprotein (ABCB/PGP) transporter, which can help plants to respond in a more dominant form to stress. We have demonstrated that the number and location of hydroxyl groups in the structure of flavonoids can determine their free radical scavenging ability and also elucidated the mechanism by which flavonoids exert free radical removal in cells. We also identified flavonoids as signaling molecules to promote rhizobial nodulation and colonization of arbuscular mycorrhizal fungi (AMF) to enhance plant–microbial symbiosis in defense to stresses. Given all this knowledge, we can foresee that the in-depth study of flavonoids will be an essential way to reveal plant tolerance and enhance plant stress resistance.
Anthocyanins contribute vibrant colors to plants, enhance a plant's ability to survive biotic and abiotic stresses, and have a positive impact on human health. Solanaceous fruit vegetables, which mainly include tomato (Solanum lycopersicum L.), eggplant (Solanum melongena L.), and pepper (Capsicum annuum L.), display natural variations of anthocyanin pigments in term of types, contents and tissue-specific patterns. Breeding anthocyanin-rich variety is the predominant avenue to improve the quality of Solanaceous fruit vegetables. Anthocyanin biosynthesis is a complicated process affected and regulated by environmental conditions and developmental cues. This article reviews the health benefits of anthocyanin, anthocyanin synthesis and transportation, transcriptional regulations, influencing factors, and strategies for increasing the anthocyanin content in Solanaceous fruit vegetables. It aims to provide a reference for further research on the biosynthesis, accumulation, and metabolism of anthocyanin, and to point out the direction for breeding and cultivation of anthocyanin-rich Solanaceous fruit vegetables.
Chitin-binding proteins are pathogenesis-related gene family, which play a key role in the defense response of plants. However, thus far, little is known about the chitin-binding family genes in pepper (Capsicum annuum L.). In current study, 16 putative chitin genes (CaChi) were retrieved from the latest pepper genome database, and were classified into four distinct classes (I, III, IV and VI) based on their sequence structure and domain architectures. Furthermore, the structure of gene, genome location, gene duplication and phylogenetic relationship were examined to clarify a comprehensive background of the CaChi genes in pepper. The tissue-specific expression analysis of the CaChi showed the highest transcript levels in seed followed by stem, flower, leaf and root, whereas the lowest transcript levels were noted in red-fruit. Phytophthora capsici post inoculation, most of the CaChi (CaChiI3, CaChiIII1, CaChiIII2, CaChiIII4, CaChiIII6, CaChiIII7, CaChiIV1, CaChiVI1 and CaChiVI2) were induced by both strains (PC and HX-9). Under abiotic and exogenous hormonal treatments, the CaChiIII2, CaChiIII7, CaChiVI1 and CaChiVI2 were upregulated by abiotic stress, while CaChiI1, CaChiIII7, CaChiIV1 and CaChiIV2 responded to hormonal treatments. Furthermore, CaChiIV1-silenced plants display weakened defense by reducing (60%) root activity and increase susceptibility to NaCl stress. Gene ontology (GO) enrichment analysis revealed that CaChi genes primarily contribute in response to biotic, abiotic stresses and metabolic/catabolic process within the biological process category. These results exposed that CaChi genes are involved in defense response and signal transduction, suggesting their vital roles in growth regulation as well as response to stresses in pepper plant. In conclusion, these finding provide basic insights for functional validation of the CaChi genes in different biotic and abiotic stresses.
The dirigent (DIR and DIR-like) proteins involved in lignification, play a pivotal role against biotic and abiotic stresses in plants. However, no information is available about DIR gene family in pepper (Capsicum annuum L.). In this study, 24 putative dirigent genes (CaDIRs) were identified, their gene structure, genome location, gene duplication and phylogenetic relationship were elucidated. Tissue- specific expression analysis displayed the highest transcription levels in flower, stem and leaf. Some CaDIRs were up-regulated by virulent (CaDIR2, 3, 6, 7, 11, 14, 16, 22 and 23) and avirulent (CaDIR3, 5, 7, 16, 20, 22, 23 and 24) Phytophthora capsici strains, as well as by Methyl jasmonate, salicylic acid, NaCl and mannitol stresses. Acid-soluble lignin content increased (103.21%) after P. capsici inoculation (48-hour). Silencing of CaDIR7 weakened plant defense by reducing (~50%) root activity and made plants more susceptible (35.7%) to P. capsici and NaCl (300 mM). Leaf discs of the CaDIR7:silenced plants exposed to NaCl and mannitol (300 mM each), exhibited a significant decrease (56.25% and 48% respectively) in the chlorophyll content. These results suggested that CaDIR7 is involved in pepper defense response against pathogen and abiotic stresses and the study will provide basic insights for future research regarding CaDIRs.
Anthocyanins are considered a stress indicator due to their involvement in the response to many stresses including high light (HL) and low temperature (LT). With the development of transcriptomics, it is necessary to find the different and common points in the mechanisms of LT-induced and HL-induced anthocyanin biosynthesis. In the present study, we determined the transcriptomes of Begonia semperflorens leaves under three different conditions (normal growing conditions (CK), HL, and LT). To validate the differentially expressed genes (DEGs), we selected four core genes involved in anthocyanin biosynthesis to perform real-time reverse transcription-quantitative PCR (RT-qPCR), and then determined anthocyanin content. In total, 94,880 unigenes with a mean length of 635 bp were assembled. The N50 values of the transcripts and unigenes were 2286 bp and 1064 bp, respectively. The functional annotations of the unigenes were analysed against five protein databases. DEGs related to anthocyanin biosynthesis, transportation, and regulation were identified. We also analysed the enrichment pathway, and the differences in mechanisms of anthocyanin induced under low-temperature and high-light conditions are discussed in this paper. This study is the first to examine broad-scale gene expression in Begonia semperflorens. By identifying DEGs regulated by both LT and HL conditions, we found that anthocyanin accumulation was employed as a common strategy by Begonia seedlings in resisting LT and HL stress. By identifying DEGs regulated differently by LT and HL conditions, we found that Begonia seedlings also had some different strategies for resisting LT and HL stresses: anthocyanins were biosynthesized under HL condition, while lignin, proanthocyanidins, and anthocyanins were biosynthesized under LT condition.
BACKGROUND/OBJECTIVES
Oxidative stress is closely related with inflammation and development of many diseases. Black soybean seed coat contains high amount of anthocyanins, which are well-known for free radical scavenging activities. This study investigated inflammatory response and action mechanism of black soybean anthocyanins with regard to antioxidant activity in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells.
MATERIALS/METHODS
RAW 264.7 cells were treated with anthocyanins extracted from black soybean seed coats in a concentration range of 12.5 to 100 µg/mL. The production of reactive oxygen species (ROS), secretion of pro-inflammatory mediators and cytokines, and the signaling in the mitogen activated protein kinases (MAPKs) pathway were examined.
RESULTS
Black soybean anthocyanins significantly decreased LPS-stimulated production of ROS, inflammatory mediators such as nitric oxide (NO) and prostaglandin E2, and pro-inflammatory cytokines, including tumor necrosis factor α and interleukin-6, in a dose-dependent manner without cytotoxicity (P < 0.001). Black soybean anthocyanins downregulated the expression of inducible NO synthase and cyclooxygenase-2 in LPS-stimulated RAW 264.7 cells (P < 0.001). Moreover, black soybean anthocyanins inhibited LPS-induced phosphorylation of MAPKs, including extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 (P < 0.001).
CONCLUSION
These results suggest that black soybean anthocyanins exert anti-inflammatory activity by inhibiting ROS generation and subsequent MAPKs signaling, thereby inhibiting inflammatory responses.
Recently the TaMYC1 gene encoding bHLH transcription factor has been isolated from the bread wheat (Triticum aestivum L.) genome and shown to co-locate with the Pp3 gene conferring purple pericarp color. As a functional evidence of TaMYC1 and Pp3 being the same, higher transcriptional activity of the TaMYC1 gene in colored pericarp compared to uncolored one has been demonstrated. In the current study, we present additional strong evidences of TaMYC1 to be a synonym of Pp3. Furthermore, we have found differences between dominant and recessive Pp3(TaMyc1) alleles. Light enhancement of TaMYC1 transcription was paralleled with increased AP accumulation only in purple-grain wheat. Coexpression of TaMYC1 and the maize MYB TF gene ZmC1 induced AP accumulation in the coleoptile of white-grain wheat. Suppression of TaMYC1 significantly reduced AP content in purple grains. Two distinct TaMYC1 alleles (TaMYC1p and TaMYC1w) were isolated from purple- and white-grained wheat, respectively. A unique, compound cis-acting regulatory element had six copies in the promoter of TaMYC1p, but was present only once in TaMYC1w. Analysis of recombinant inbred lines showed that TaMYC1p was necessary but not sufficient for AP accumulation in the pericarp tissues. Examination of larger sets of germplasm lines indicated that the evolution of purple pericarp in tetraploid wheat was accompanied by the presence of TaMYC1p. Our findings may promote more systematic basic and applied studies of anthocyanins in common wheat and related Triticeae crops.
Anthocyanins are colored water-soluble pigments belonging to the phenolic group. The pigments are in glycosylated forms. Anthocyanins responsible for the colors, red, purple, and blue, are in fruits and vegetables. Berries, currants, grapes, and some tropical fruits have high anthocyanins content. Red to purplish blue-colored leafy vegetables, grains, roots, and tubers are the edible vegetables that contain a high level of anthocyanins. Among the anthocyanin pigments, cyanidin-3-glucoside is the major anthocyanin found in most of the plants. The colored anthocyanin pigments have been traditionally used as a natural food colorant. The color and stability of these pigments are influenced by pH, light, temperature, and structure. In acidic condition, anthocyanins appear as red but turn blue when the pH increases. Chromatography has been largely applied in extraction, separation, and quantification of anthocyanins. Besides the use of anthocyanidins and anthocyanins as natural dyes, these colored pigments are potential pharmaceutical ingredients that give various beneficial health effects. Scientific studies, such as cell culture studies, animal models, and human clinical trials, show that anthocyanidins and anthocyanins possess antioxidative and antimicrobial activities, improve visual and neurological health, and protect against various non-communicable diseases. These studies confer the health effects of anthocyanidins and anthocyanins, which are due to their potent antioxidant properties. Different mechanisms and pathways are involved in the protective effects, including free-radical scavenging pathway, cyclooxygenase pathway, mitogen-activated protein kinase pathway, and inflammatory cytokines signaling. Therefore, this review focuses on the role of anthocyanidins and anthocyanins as natural food colorants and their nutraceutical properties for health.
Abbreviations: CVD: Cardiovascular disease VEGF: Vascular endothelial growth factor
The apple (Malus domestica) MdMYB1 gene encodes an R2R3-MYB transcription factor (TF) that regulates anthocyanin biosynthesis. The transient and stable expression of the apple basic helix-loop-helix 3 gene, MdbHLH3, produces anthocyanin pigmentation in apple fruit and calli. In order to understand the MdMYB1- and MdbHLH3-related regulatory mechanisms of anthocyanin biosynthesis, we established and characterized the expression of those two genes in stably transformed tobacco (Nicotiana tabacum NC89) plants, including the phenotypic differences in anthocyanin-containing flowers, ovaries, anthers and anthocyanin-free leaves. A real-time PCR analysis showed that the expression levels of the MdMYB1 and MdbHLH3 transgenes were up-regulated in all tobacco organs. However, the bHLH and MYB TFs, NtAN1 and NtAN2, were not detected in tobacco leaves, respectively, indicating that neither can individually activate anthocyanin biosynthesis. However, MdMYB1 induced pigment accumulation in ovaries because NtAN1 is active in that organ. Thus, the endogenous bHLH TF is necessary for anthocyanin biosynthesis in tobacco. Furthermore, MdMYB1 binding to the promoters of the anthocyanin biosynthesis genes MdDFR and MdUFGT activated their expression in apple. However, because NtAN2 is not activated in leaves or ovaries, the pigmentation patterns were not significantly altered in these organs in MdbHLH3-overexpressing tobacco. Thus, the data indicated that MdMYB1 regulated anthocyanin biosynthesis directly by binding the MdDFR and MdUFGT promoters, and interacted with the tobacco bHLH TF, triggering anthocyanin accumulation.
The epidermis of swollen storage roots in purple cultivars of turnip “Tsuda” (Brassica rapa) accumulates anthocyanin in a light-dependent manner, especially in response to UV-A light, of which the mechanism is unclear. In this study, we mutagenized 15,000 seeds by 0.5% (v/v) ethyl methane sulfonate (EMS) and obtained 14 mutants with abnormal anthocyanin production in their epidermis of swollen storage roots. These mutants were classified into two groups: the red mutants with constitutive anthocyanin accumulation in their epidermis of storage roots even in underground parts in darkness and the white mutants without anthocyanin accumulation in the epidermis of storage roots in aboveground parts exposed to sunlight. Test cross analysis demonstrated that w9, w68, w204, r15, r21, r30 and r57 contained different mutations responsible for their phenotypic variations. Further genetic analysis of four target mutants (w9, w68, w204 and r15) indicated that each of them was controlled by a different recessive gene. Intriguingly, the expression profiles of anthocyanin biosynthesis genes, including structural and regulatory genes, coincided with their anthocyanin levels in the epidermis of storage roots in the four target mutants. We proposed that potential genes responsible for the mutations should be upstream factors of the anthocyanin biosynthesis pathway in turnips, which provided resources to further investigate the mechanisms of light-induced anthocyanin accumulation.
Pigments are important in vegetable kingdom. However, little is known regarding role of their combination in pepper. In this study, three pepper strains were crossed to get F1 pepper plants, and exposed to whitefly attack at 6–8 leaves stage. Populations (M1–M9) differing in leaf color divulged that leaf color inherited to progenies at different extent and intensively showed matrocliny. Furthermore, in purple-colored population, the accumulation of anthocyanin improved the physiology and resistance to whitefly, with increased photosynthetic rate (Pn), Fv/Fm as well as root activity and reduction in superoxide dismutase (SOD), peroxidase (POD), malonaldehyde (MDA) and pest index (PI). Best manifestation of these parameters was observed in M3 population, indicating plant development be related to the optimum combination of anthocyanin and chlorophyll in the investigated leaves. Interestingly, M3 population possessed highest resistance to whitefly, while level of its anthocyanin and chlorophyll was in moderate level. Besides, no significant difference was observed in the expression levels of genes related to carotenoid metabolism in all populations. significant correlation was observed between PI and expression of genes related to pigment, namely CaGLK and CaCHS genes (R2 > 0.8), while the correlation was also found between ANS and GUN4 genes. These results suggested that the combination of anthocyanin and chlorophyll could enhance the resistance of pepper plants against whitefly attack.
Homeodomain-leucine zipper class I (HD-Zip I) transcription factors (TFs) have been functionally characterized in plant responses to abiotic stresses, but their roles in plant immunity are poorly understood. Here, a HD-Zip I gene, CaHZ27, was isolated from pepper (Capsicum annum) and characterized for its role in pepper immunity. Quantitative real-time PCR showed that CaHDZ27 was transcriptionally induced by Ralstonia solanacearum inoculation and exogenous application of methyl jasmonate (MeJA), salicylic acid (SA), or ethephon (ETH). The CaHDZ27-GFP (green fluorescent protein) fused protein was targeted exclusively to the nucleus. Chromatin immunoprecipitation (ChIP) demonstrated that CaHDZ27 bound to the 9-bp pseudopalindromic element (CAATAATTG) and triggered GUS expression in a CAATAATTG-dependent manner. Virus-induced gene silencing (VIGS) of CaHDZ27 significantly attenuated the resistance of pepper plants against R. solanacearum and downregulated defense-related marker genes including CaHIR1, CaACO1, CaPR1, CaPR4, CaPO2, and CaBPR1. By contrast, transient overexpression of CaHDZ27 triggered strong cell death mediated by the hypersensitive response (HR), and upregulated the tested immunity-associated marker genes. Ectopic CaHDZ27 expression in tobacco enhances its resistance against R. solanacearum. These results collectively suggest that CaHDZ27 functions as a positive regulator in pepper resistance against R. solanacearum. Bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (CoIP) assays indicate that CaHDZ27 monomers bind with each other, and this binding is enhanced significantly by R. solanacearum inoculation. We speculate that homodimerization of CaHZ27 might play a role in pepper response to R. solanacearum, further direct evidence is required to confirm it.
