MYB Transcription Factor OsC1PLSr Involves the Regulation of Purple Leaf Sheath in Rice.

Although several regulators associated with purple traits in rice have been identified, the genetic basis of the purple sheath remains unclear. In the present study, F2-1 and F2-2 populations were constructed using purple sheath (H93S) and green sheath (R1173 and YHSM), respectively. In order to identify QTL loci in purple sheaths, BSA analyses were performed on the two F2 populations. A crucial QTL for purple sheath was identified, tentatively named qPLSr6, and was located in the 4.61 Mb to 6.03 Mb region of chromosome 6. Combined with expression pattern analysis of candidate genes, LOC_Os06g10350 (OsC1PLSr) was suggested as a candidate gene. The homozygous mutant KO-1 and KO-2 created through CRISPR/Cas9 editing, lost their purple leaf sheath. The RT-PCR revealed that OsC1PLSr, anthocyanin synthase (ANS), diflavonol-4-reductase (DFR), flavanone-3-hydroxylase (F3H), and flavanone-3'-hydroxylase (F3'H) expression levels were dramatically down-regulated in the mutants. The yeast report system indicated that the 145-272 aa region at the C-terminal of OsC1PLSr is a positive transcriptional activation domain. The results indicated that OsC1PLSr synthesized anthocyanins by regulating the expression of ANS, DFR, F3H, and F3'H. This study provides new insights into the genetic basis of the purple sheath.

Integrative Analysis of Metabolomics and Transcriptomics Reveals Molecular Mechanisms of Anthocyanin Metabolism in the Zikui Tea Plant (Camellia sinensis cv. Zikui).

In this study, we performed an association analysis of metabolomics and transcriptomics to reveal the anthocyanin biosynthesis mechanism in a new purple-leaf tea cultivar Zikui (Camellia sinensis cv. Zikui) (ZK). Three glycosylated anthocyanins were identified, including petunidin 3-O-glucoside, cyanidin 3-O-galactoside, and cyanidin 3-O-glucoside, and their contents were the highest in ZK leaves at 15 days. This is the first report on petunidin 3-O-glucoside in purple-leaf tea. Integrated analysis of the transcriptome and metabolome identified eleven dependent transcription factors, among which CsMYB90 had strong correlations with petunidin 3-O-glucoside, cyanidin 3-O-galactoside, and cyanidin 3-O-glucoside (PCC > 0.8). Furthermore, we also identified key correlated structural genes, including two positively correlated F3'H (flavonoid-3'-hydroxylase) genes, two positively correlated ANS (anthocyanin synthase) genes, and three negatively correlated PPO (polyphenol oxidase) genes. Overexpression of CsMYB90 in tobacco resulted in dark-purple transgenic calluses. These results showed that the increased accumulation of three anthocyanins in ZK may promote purple-leaf coloration because of changes in the expression levels of genes, including CsMYB90, F3'Hs, ANSs, and PPOs. These findings reveal new insight into the molecular mechanism of anthocyanin biosynthesis in purple-leaf tea plants and provide a series of candidate genes for the breeding of anthocyanin-rich cultivars.

RNA-Seq-Based Profiling of pl Mutant Reveals Transcriptional Regulation of Anthocyanin Biosynthesis in Rice (Oryza sativa L.).

Purple-colored leaves in plants attain much interest for their important biological functions and could be a potential source of phenotypic marker in selecting individuals in breeding. The transcriptional profiling helps to precisely identify mechanisms of leaf pigmentation in crop plants. In this study, two genetically unlike rice genotypes, the mutant purple leaf (pl) and wild (WT) were selected for RNA-sequencing and identifying the differentially expressed genes (DEGs) that are regulating purple leaf color. In total, 609 DEGs were identified, of which 513 and 96 genes were up- and down-regulated, respectively. The identified DEGs are categorized into metabolic process, carboxylic acid biosynthesis, phenylpropanoids, and phenylpropanoid biosynthesis process enrichment by GO analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) confirmed their association with phenylpropanoid synthesis, flavonoid synthesis, and phenylalanine metabolism. To explore molecular mechanism of purple leaf color, a set of anthocyanin biosynthetic and regulatory gene expression patterns were checked by qPCR. We found that OsPAL (Os02g0626100, Os02g0626400, Os04g0518400, Os05g0427400 and Os02g0627100), OsF3H (Os03g0122300), OsC4HL (Os05g0320700), and Os4CL5 (Os08g0448000) are associated with anthocyanin biosynthesis, and they were up-regulated in pl leaves. Two members of regulatory MYB genes (OsMYB55; Os05g0553400 and Os08g0428200), two bHLH genes (Os01g0196300 and Os04g0300600), and two WD40 genes (Os11g0132700 and Os11g0610700) also showed up-regulation in pl mutant. These genes might have significant and vital roles in pl leaf coloration and could provide reference materials for further experimentation to confirm the molecular mechanisms of anthocyanin biosynthesis in rice.

Study of the Relationship between Leaf Color Formation and Anthocyanin Metabolism among Different Purple Pakchoi Lines.

Purple pakchoi (Brassica rapa ssp. Chinensis) is particularly appreciated due to its high edible quality and ornamental value, but there are few studies on the underlying mechanisms of leaf color formation. To comprehensively assess the differences in purple formation in pakchoi, four lines of pakchoi with different purple leaves were used in this experiment to determine the pigment content and to investigate the distribution and components of anthocyanin using LCMS (Liquid Chromatography Mass Spectrometry) and leaf cross-sections. Moreover, the expression levels of anthocyanin synthesis-related genes in four lines were calculated by qRT-PCR. The results showed that three new purple lines rich in anthocyanin and of high-quality were bred, and the anthocyanin were mainly distributed in both the upper epidermis and lower epidermis of leaves. Thirteen anthocyanin components were separated and identified, all the anthocyanins were acylated and glycosylated cyanidins; the main anthocyanins in purple pakchoi were a diacylated form of cyanidin 3-trans-(feruloyl)diglucoside-5-(malonyl)glucoside. Both the ratio of non-aromatic acylated cyanidin to aromatic acylated cyanidin and the ratio of anthocyanin content to chlorophyll content were responsible for the color formation in different purple pakchoi lines. When the ratio was high, the leaf appeared reddish purple, and when the ratio was low, the leaf appeared deep purple, even blackish purple. The expression level of BrF3H was significantly correlated with the content of anthocyanin through the correlation coefficient, which was speculated to be the main anthocyanin synthesis-related gene resulting in color differences among the four purple pakchoi lines. These results will enhance our understanding for the cultivation of new purple pakchoi varieties.

Mapping and Identifying a Candidate Gene Plr4, a Recessive Gene Regulating Purple Leaf in Rice, by Using Bulked Segregant and Transcriptome Analysis with Next-Generation Sequencing.

The anthocyanin biosynthesis of rice is a major concern due to the potential nutritional value. Purple appears in various organs and tissues of rice such as pericarp, flower organs, leaves, leaf sheaths, internodes, ligules, apex, and stigma. At present, there are many studies on the color of rice pericarp, but the gene and mechanism of other organs such as leaves are still unclear, and the gene regulatory network of specific organ coloring has not been systematically understood. In this study, genetic analysis demonstrated that the purple leaf traits of rice were regulated by a recessive gene. The green leaf cultivar Y58S and purple leaf cultivar XianHongB were used to construct the mapping population. A set of near isogenicline (NIL) (BC3F1) was bred via crossing and back-crossing. The generations of BC3F2 appeared to separate four phenotypes, pl1, pl2, pl3, and pl4, due to the occurrence of a purple color in different organs. We constructed three bulked segregant analysis (BSA) pools (pl1-pl2, pl1-pl3, and pl1-pl4) by using the separated generations of BC3F5 and mapped the purple leaf gene plr4 to the vicinity of 27.9-31.1 Mb on chromosome 4. Subsequently, transcriptome sequencing (RNA-Seq) for pl3 and pl2 was used to analyze the differentially expressed genes in the localization interval, where 12 unigenes exhibited differential expression in which two genes (Os04g0577800, Os04g0616400) were downregulated. The two downregulated genes (Os04g0577800 and Os04g0616400) are possible candidate genes because of the recessive genetic characteristics of the purple leaf genes. These results will facilitate the cloning of plr4 and illustrate the molecular mechanisms of the anthocyanin synthesis pathway.

Genetics and fine mapping of a purple leaf gene, BoPr, in ornamental kale (Brassica oleracea L. var. acephala).

BACKGROUND: Due to its variegated and colorful leaves, ornamental kale (Brassica oleracea L. var. acephala) has become a popular ornamental plant. In this study, we report the fine mapping and analysis of a candidate purple leaf gene using a backcross population and an F2 population derived from two parental lines: W1827 (with white leaves) and P1835 (with purple leaves). RESULTS: Genetic analysis indicated that the purple leaf trait is controlled by a single dominant gene, which we named BoPr. Using markers developed based on the reference genome '02-12', the BoPr gene was preliminarily mapped to a 280-kb interval of chromosome C09, with flanking markers M17 and BoID4714 at genetic distances of 4.3 cM and 1.5 cM, respectively. The recombination rate within this interval is almost 12 times higher than the usual level, which could be caused by assembly error for reference genome '02-12' at this interval. Primers were designed based on 'TO1000', another B. oleracea reference genome. Among the newly designed InDel markers, BRID485 and BRID490 were found to be the closest to BoPr, flanking the gene at genetic distances of 0.1 cM and 0.2 cM, respectively; the interval between the two markers is 44.8 kb (reference genome 'TO1000'). Seven annotated genes are located within the 44.8 kb genomic region, of which only Bo9g058630 shows high homology to AT5G42800 (dihydroflavonol reductase), which was identified as a candidate gene for BoPr. Blast analysis revealed that this 44.8 kb interval is located on an unanchored scaffold (Scaffold000035_P2) of '02-12', confirming the existence of assembly error at the interval between M17 and BoID4714 for reference genome '02-12'. CONCLUSIONS: This study identified a candidate gene for BoPr and lays a foundation for the cloning and functional analysis of this gene.

Fine mapping and candidate gene analysis of an anthocyanin-rich gene, BnaA.PL1, conferring purple leaves in Brassica napus L.

Because of the advantages of anthocyanins, the genetics and breeding of crops rich in anthocyanins has become a hot research topic. However, due to the lack of anthocyanin-related mutants, no regulatory genes have been mapped in Brassica napus. In this study, we first report the characterization of a B. napus line with purple leaves and the fine mapping and candidate screening of the BnaA.PL1 gene. The amount of anthocyanins in the purple leaf line was six times higher than that in a green leaf line. A genetic analysis indicated that the purple character was controlled by an incomplete dominant gene. Through map-based cloning, we localized the BnaA.PL1 gene to a 99-kb region at the end of B. napus chromosome A03. Transcriptional analysis of 11 genes located in the target region revealed that the expression level of only the BnAPR2 gene in seedling leaves decreased from purple to reddish green to green individuals, a finding that was consistent with the measured anthocyanin accumulation levels. Molecular cloning and sequence analysis of BnAPR2 showed that the purple individual-derived allele contained 17 variants. Markers co-segregating with BnaA.PL1 were developed from the sequence of BnAPR2 and were validated in the BC4P2 population. These results suggested that BnAPR2, which encodes adenosine 5'-phosphosulfate reductase, is likely to be a valuable candidate gene. This work may lay the foundation for the marker-assisted selection of B. napus vegetables that are rich in anthocyanins and for an improved understanding of the molecular mechanisms controlling anthocyanin accumulation in Brassica.

Metabolomic and transcriptomic analyses reveal a MYB gene, CsAN1, involved in anthocyanins accumulation separation in F1 between 'Zijuan' (Camellia sinensis var. assamica) and 'Fudingdabaicha' (C. sinensis var. sinensis) tea plants.

'Zijuan' (Camellia sinensis var. assamica), a somatic mutant with purple foliage and stem selected from the Yunnan Daye cultivar, has been well developed owing to abnormal pattern of anthocyanin accumulation. However, the genetic basis for the specific accumulation of phloem glycosides is not clear. Tea plants are self-incompatible, so parents with large differences in foliage color were used for crosses to investigate the genetic mechanism of anthocyanins. In this study, 'Zijuan' and green foliage cultivar 'Fudingdabaicha' (C. sinensis var. sinensis) were used as female and male parents, respectively, to generated F1 hybrid progenies with various anthocyanin contents. In order to decipher the genetic rules of anthocyanins accumulation, we performed widely targeted metabolic and transcriptomic profiling. The results showed that cyanidin-3-O-galactoside, delphinidin-3-O-galactoside and petunidin-3-O-galactoside were the major types of anthocyanins and factors directly led to the color variation between parents and F1 plants. Transcriptomic analyses suggested the significant up-regulation of anthocyanidin synthase gene (CsANS1) and CsAN1, a MYB family gene positively regulated the expression of CsANS1, in anthocyanin-rich tea plants. Furthermore, the deletion mutation of CsAN1 was found by cloning and alignment in anthocyanin-lacking cultivars. Taken together, the function deficiency of CsAN1 is predominantly responsible for the inability of anthocyanins accumulation, and this trait is heritable in progenies through hybridization. The present study elucidated the molecular basis of leaf purple trait formation in 'zijuan' and 'Fudingdabaicha' and their F1 plants, which helps to elucidate the genetic mechanism of leaf anthocyanin accumulation regulation in tea plants, and the results provide a research reference for the selection and breeding of high anthocyanin type tea varieties.

Integrated Analysis of Transcriptome and Metabolome Reveals New Insights into the Formation of Purple Leaf Veins and Leaf Edge Cracks in Brassica juncea.

Purple leaf veins and leaf edge cracks comprise the typical leaf phenotype of Brassica juncea; however, the molecular mechanisms and metabolic pathways of the formation of purple leaf veins and leaf edge cracks remain unclear. In this study, transcriptome and metabolome analyses were conducted to explore the regulation pathway of purple leaf vein and leaf edge crack formation based on four mustard samples that showed different leaf colors and degrees of cracking. The results showed genes with higher expression in purple leaf veins were mainly enriched in the flavonoid biosynthesis pathway. Integrating related genes and metabolites showed that the highly expressed genes of ANS (BjuA004031, BjuB014115, BjuB044852, and BjuO009605) and the excessive accumulation of dihydrokaempferol and dihydroquercetin contributed to the purple leaf veins by activating the synthetic pathways of pelargonidin-based anthocyanins and delphinidin-based anthocyanins. Meanwhile, "alpha-farnesene synthase activity" and "glucan endo-1, 3-beta-D-glucosidase activity" related to the adversity were mainly enriched in the serrated and lobed leaves, indicating that the environmental pressure was the dominant factor controlling the change in leaf shape. Overall, these results provided new insights into the regulation pathways for formation of purple leaf veins and leaf edge cracks, which could better accelerate the theoretical research on purple leaf vein color and leaf edge cracks in mustard.

Metabolic Profiling and Gene Expression Analyses of Purple-Leaf Formation in Tea Cultivars (Camellia sinensis var. sinensis and var. assamica).

Purple-leaf tea cultivars are known for their specific chemical composition that greatly influences tea bioactivity and plant resistance. Some studies have tried to reveal the purple-leaf formation mechanism of tea by comparing the purple new leaves and green older leaves in the same purple-leaf tea cultivar. It has been reported that almost all structural genes involved in anthocyanin/flavonoid biosynthesis were down-regulated in purple-leaf tea cultivars when the purple new leaves become green older leaves. However, anthocyanin/flavonoid biosynthesis is also affected by the growth period of tea leaves, gradually decreasing as new tea leaves become old tea leaves. This leads to uncertainty as to whether the purple-leaf formation is attributed to the high expression of structural genes in anthocyanin/flavonoid biosynthesis. To better understand the mechanisms underlying purple-leaf formation, we analyzed the biosynthesis of three pigments (chlorophylls, carotenoids, and anthocyanins/flavonoids) by integrated metabolic and gene expression analyses in four purple-leaf tea cultivars including Camellia sinensis var. sinensis and var. assamica. Green-leaf and yellow-leaf cultivars were employed for comparison. The purple-leaf phenotype was mainly attributed to high anthocyanins and low chlorophylls. The purple-leaf phenotype led to other flavonoid changes including lowered monomeric catechin derivatives and elevated polymerized catechin derivatives. Gene expression analysis revealed that 4-coumarate: CoA ligase (4CL), anthocyanidin synthase (ANS), and UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) genes in the anthocyanin biosynthetic pathway and the uroporphyrinogen decarboxylase (HEME) gene in the chlorophyll biosynthetic pathway were responsible for high anthocyanin and low chlorophyll, respectively. These findings provide insights into the mechanism of purple-leaf formation in tea cultivars.

Purple-leaf tea (Camellia sinensis L.) ameliorates high-fat diet induced obesity and metabolic disorder through the modulation of the gut microbiota in mice.

BACKGROUND: Obesity and its associated diseases have become a major world-wide health problem. Purple-leaf Tea (Camellia sinensis L.) (PLT), that is rich of anthocyanins, has been shown to have preventive effects on obesity and metabolic disorders. The intestinal microbiota has been shown to contribute to inflammation, obesity, and several metabolic disorders. However, whether PLT consumption could prevent obesity and diet-induced metabolic diseases by modulating the gut microbiota, is not clearly understood. METHODS: In this study, six-week-old male C57BL/6 J mice were fed a normal diet (ND) or a high fat diet (HFD) without or with PLT for 10 weeks. RESULTS: PLT modulated the gut microbiota in mice and alleviated the symptoms of HFD-induced metabolic disorders, such as insulin resistance, adipocyte hypertrophy, and hepatic steatosis. PLT increased the diversity of the microbiota and the ratio of Firmicutes to Bacteroidetes. f_Barnesiellaceae, g_Barnesiella, f_Ruminococcaceae, and f_Lachnospiraceae were discriminating faecal bacterial communities of the PLT mice that differed from the HFD mice. CONCLUSIONS: These data indicate that PLT altered the microbial contents of the gut and prevented microbial dysbiosis in the host, and consequently is involved in the modulation of susceptibility to insulin resistance, hepatic diseases, and obesity that are linked to an HFD.

The Purple Leaf (pl6) Mutation Regulates Leaf Color by Altering the Anthocyanin and Chlorophyll Contents in Rice.

The anthocyanin biosynthesis attracts strong interest due to the potential antioxidant value and as an important morphological marker. However, the underlying mechanism of anthocyanin accumulation in plant tissues is not clearly understood. Here, a rice mutant with a purple color in the leaf blade, named pl6, was developed from wild type (WT), Zhenong 41, with gamma ray treatment. By map-based cloning, the OsPL6 gene was located on the short arm of chromosome 6. The multiple mutations, such as single nucleotide polymorphism (SNP) at -702, -598, -450, an insertion at -119 in the promoter, three SNPs and one 6-bp deletion in the 5'-UTR region, were identified, which could upregulate the expression of OsPL6 to accumulate anthocyanin. Subsequently, the transcript level of structural genes in the anthocyanin biosynthesis pathway, including OsCHS, OsPAL, OsF3H and OsF3'H, was elevated significantly. Histological analysis revealed that the light attenuation feature of anthocyanin has degraded the grana and stroma thylakoids, which resulted in poor photosynthetic efficiency of purple leaves. Despite this, the photoabatement and antioxidative activity of anthocyanin have better equipped the pl6 mutant to minimize the oxidative damage. Moreover, the contents of abscisic acid (ABA) and cytokanin (CK) were elevated along with anthocyanin accumulation in the pl6 mutant. In conclusion, our results demonstrate that activation of OsPL6 could be responsible for the purple coloration in leaves by accumulating excessive anthocyanin and further reveal that anthocyanin acts as a strong antioxidant to scavenge reactive oxygen species (ROS) and thus play an important role in tissue maintenance.

A rice gene, OsPL, encoding a MYB family transcription factor confers anthocyanin synthesis, heat stress response and hormonal signaling.

Plants with purple leave attain interest because of their biological importance. A new rice mutant, purple leaf (pl) was isolated from an indicia cultivar Zhenong 34, which was induced by ethyl methane sulfonate (EMS) mutagenesis. The genetic analyses substantiated that pl was corroborated by one recessive allele and confirmed by map based cloning using Insertion-Deletion (InDel) markers located on the long arm of chromosome 5. DNAseq data of the candidate part showed one bp insertion ('C' insertion) at +901 bp position in the 3rd exon of OsPL gene. The pl was characterized as purple leaves, sheaths and leaf senescence phenotype at late grain filling stage of growth cycle. It possessed abnormal cell with distorted chloroplasts, less chlorophyll, and increased anthocyanin content in leaves. The anthocyanin biosynthesis genes, OsPAL, OsCHS, OsANS, and OsMYB55 showed up-regulation in pl plants compared to wild type (WT). High super oxide dismutase enzyme (SOD), catalase enzyme activity (CAT), total soluble sugar (TSS) and malondialdehyde activity (MDA) were detected in the pl; contrastingly, photosynthesis linked genes were down-regulated. The germinated pl seeds showed comparatively higher temperature stress tolerance than WT. The phytohormones abscicic acid (ABA), jasmonic acid (JA) and indole acetic acid (IAA) content were increased significantly in the pl plants. This research work will be provided information on better understanding of the molecular mechanism toward the anthocyanin biosynthetic pathway in rice. Therefore, OsPL gene could be a good genetic tool in marker aided backcrossing or gene editing for improving the rice cultivation in future.

Genetic and Evolutionary Analysis of Purple Leaf Sheath in Rice.

BACKGROUND: Anthocyanin accumulates in many plant tissues or organs, in rice for example leading to red, purple red and purple phenotypes for protection from damage by biotic and abiotic stresses and for reproduction. Purple leaf, leaf sheath, stigma, pericarp, and apiculus are common in wild rice and landraces and occasionally found in modern cultivars. No gene directly conferring anthocyanin deposited in a purple leaf sheath has yet been isolated by using natural variants. An F2 population derived from ssp. japonica cv. Tainung 72 (TNG72) with purple leaf sheath (PSH) crossed with ssp. indica cv. Taichung Sen 17 (TCS17) with green leaf sheath (GSH) was utilized to isolate a gene conferring leaf sheath color. RESULTS: By positional cloning, 10-and 3-bp deletions in the R2R3 Myb domain of OsC1 were uncovered in GSH varieties TCS17 and Nipponbare, respectively. Allelic diversity, rather than gene expression levels of OsC1, might be responsible for anthocyanin accumulation. Parsimony-based analysis of genetic diversity in 50 accessions, including cultivars, landraces, and A-genome wild rice, suggests that independent mutation occurred in Asian, African, South American, and Australian species, while O. meridionalis had a divergent sequence. OsC1 was thought of as a domestication related gene, with up to 90 % reduction of genetic diversity in GSH; however, no values from three tests showed significant differences from neutral expectations, implying that OsC1 had not been subjected to recent selection. Haplotype network analysis revealed that species from different continents formed unique haplotypes with no gene flow. Two major groups of haplotypes corresponding to 10-bp deletion and other sequences were formed in Asian rice, including O. rufipogon, O. nivara and O. sativa. Introgressions of OsC1 between subspecies through natural and artificial hybridization were not rare. Because artificial and natural selection imposed admixture on rice germplasm in Taiwan, the genealogy of OsC1 might not be congruent with the current distribution of alleles through lineage diversification. CONCLUSION: OsC1 is responsible for purple leaf sheath, and much new information about OsC1 is provided e.g., new alleles, non-domestication syndrome, and incongruence of genealogy with geographic distribution.