Anthocyanin Biosynthesis-related Genes Research Articles

The Combined Application of Urea and Fulvic Acid Regulates Apple Tree Carbon and Nitrogen Metabolism and Improves Anthocyanin Biosynthesis

Improving apple peel color has been an important objective in apple production. To better understand the effect and mechanism of the combined application of urea and FA (fulvic acid) regulation of anthocyanin biosynthesis, a field experiment was performed in 2022 and 2023, respectively, under five treatments of urea + FA (CK, urea only; FA50, urea + 50 kg ha−1 FA; FA100, urea + 100 kg ha−1 FA; FA150, urea + 150 kg ha−1 FA; FA200, urea + 200 kg ha−1 FA), using isotope (13C and 15N) marking to analyze the changes in carbon (C) and nitrogen (N) nutrient distribution as well as anthocyanin biosynthesis in fruits. We observed that, under FA application conditions, anthocyanin content in the peel was elevated in both years, with increases of 15.98~52.88% in 2022 and 15.93~52.94% in 2023. The best promotion effects were observed under FA150 treatment. Apart from the expression levels of anthocyanin biosynthesis-related genes and transcription factors in the apple peel, this positive effect on anthocyanin content induced by FA addition was also found to be associated with the optimization of C and N distribution in leaves and fruits. On the one hand, the application of FA not only enhanced leaf photosynthetic-related indexes (such as Pn, Gs, and Rubisco activity) and influenced (increased) S6PDH, SPS, and SS activities in leaves, but also elevated fruit sugar metabolism-related enzyme (SDH, SS-c, AI, and NI) activity and upregulated fruit stalk sugar transporter (MdSOT1, MdSOT3, MdSUT1 and MdSUT4) gene expression, which ultimately promoted the synthesis and the leaf to fruit transport of photosynthates, thus promoting 13C-photosynthate accumulation in fruits. On the other hand, FA application elevated leaves’ N metabolism-related enzyme (GS and GOGAT) activity and optimized 15N distribution in leaves and fruits. Moreover, we also observed that FA application altered the fate of N fertilizer in apple orchards, showed an elevation in apple tree 15NUE and soil 15N residuals and showed a decrease in soil 15N loss. In summary, the appropriate application of FA150 (urea + 150 kg ha−1) synergistically optimized C and N nutrient distribution, and promoted anthocyanin biosynthesis in apple trees.

Functional identification of AaMYB113 and AaMYB114 from Aeonium arboreum ‘Halloween’ in model plants

Aeonium arboreum ‘Halloween’, a popular indoor ornamental succulent in China, changes its leaf colour to red on light exposure. However, the underlying molecular mechanisms is still vague. Comparative analysis of transcriptome data from ‘Halloween’ leaves treated under dark and light conditions revealed two R2R3-MYB transcription factors, AaMYB113 and AaMYB114, that may mediate anthocyanin accumulation. In this study, we cloned the AaMYB113 and AaMYB114 genes, encoding proteins of 279 and 248 amino acids, respectively. Transcriptional activity analysis revealed that AaMYB113 exhibits strong transcriptional activity, in contrast to AaMYB114, which demonstrates minimal activity. Transient expression studies in tobacco leaves demonstrated that AaMYB113 induced red pigmentation, whereas AaMYB114 did not. Subsequent stable overexpression in Arabidopsis thaliana confirmed that AaMYB113, but not AaMYB114, could similarly turn Arabidopsis leaves red. Further stable transformation of AaMYB113 in tobacco affected multiple floral components, including leaves, petals, calyx, flower tubes, and filaments, turning them red. Quantitative real-time PCR (qRT-PCR) assay in leaves of AaMYB113 stably transformed tobacco and Arabidopsis revealed upregulation of anthocyanin biosynthesis-related structural genes and TT8-like transcription factors. Moreover, the dual luciferase analysis confirmed that AaMYB113 can activate the promoters of ‘Halloween’ anthocyanin synthesis structural genes, AaCHS, AaCHI, AaF3H, AaDFR and AaANS. The above results indicate that AaMYB113 can promote anthocyanin synthesis, while AaMYB114 does not have this function. This study contributes significantly to the limited body of research on the molecular mechanisms of anthocyanin synthesis in succulents, advancing our understanding of how these pathways are regulated in ‘Halloween’ succulents and potentially other species.

Csn-miR156d-CsSPL1 regulates flowering and anthocyanin metabolism.

MiR156 play important roles in regulation of plant growth and development, secondary metabolite synthesis, and other biological processes by targeting the SQUAMOSA promoter binding protein-like (SPL) family. Our previous sequencing data analysis suggested that Csn-miR156d may regulate flowering and anthocyanin accumulation by cleavage and degradation of the expression of the SPL in tea plant, but it remains to be elucidated. In this study, 5'RLM-RACE experiment, tobacco transient transformation, qRT-PCR, and antisense oligonucleotide (asODN) were used to verify that CsSPL1 is the target gene of Csn-miR156d. Stable transformation of Arabidopsis revealed that Csn-miR156d could delay flowering by negatively regulating the transcript levels of FT, AP1, FUL, and SOC1, while overexpression of CsSPL1 showed an opposite effect. Additionally, overexpression of Csn-miR156d in Arabidopsis could enhance the transcription of the anthocyanin biosynthesis-related structural genes DFR, ANS, F3H, UGT78D2, and LDOX, as well as regulatory genes PAP1, MYB113, GL3, MYB11, and MYB12, leading to anthocyanin accumulation. Moreover, asODN experiment revealed that Csn-miR156d could increase the anthocyanin content in tea plant. These results suggest that Csn-miR156d regulates flowering and anthocyanin accumulation in tea plant by suppressing the expression of CsSPL1. Our study provides new insights into the development and anthocyanin accumulation in tea plant and lays a theoretical foundation for further research on the molecular mechanism of miRNAs in regulating tea plant growth and secondary metabolism.

Genome-Wide Identification and Expression Analysis of Fifteen Gene Families Involved in Anthocyanin Synthesis in Pear

Anthocyanins play a crucial role in imparting red coloration to pear fruits. However, the specific number and expression patterns of each member within the anthocyanin biosynthesis-related gene families in pears require systematic exploration. In this study, based on the pear genome we identified 15 gene families involved in the anthocyanin biosynthesis pathway using the BLASTP and Hidden Markov Model search methods, comprising a total of 94 enzyme genes. Through phylogenetic analysis, conserved domains, motif, and gene structure analysis, these gene families were further categorized into eight distinct lineages. Subsequent collinearity analysis revealed that the expansion of anthocyanin synthesis-related gene families primarily originated from segmental duplications. Analysis of cis-element in the promoter regions of genes related to anthocyanin synthesis unveiled the presence of light-responsive elements and various hormone-responsive elements. This suggests that changes in light stimulation and hormone levels may influence anthocyanin synthesis. RNA-Seq and qRT-PCR analyses indicated differential expression of anthocyanin biosynthesis-related genes between the peel and flesh tissues. During the accumulation of anthocyanins in red-fleshed pears, upstream genes in the anthocyanin biosynthesis pathway such as PbrPAL2, PbrC4H2, PbrC4H3, Pbr4CL2, Pbr4CL17, PbrF3H5, and PbrF3H6 exhibited high expression levels, likely contributing significantly to the red coloration of pear flesh. In summary, we have identified the number of gene family members involved in pear anthocyanin biosynthesis and analyzed the expression patterns of the genes related to pear anthocyanin biosynthesis. These findings provide a solid foundation for further research on the regulatory mechanisms underlying pear anthocyanin biosynthesis and the breeding of red pear varieties.

The CsMYB123 and CsbHLH111 are involved in drought stress-induced anthocyanin biosynthesis in Chaenomeles speciosa

Drought stress has been demonstrated to enhance the biosynthesis of anthocyanins in the leaves, resulting in an increased aesthetic appeal. However, the molecular mechanisms underlying drought-induced anthocyanin biosynthesis in Chaenomeles speciosa remain unclear. In this study, the metabolites of C. speciosa leaves were analyzed, and it was found that the content of cyanidin-3-O-rutinoside increased significantly under drought stress. The differentially expressed genes CsMYB123 and CsbHLH111 were isolated by transcriptomics data analysis and gene cloning, and gene overexpression and VIGS experiments verified that both play important roles in anthocyanin biosynthesis. Subsequently, Y1H and Dual-luciferase reporter assay showed that CsMYB123 binds to the promoters of anthocyanin biosynthesis-related structural genes (such as CsCHI, CsF3H, and CsANS), while CsbHLH111 was shown to bind to the promoter of CsCHI, positively regulating its activity. Furthermore, BIFC and Y2H assays unveiled potential protein–protein interactions between CsMYB123 and CsbHLH111 at the cell nucleus. Collectively, these results shed light on the critical roles played by CsMYB123 and CsbHLH111 in anthocyanin biosynthesis, thus providing a valuable insight into understanding the molecular mechanisms of how the MYB and bHLH genes regulate anthocyanin biosynthesis in the process of leaf coloration in C. speciosa.Graphical

A Novel R2R3–MYB Transcription Factor FaMYB10-like Promotes Light-Induced Anthocyanin Accumulation in Cultivated Strawberry

Anthocyanins widely accumulate in the vegetative and reproductive tissues of strawberries and play an important role in stress resistance and fruit quality. Compared with other fruits, little is known about the molecular mechanisms regulating anthocyanin accumulation in strawberry vegetative tissues. In this study, we revealed an R2R3–MYB transcription factor, FaMYB10-like (FaMYB10L), which positively regulated anthocyanin accumulation and was induced by light in the petiole and runner of cultivated strawberry. FaMYB10L is a homologue of FveMYB10-like and a nuclear localization protein. Transient overexpression of FaMYB10L in a white fruit strawberry variety (myb10 mutant) rescued fruit pigmentation, and further qR–PCR analysis revealed that FaMYB10L upregulated the expression levels of anthocyanin biosynthesis-related genes and transport gene. A dual luciferase assay showed that FaMYB10L could activate the anthocyanin transport gene FaRAP. Anthocyanin accumulation was observed in FaMYB10L-overexpressing strawberry calli, and light treatment enhanced anthocyanin accumulation. Furthermore, transcriptomic profiling indicated that the DEGs involved in the flavonoid biosynthesis pathway and induced by light were enriched in FaMYB10L-overexpressing strawberry calli. In addition, yeast two-hybrid assays and luciferase complementation assays indicated that FaMYB10L could interact with bHLH3. These findings enriched the light-involved regulatory network of anthocyanin metabolism in cultivated strawberries.

Genome-wide identification of IbGRFs in sweetpotato and functional analysis of IbGRF9 in anthocyanin biosynthesis

Growth-regulating factors (GRFs), a small transcription factor family in plant, hold vital biological roles during developmental processes and plant responses. However, little is known about GRF genes in sweetpotato (Ipomoea batatas L.). Here, 10 IbGRFs were identified and cloned from the sweetpotato genome. Four IbGRF genes contained miR396-binding sites. IbGRF9 had a higher expression level in the transgenic sweetpotato line MIM396. The anthocyanin contents increased in both MIM396 and transgenic Arabidopsis and in the hairy roots of sweetpotato overexpressing IbGRF9. The IbGRF9 protein bound to the promoters of anthocyanin-biosynthesis related genes to increase anthocyanin content. These results raise new insights for the biological function of GRF genes in regulating anthocyanin biosynthesis in sweetpotato.

Differential Coloration, Pigment Biosynthesis-Related Gene Expression, and Accumulation According to Developmental Stage in the ‘Enbu’ Apple

Coloration, a major factor contributing to apple quality, attracts consumer attraction, thereby increasing the sale of fruits in the market. Among the fruit pigments, anthocyanins and carotenoids are the most important pigments that impart coloration to apples. Pigment accumulation, which is regulated by transcriptional factors in the anthocyanin and carotenoid biosynthesis pathways, varies according to genetics, transcription, and developmental stage. To understand the regulation of color development in apples, we evaluated the expression of the genes in the anthocyanin and carotenoid biosynthetic pathways and analyzed the accumulation of pigment, including anthocyanin and carotenoid in the skin and flesh of the red-fleshed apple ‘Enbu’ cultivar at different fruit development stages (30, 60, 90, 120, and 150 days after full bloom, and ripe). Color development increased according to the developmental stage, with considerable variation in both the transcript levels and pigment concentrations observed in tissues, skin, and flesh. Moreover, we identified key transcription factors among the anthocyanin and carotenoid biosynthesis genes that regulated pigment accumulation. Pearson’s correlation analysis showed a strong correlation between the coloration patterns and the expression levels of anthocyanin biosynthesis-related genes (MdPAL, MdCHI, and MdF3H) and carotenoid biosynthesis-related genes (MdGGPPS, MdPSY, MdZDS, MdCRTISO, MdCRHβ, and MdZEP). This study provides insight into the molecular mechanisms underlying pigment biosynthesis for breeding high-quality red-fleshed apple varieties to cater to consumer attention and preference.

The Synergistic Effects of Environmental and Genetic Factors on the Regulation of Anthocyanin Accumulation in Plant Tissues.

Anthocyanin accumulation is responsible for the coloration of apple fruit, and their accumulation depends on the expression of anthocyanin biosynthesis-related genes. Light is an environmental stimulus that induces fruit color by regulating genes involved in the anthocyanin biosynthesis pathway. In this study, the roles of light and genetic factors on fruit coloration and anthocyanin accumulation in apple fruit were investigated. Three genes in the anthocyanin biosynthesis pathway, MdCHS, MdANS, and MdUFGT1, were synthesized and cloned into a viral-based expression vector system for transient expression in 'Ruby S' apple fruits. Apple fruits were agroinfiltrated with expression vectors harboring MdCHS, MdANS, and MdUFGT1. Agroinfiltrated apple fruits were then either kept in the dark (bagged fruits) or exposed to light (exposed fruits). The agroinfiltrated fruits showed significantly different coloration patterns, transcript expression levels, and anthocyanin accumulation compared to the control fruits. Moreover, these parameters were higher in exposed fruits than in bagged fruits. For stable expression, MdCHS was introduced into a binary vector under the control of the rice α-amylase 3D (RAmy3D) promoter. The ectopic overexpression of MdCHS in transgenic rice calli showed a high accumulation of anthocyanin content. Taken together, our findings suggest that light, together with the overexpression of anthocyanin biosynthesis genes, induced the coloration and accumulation of anthocyanin content in apple fruits by upregulating the expression of the genes involved in the anthocyanin biosynthesis pathway.

Comparative transcriptome profiling unveils the potential key genes involved in peel coloration between Ziziphus mauritiana Lam. and Z. jujuba Mill

Ber (Ziziphus mauritiana Lam.) is one of the important tropical and subtropical fruit trees of the genus Ziziphus Mill. Most cultivars of Ber keep green throughout fruit development while its close relative Chinese jujube (Z. jujuba Mill.) changes the color from green to red in all cultivars. However, the lack of transcriptomic and genomic information limits our insights into the molecular mechanisms underpinning fruit coloration in Ber. Here, high-throughput RNA sequencing (RNA-Seq) was used for the first time to de novo assemble and characterize the transcriptome of Ber fruit. Over 20 million clean reads were produced, and 62,029 unigenes with an average length of 1078.76 bp were assembled. The differentially regulated genes that are responsive to fruit color were also studied in Ber. Eight structural genes, ZmC4H1, ZmBIA1, ZmLDOX, and ZmUGT (UTG6, UFGT13 UFGT14, UFGT15, and UFGT16), were found to be significantly differentially expressed and downregulated in Ber fruit peel, indicating that these genes may be involved in no color formation in the fruit peel of Ber. Furthermore, transcription factors such as bHLH and ZmMYB (MYB13, 14 15, and 16) were found to be downregulated at five developmental stages of Ber fruit, suggesting an important interconnection with flavonoids like anthocyanin biosynthesis-related genes. The expression of these selected genes was also checked at all fruit developmental stages in the Chinese jujube transcriptome. All the selected genes were significantly up-regulated in the fruit developmental stages of Jujube. The expression pattern of these selected genes encoding flavonoid or anthocyanin biosynthetic enzymes and transcription factors in the Ber and Jujube fruit peels were confirmed using quantitative real-time PCR. This study provided insights into the diversity of fruit coloration in Ziziphus Mill. and will serve as a new starting point for further functional investigation of these genes.

Exogenously Induced Silencing of Four MYB Transcription Repressor Genes and Activation of Anthocyanin Accumulation in Solanum lycopersicum.

RNA interference (RNAi) is a natural post-transcriptional regulatory mechanism that can be artificially induced by exogenous application of double-stranded RNAs (dsRNAs) to the plant surfaces. Recent studies show that it is possible to silence plant genes and change plant properties using plant RNA spraying and other approaches for dsRNA delivery. In this study, we investigated the effect of exogenous gene-specific dsRNAs on the silencing of four tomato genes encoding MYB-family transcription repressors of anthocyanin biosynthesis in the leaves of tomato Solanum lycopersicum L. We found that the exogenous application of dsRNAs encoding for the SlMYBATV1, SlMYB32, SlMYB76, and SlTRY genes downregulated mRNA levels of these endogenous repressors of anthocyanin production, upregulated the expression of anthocyanin biosynthesis-related genes, and enhanced anthocyanin content in the leaves of S. lycopersicum. The data demonstrated that exogenous gene-specific dsRNAs can induce post-transcriptional gene silencing in tomato leaves by direct foliar application of dsRNAs. This approach may be used for plant secondary metabolism induction and as a silencing tool for gene function studies without the need to produce genetically modified plants.

Modulation of Anthocyanin Biosynthesis-Related Genes during the Ripening of Olea europaea L. cvs Carolea and Tondina Drupes in Relation to Environmental Factors.

Anthocyanins protect plants against various biotic and abiotic stresses, and anthocyanin-rich foods exert benefits on human health due to their antioxidant activity. Nevertheless, little information is available on the influence of genetic and environmental factors on the anthocyanin content in olive fruits. Based on this consideration, the total anthocyanin content, the genes involved in anthocyanin biosynthesis, and three putative R2R3-MYB transcription factors were evaluated at different ripening stages in the drupes of the Carolea and Tondina cultivars, sampled at different altitudes in the Calabria region, Italy. During drupe ripening, the total anthocyanin content and the transcript levels of analyzed genes gradually increased. In line with the anthocyanin content, a different level of expression of anthocyanin structural genes was observed in 'Carolea' compared to 'Tondina', and in relation to the cultivation area. Furthermore, we identified Oeu050989.1 as a putative R2R3-MYB involved in the regulation of anthocyanin structural genes correlated with the environmental temperature change response. We conclude that anthocyanin accumulation is strongly regulated by development, genotype, and also by environmental factors such as temperature, associated with the altitude gradient. The obtained results contribute to reducing the current information gap regarding the molecular mechanisms on anthocyanin biosynthesis regulation related to the environmental conditions in Olea europaea.

Comparative Transcriptome and Metabolome Analyses of Broccoli Germplasms with Purple and Green Curds Reveal the Structural Genes and Transitional Regulators Regulating Color Formation.

Owing to the high anthocyanin content, broccoli varieties with purple curds have become more popular in food inventories, while the genetic mechanisms of anthocyanin biosynthesis pathways remain largely unknown. We bred a pair of near-isogenic lines (NILs), GB767 and PB767, whose curds exhibited green and purple colors, respectively, due to the purple sepals of florets. RNA sequencing and widely targeted metabolic analyses were conducted. Compared with GB767, eighteen anthocyanin biosynthesis-related genes exhibited significantly higher expressions in PB767, and in turn, the expression level of BolMYBL2.1 was attenuated. A comparison of the metabolites in the flavonoid biosynthetic pathways revealed 142 differentially accumulated metabolites, among which higher content of anthocyanins was responsible for the purple color of PB767. Interestingly, the total cyanidin contents were similar between the curds of NILs, whereas total delphinidin contents were increased by more than 170 times in purple curds, presumably due to a non-canonical F3'H/CYP75B gene, BolC02g015480.2J, with elevated expression in PB767. Furthermore, correlation analysis further confirmed that the identified nineteen DEGs were significantly correlated with seven differentially accumulated anthocyanins in PB767. Together, these results identified the metabolic factors and genes that contribute to the purplish curds, which could lay foundations for the breeding programs of purple broccoli.

Persulfidation of transcription factor MYB10 inhibits anthocyanin synthesis in red-skinned pear.

Hydrogen sulfide (H2S) is a gaseous signaling molecule that delays color change during fruit ripening. Whether H2S affects anthocyanin biosynthesis in red-skinned pears (Pyrus L.) remains unclear. Here, we found that H2S substantially inhibits anthocyanin accumulation in red-skinned pears and the expression of several genes encoding transcription factors is affected in response to H2S signaling. For example, PyMYB10 and PyMYB73 were down-regulated, whereas PyMYB114 and PyMYB6 were up-regulated. Bioinformatics analysis showed that PyMYB73 and PyMYB6, each containing an EAR motif, may negatively regulate anthocyanin accumulation. Transient expression analysis showed that PyMYB73 substantially promotes anthocyanin biosynthesis by co-transforming with PyMYB10/PyMYB114 + PybHLH3; however, PyMYB6 inhibited anthocyanin biosynthesis in strawberry (Fragaria vesca) receptacles and pear fruits, and PyMYB73 interacted with PyMYB10 and PyMYB6 but not PyMYB114 or PybHLH3. Further investigation showed that Cys194 and Cys218 of PyMYB10 were modified by persulfidation and that PyMYB10Cys218Ala substantially increased anthocyanin accumulation by a transient transformation system. Co-transformation of PyMYB10Cys218Ala + PyMYB73/PyMYB6 also promoted anthocyanin accumulation in pear fruits. Yeast two-hybrid assays showed that the mutation of PyMYB10 did not affect the interaction between PyMYB10 and PyMYB73, but it inhibited interaction with PyMYB6. Moreover, H2S weakened the interaction between PyMYB10 and PyMYB73 but enhanced the interaction with PyMYB6. Thus, we provided a model in which PyMYB10 undergoes persulfidation at Cys218, enhancing the interaction with PyMYB6 and reducing the interaction with PyMYB73. These subsequently results in lower expression of the anthocyanin biosynthesis-related genes Pyrus dihydroflavonol 4-reductase (PyDFR), Pyrus anthocyanidin synthase (PyANS), Pyrus UDP-glucose: flavonoid 3-glucosyl transferase (PyUFGT) and Pyrus glutathione S-transferase (PyGST), thereby inhibiting anthocyanin accumulation in red-skinned pears. Our findings provided a molecular mechanism for H2S-mediated anthocyanin biosynthesis in red-skinned pears.

Ethylene Signal Is Involved in the Regulation of Anthocyanin Accumulation in Flesh of Postharvest Plums (Prunus salicina Lindl.)

Ethylene is positively correlated with the anthocyanin accumulation in postharvest plum fruit, but the regulation mechanism has not been fully clarified. In this work, the 'Friar' plum fruit under different storage temperatures (0, 10 and 25 °C) and treatments (100.0 μL L-1 ethylene and 1.0 μL L-1 1-MCP) were applied to study the relationship between anthocyanin accumulation and ethylene signal pathway. The fruits stored at 10 °C had higher ethylene production rate and more anthocyanin in flesh than those stored at 0 °C and 25 °C. Ten ethylene biosynthesis associated genes and forty-one ethylene signal transduction related genes were obtained from the previous transcriptome data. Among them, the expression levels of ethylene biosynthesis associated genes (PsACS1, PsACS4 and PsACO1), and ethylene signal transduction related genes (PsERS1s, PsETR2, PsERF1a, and PsERF12) were markedly higher in the fruits stored at 10 °C than those at 0 °C and 25 °C. Exogenous ethylene treatment enhanced while 1-MCP treatment inhibited the anthocyanin accumulation in the flesh under storage at 10 °C. In addition, exogenous ethylene treatment markedly increased the expression levels of PsACS1, PsACS4, PsACO1, PsETR2, PsERF1a, and PsERF12 in the flesh once it turning red, as well as the anthocyanin biosynthesis related genes (PsPAL, PsCHS, PsF3H, PsDRF, PsANS, PsUFGT and PsMYB10), whereas 1-MCP treatment manifested the contrary effects. Correlation analysis indicated that there was a significant positive correlation between genes expression related to ethylene signal pathway and anthocyanin biosynthesis, except for PsERF11. In conclusion, ethylene signal pathway is involved in the flesh reddening by up-regulating the anthocyanin biosynthesis related genes.

Investigation and Expression Analysis of R2R3-MYBs and Anthocyanin Biosynthesis-Related Genes during Seed Color Development of Common Bean (Phaseolus vulgaris).

Anthocyanins are responsible for the coloration of common bean seeds, and their accumulation is positively correlated with the expression level of anthocyanin biosynthetic genes. The MBW (MYB-bHLH-WD40) complex is thought to regulate the expression of these genes, and MYB proteins, which are a key factor in activating anthocyanin pathway genes, have been identified in several plants. This study demonstrated gene structures, chromosomal placements, gene duplications of R2R3-MYBs, miRNAs associated with R2R3-MYBs, and the interaction of these genes with other flavonoid regulatory genes. qRT-PCR was used to investigate the role of specific R2R3-MYBs and flavonoid genes in common bean seed color development. As a result of a comprehensive analysis with the help of in silico tools, we identified 160 R2R3-MYB genes in the common bean genome. We divided these genes into 16 classes on the basis of their intron-exon and motif structures. Except for three, the rest of the common bean R2R3-MYB members were distributed to all chromosomes with different densities, primarily located on chromosomes 3 and 8. We identified a total of 44 duplicated gene pairs dispersed across 11 chromosomes and evolved under purifying selection (Ka/Ks < 1), 19 of which were derived from a whole-genome duplication. Our research uncovered 25 putative repressor PvMYB proteins that contain the EAR motif. Additionally, fifty different cis-regulatory elements regulated by light, stress, and hormone were identified. Within the genome of the common bean, we discovered a total of 36 microRNAs that target a total of 72 R2R3-MYB transcripts. The effect of 16 R2R3-MYB genes and 16 phenylpropanoid pathway genes, selected on the basis of their interaction in the protein-protein interaction map, playing role in the regulation of seed coat color development was evaluated using qRT-PCR in 5 different tissues at different developmental stages. The results revealed that these specific genes have different expression levels during different developmental periods, with higher levels in the pod filling and early pod stages than in the rest of the developmental periods. Furthermore, it was shown that PvTT8 (bHLH), PvTT2 (PvMYB42), PvMYB113, PvTTG1, and PvWD68 genes have effects on the regulation of seed coat color. The findings of this study, which is the first to use whole-genome analysis to identify and characterize the R2R3-MYB genes in common bean, may serve as a reference for future functional research in the legume.

Purple stem Brassica napus exhibits higher photosynthetic efficiency, antioxidant potential and anthocyanin biosynthesis related genes expression against drought stress.

Purple-stem Brassica napus (B. napus) is a phenotype with unique color because of its high anthocyanins content. Anthocyanins are naturally occurring plant pigments that have antioxidants activity and play important role in plant defense against abiotic and biotic stresses. In the present study, drought induced effects on plants were investigated in hydroponically grown seedlings of green stem (GS) and purple stem (PS) genotypes of B. napus. The results of this study showed that the major function of anthocyanins accumulation during drought was to enhance the antioxidant capability and stress tolerance in B. napus plants. Our results showed that drought significantly inhibited the plant growth in terms of decreased biomass accumulation in both genotypes, although marked decline was observed in GS genotype. The reduction in photosynthetic attributes was more noticeable in the GS genotype, whereas the PS genotype showed better performance under drought stress. Under stressful conditions, both the genotype showed excessive accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), as well as higher levels of antioxidant enzymes activities. Under drought conditions, the GS genotype showed apparent damages on chloroplast deformation like in thylakoid membrane and grana structural distortion and fewer starch grains and bigger plastoglobuli. Moreover, during drought stress, the PS genotype exhibited maximum expression levels of anthocyanins biosynthesis genes and antioxidant enzymes accompanied by higher stress tolerance relative to GS genotype. Based on these findings, it can be concluded that GS genotype found more sensitive to drought stress than the PS genotype. Furthermore this research paper also provides practical guidance for plant biologists who are developing stress-tolerant crops by using anthocyanin biosynthesis or regulatory genes.

Expression of anthocyanin biosynthesis-related genes during flower development in Lilium spp.

Sterility of hybrids produced from interspecific hybridization in lilies (Lilium, Liliaceae) is a great limitation in the breeding program, especially for Lilium longiflorum, which only has white-flowered cultivars. Because modification of flower colour in L. longiflorum by conventional breeding is limited by pre- and post-fertilization barriers, we think genetic modification could be used as an alternative in the future. For this, we need to understand what determines white colouration in L. longiflorum and other species and identify the molecular mechanisms regulating flower colour. In this study, we determined the accumulation of anthocyanins and related compounds in flower tissues during flower developmental stages in L. longiflorum cultivar ‘Lincoln’ and in the Oriental hybrid lily cultivars ‘Rialto’, ‘Perth’ and ‘Gran Tourismo’, respectively with white, pink and red flowers. Furthermore, the presence/absence and the expression of eight structural genes (CHSa, CHSb, CHIa, CHIb, F3H, F3’H, DFR, ANS) and three transcription factor genes (MYB12, MYB15, bHLH2) in flower tissues were investigated. Two structural genes (LlLinF3’H and LlLinDFR) and one transcription factor gene (LlLinbHLH2) were not detected in ‘Lincoln’ flowers. In ‘Rialto’, an amino acid substitution in the R2 repeat of LhRiaMYB12 which was previously reported to be responsible for the white flower colour is also found in the LhPerMYB12 of the pink ‘Perth’ flowers. Moreover, LhRiaDFR is present but not expressed in ‘Rialto’ flowers. Accumulation of cyanidin was observed in the flowers of ‘Perth’ and ‘Gran Tourismo’. High amounts of dihydrokaempferol accumulated in flowers of all four lily cultivars confirming the expression and functionality of early structural genes in the pathway. The elevated expression of the structural genes is strongly correlated with the expression of LhMYB12 and LhMYB15. This information can be used in the future to generate new L. longiflorum or Oriental lily hybrid cultivars with novel flower colours.

A highly efficient mesophyll protoplast isolation and PEG-mediated transient expression system in eggplant

Protoplasts are widely used in transient expression systems that are a simple, robust, and powerful method for gene function verification. However, such a versatile tool has not been developed using eggplant. We here established an efficient protocol for protoplast isolation from plantlet leaves of eggplant by optimizing enzymatic conditions. This isolation required an optimal D-mannitol concentration (0.5 M), enzyme concentration (1.25% (w/v) cellulose and 0.4% (w/v) macerozyme), digestion time (3 h), and temperature (26 ℃). This protocol provided the highest yield (1.2 × 107/g fresh weight (FW)) and viability (96%) of protoplasts. Furthermore, the polyethylene glycol (PEG)-mediated transient transformation of eggplant protoplasts with the marker gene yellow fluorescent protein (YFP) was investigated by optimizing different crucial factors affecting transfection efficiency, including plasmid DNA amount, PEG concentration, and transfection duration. More than 50% transfection efficiency could be obtained with the optimized system. In addition, subcellular localization of several fusion proteins was verified, and the bimolecular fluorescence complementation assay was used to detect protein–protein interaction in this system. Dual-luciferase assays in eggplant protoplasts showed that the expression of anthocyanin biosynthesis-related structural genes (SmCHS and SmDFR) was activated by the transcription factor SmMYB1. Taken together, we developed a highly efficient and stable protocol for protoplast isolation and transfection in eggplant, thereby facilitating gene function characterization in molecular breeding.

Anthocyanins identification and transcriptional regulation of anthocyanin biosynthesis in purple Brassica napus.

The main anthocyanin components were identified, and the transcriptional regulation pattern of anthocyanin related genes in leaves and stem bark was elucidated in a purple B. napus. Brassica napus is one of the most important oil crops planted worldwide, and developing varieties of dual-purpose for oil and vegetable is beneficial to improve economic benefits. Anthocyanins are a class of secondary metabolites that not only make plants present beautiful colors, but have a variety of important physiological functions and biological activities. Therefore, increasing the accumulation of anthocyanin in vegetative organs can improve vegetable value of rapeseed. However, anthocyanin enriched varieties in vegetative organs are rare, and there are few studies on category identification and accumulation mechanism of anthocyanin, which limits the utilization of anthocyanins in B. napus. In this study, 157 anthocyanin biosynthesis related genes (ABGs) were identified in B. napus genome by homology comparison and collinearity analysis of genes related to anthocyanin synthesis and regulation in Arabidopsis. Moreover, five anthocyanins were identified in the stem bark and leaves of the purple B. napus PR01 by high performance liquid chromatography-mass spectrometry (HPLC-MS), and the expression characteristics of ABGs in the leaves and stem bark of PR01 were analyzed and compared with the green cultivar ZS11 by RNA-Seq. Combining further weighted gene co-expression network analysis (WGCNA), the up-regulation of transcript factors BnaA07. PAP2 and BnaC06. PAP2 were identified as the key to the up-regulation of most of anthocyanin synthesis genes that promoted anthocyanin accumulation in PR01. This study is helpful to understand the transcriptional regulation of anthocyanin biosynthesis in B. napus and provides the theoretical basis for breeding novel varieties of dual-purpose for oil and vegetable.