Construction of yeast one-hybrid library and screening of transcription factors regulating LhMYBSPLATTER expression in Asiatic hybrid lilies (Lilium spp.)

Background Anthocyanins, which belong to flavonoids, are widely colored among red-purple pigments in the Asiatic hybrid lilies (Lilium spp.). Transcription factor (TF) LhMYBSPLATTER (formerly known as LhMYB12-Lat), identified as the major kernel protein, regulating the anthocyanin biosynthesis pathway in ‘Tiny Padhye’ of Tango Series cultivars, which the pigmentation density is high in the lower half of tepals and this patterning is of exceptional ornamental value. However, the research on mechanism of regulating the spatial and temporal expression differences of LhMYBSPLATTER, which belongs to the R2R3-MYB subfamily, is still not well established. To explore the molecular mechanism of directly related regulatory proteins of LhMYBSPLATTER in the anthocyanin pigmentation, the yeast one-hybrid (Y1H) cDNA library was constructed and characterized. Results In this study, we describe a yeast one-hybrid library to screen transcription factors that regulate LhMYBSPLATTER gene expression in Lilium, with the library recombinant efficiency of over 98%. The lengths of inserted fragments ranged from 400 to 2000 bp, and the library capacity reached 1.6 × 106 CFU of cDNA insert, which is suitable to fulfill subsequent screening. Finally, seven prey proteins, including BTF3, MYB4, IAA6-like, ERF4, ARR1, ERF WIN1-like, and ERF061 were screened by the recombinant bait plasmid and verified by interaction with the LhMYBSPLATTER promoter. Among them, ERFs, AUX/IAA, and BTF3 may participate in the negative regulation of the anthocyanin biosynthesis pathway in Lilium. Conclusion A yeast one-hybrid library of lily was successfully constructed in the tepals for the first time. Seven candidate TFs of LhMYBSPLATTER were screened, which may provide a theoretical basis for the study of floral pigmentation.

Solanum galapagense-derived purple tomato fruit color is conferred by novel alleles of the Anthocyanin fruit and atroviolacium loci

One hypothesis for the origin of endemic species of tomato on the Galápagos islands postulates a hybridization of Solanum pimpinellifolium and S. habrochaites. S. galapagense accession LA1141 has purple fruit pigmentation which has previously been described in green-fruited wild tomatoes such as S. habrochaites. Characterization of LA1141 derived purple pigmentation provides a test of the hybridization hypothesis. Purple pigmentation was recovered in progenies derived from LA1141 and the anthocyanins malvidin 3(coumaroyl)rutinoside-5-glucoside, petunidin 3-(coumaroyl) rutinoside-5-glucoside, and petunidin 3-(caffeoyl)rutinoside-5-glucoside were abundant. Fruit color was evaluated in an introgression population and three quantitative trait loci (QTLs) were mapped and validated in subsequent populations. The loci atroviolacium on chromosome 7, Anthocyanin fruit on chromosome 10, and uniform ripening also on chromosome 10, underly these QTLs. Sequence analysis suggested that the LA1141 alleles of Aft and atv are unique relative to those previously described from S. chilense accession LA0458 and S. cheesmaniae accession LA0434, respectively. Phylogenetic analysis of the LA1141 Aft genomic sequence did not support a green-fruited origin and the locus clustered with members of the red-fruited tomato clade. The LA1141 allele of Aft is not the result of an ancient introgression and underlies a gain of anthocyanin pigmentation in the red-fruited clade.

Prey Capture in Anthocyanin-free Sarracenia leucophylla (Sarraceniaceae) Is Associated with Leaf Size, But Not Red Pigmentation

Anthocyanin pigmentation is a significant horticultural feature in plants and can be a crucial mediator of plant–insect interactions. In carnivorous plants, the modified leaves that capture prey can be visually striking and are traditionally considered prey attractants. Nevertheless, the question of whether bold color and venation patterns function as lures for insect prey remains ambiguous, and appears to vary across taxa. Furthermore, vegetative pigments can have alternate functions as protectants against thermal and oxidative damage. Our dual-year study compares the wild-type pitcher phenotype with a true-breeding anthocyanin-free mutant of the white-topped pitcher plant (Sarracenia leucophylla Raf.). We bred full-sibling crosses of S. leucophylla carrying either the wild-type anthocyanin gene or the anthocyanin-free variant. In both experimental years, growth points were established in outdoor plots and pitchers were allowed to capture prey before harvest at the end of each growing season. Dry weight of prey biomass was measured from pitchers of both pigment morphs, along with nectary counts, pitcher size, and internal temperature. The presence of anthocyanins in trapping leaves did not affect the biomass of insects captured. Nor did wild-type or anthocyanin-free pitcher morphs differ in size, temperature, or nectary counts. Instead, pitcher height, and, nominally, mouth diameter were better predictors of prey biomass. Despite striking visual differences in pitcher color, wild-type and anthocyanin-free plants did not catch significantly different quantities of prey. Our study provides empirical data that anthocyanin pigmentation in S. leucophylla does not affect the capture of prey biomass, and supports a growing body of literature showing that pigmentation traits serve in multiple contexts.

Genetic and Transcription Profile Analysis of Tissue-Specific Anthocyanin Pigmentation in Carrot Root Phloem

In purple carrots, anthocyanin pigmentation can be expressed in the entire root, or it can display tissue specific-patterns. Within the phloem, purple pigmentation can be found in the outer phloem (OP) (also called the cortex) and inner phloem (IP), or it can be confined exclusively to the OP. In this work, the genetic control underlying tissue-specific anthocyanin pigmentation in the carrot root OP and IP tissues was investigated by means of linkage mapping and transcriptome (RNA-seq) and phylogenetic analyses; followed by gene expression (RT-qPCR) evaluations in two genetic backgrounds, an F2 population (3242) and the inbred B7262. Genetic mapping of ‘root outer phloem anthocyanin pigmentation’ (ROPAP) and inner phloem pigmentation (RIPAP) revealed colocalization of ROPAP with the P1 and P3 genomic regions previously known to condition pigmentation in different genetic stocks, whereas RIPAP co-localized with P3 only. Transcriptome analysis of purple OP (POP) vs. non-purple IP (NPIP) tissues, along with linkage and phylogenetic data, allowed an initial identification of 28 candidate genes, 19 of which were further evaluated by RT-qPCR in independent root samples of 3242 and B7262, revealing 15 genes consistently upregulated in the POP in both genetic backgrounds, and two genes upregulated in the POP in specific backgrounds. These include seven transcription factors, seven anthocyanin structural genes, and two genes involved in cellular transport. Altogether, our results point at DcMYB7, DcMYB113, and a MADS-box (DCAR_010757) as the main candidate genes conditioning ROPAP in 3242, whereas DcMYB7 and MADS-box condition RIPAP in this background. In 7262, DcMYB113 conditions ROPAP.

Comparative Transcriptome Analysis of the Accumulation of Anthocyanins Revealed the Underlying Metabolic and Molecular Mechanisms of Purple Pod Coloration in Okra (Abelmoschus esculentus L.)

Color is an essential agronomic trait and the consumption of high anthocyanin containing vegetables in daily diet does provide benefits to human health, but the mechanisms on anthocyanin accumulation in tender pods of okra (Abelmoschus esculentus L.) were totally unknown. In this study, a wide characterization and quantitation of anthocyanins and flavonols in tender pods of 15 okra varieties were performed by UHPLC-Q-Orbitrap HRMS for the first time. Two major anthocyanins (delphinidin 3-O-sambubioside and cyanidin 3-O-sambubioside) and six kinds of flavonol glycosides (most are quercetin-based) were identified and quantified. The coloration of the purple okra pod mainly arises from the accumulation of both delphinidin 3-O-sambubioside and cyanidin 3-O-sambubioside in most of purple varieties (Hong Yu, Bowling Red and Burgundy), except Jing Orange. The significant differences in the compositions and contents of anthocyanins are responsible for the pod color ranging from brick-red to purplish-red among the various okra cultivars. Furthermore, four representative okra cultivars exhibiting obvious differences in anthocyanin accumulation were further analyzed with transcriptome and more than 4000 conserved differentially expressed genes were identified across the three compared groups (B vs. BR, B vs. HY and B vs. JO). Based on the comprehensive analysis of transcriptomic data, it was indicated that MBW complex consisting of AeMYB114, AeTT8, and AeTTG1 and other transcriptional factors coordinately regulate the accumulation of anthocyanins via the transcriptional regulation of structural genes. Moreover, four independent working models explaining the diversities of anthocyanin pigmentation in okra pods were also proposed. Altogether, these results improved our understanding on anthocyanin accumulation in okra pods, and provided strong supports for the development of okra pod as a functional food in the future.

Construction of Yeast One-hybrid Library and Screening of Transcription Factors Regulating LhMYB12-Lat Expression in Asiatic Hybrid Lilies (Lilium spp.)

Background: Anthocyanins, which belong to flavonoids, are widely colored among red-purple pigments in the Asiatic hybrid lilies (Lilium spp.). Transcription factor (TFs) LhMYB12-Lat, identified as the major kernel protein, regulating the anthocyanin biosynthesis pathway in ‘Tiny Padhye’ of Tango series cultivars, which the pigmentation density is high in the lower half of tepals and this patterning is of exceptional ornamental value. However, the research on mechanism of regulating the spatial and temporal expression differences of LhMYB12-Lat, which belongs to the R2R3-MYB subfamily, is still not well established. To explore the molecular mechanism of directly related regulatory proteins of LhMYB12-Lat in the anthocyanin pigmentation, the yeast one-hybrid (Y1H) cDNA library was constructed and characterized. Results: In this study, we describe a yeast one-hybrid library to screen transcription factors that regulate LhMYB12-Lat gene expression in Lilium, with the library recombinant efficiency of over 98%. The lengths of inserted fragments ranged from 400-2000 bp, and the library capacity reached 1.6 × 106 CFU of cDNA insert, which is suitable to fulfill subsequent screening. Finally, seven prey proteins, including BTF3, MYB4, IAA6-like, ERF4, ARR1, ERF WIN1-like, and ERF061 were screened by the recombinant bait plasmid and verified by interaction with the LhMYB12-Lat promoter. Among them, ERF, AUX/IAA, and BTF3 may participate in the negative regulation of the anthocyanin biosynthesis pathway in Lilium.Conclusion: A yeast one-hybrid library of lily was successfully constructed in the tepals for the first time. Seven candidate TFs of LhMYB12-Lat were screened, which may provide a theoretical basis for the study of floral pigmentation.

Anthocyanin Biosynthesis Genes as Model Genes for Genome Editing in Plants

CRISPR/Cas, one of the most rapidly developing technologies in the world, has been applied successfully in plant science. To test new nucleases, gRNA expression systems and other inventions in this field, several plant genes with visible phenotypic effects have been constantly used as targets. Anthocyanin pigmentation is one of the most easily identified traits, that does not require any additional treatment. It is also associated with stress resistance, therefore plants with edited anthocyanin genes might be of interest for agriculture. Phenotypic effect of CRISPR/Cas editing of PAP1 and its homologs, DFR, F3H and F3′H genes have been confirmed in several distinct plant species. DFR appears to be a key structural gene of anthocyanin biosynthesis, controlled by various transcription factors. There are still many promising potential model genes that have not been edited yet. Some of them, such as Delila, MYB60, HAT1, UGT79B2, UGT79B3 and miR156, have been shown to regulate drought tolerance in addition to anthocyanin biosynthesis. Genes, also involved in trichome development, such as TTG1, GLABRA2, MYBL2 and CPC, can provide increased visibility. In this review successful events of CRISPR/Cas editing of anthocyanin genes are summarized, and new model genes are proposed. It can be useful for molecular biologists and genetic engineers, crop scientists, plant genetics and physiologists.

The report of anthocyanins in the betalain-pigmented genus Hylocereus is not well evidenced and is not a strong basis to refute the mutual exclusion paradigm

Here we respond to the paper entitled “Contribution of anthocyanin pathways to fruit flesh coloration in pitayas” (Fan et al., BMC Plant Biol 20:361, 2020). In this paper Fan et al. 2020 propose that the anthocyanins can be detected in the betalain-pigmented genus Hylocereus, and suggest they are responsible for the colouration of the fruit flesh. We are open to the idea that, given the evolutionary maintenance of fully functional anthocyanin synthesis genes in betalain-pigmented species, anthocyanin pigmentation might co-occur with betalain pigments, as yet undetected, in some species. However, in absence of the LC-MS/MS spectra and co-elution/fragmentation of the authentic standard comparison, the findings of Fan et al. 2020 are not credible. Furthermore, our close examination of the paper, and re-analysis of datasets that have been made available, indicate numerous additional problems. Namely, the failure to detect betalains in an untargeted metabolite analysis, accumulation of reported anthocyanins that does not correlate with the colour of the fruit, absence of key anthocyanin synthesis genes from qPCR data, likely mis-identification of key anthocyanin genes, unreproducible patterns of correlated RNAseq data, lack of gene expression correlation with pigmentation accumulation, and putative transcription factors that are weak candidates for transcriptional up-regulation of the anthocyanin pathway.

Demonstration of local adaptation of maize landraces by reciprocal transplantation

Populations are locally adapted when they exhibit higher fitness than foreign populations in their native habitat. Maize landrace adaptations to highland and lowland conditions are of interest to researchers and breeders. To determine the prevalence and strength of local adaptation in maize landraces, we performed a reciprocal transplant experiment across an elevational gradient in Mexico. We grew 120 landraces, grouped into four populations (Mexican Highland, Mexican Lowland, South American Highland, South American Lowland), in Mexican highland and lowland common gardens and collected phenotypes relevant to fitness, as well as reported highland-adaptive traits such as anthocyanin pigmentation and macrohair density. 67k DArTseq markers were generated from field specimens to allow comparison between phenotypic patterns and population genetic structure. We found phenotypic patterns consistent with local adaptation, though these patterns differ between the Mexican and South American populations. While population genetic structure largely recapitulates drift during post-domestication dispersal, landrace phenotypes reflect adaptations to native elevation. Quantitative trait QST was greater than neutral FST for many traits, signaling divergent directional selection between pairs of populations. All populations exhibited higher fitness metric values when grown at their native elevation, and Mexican landraces had higher fitness than South American landraces when grown in our Mexican sites. Highland populations expressed generally higher anthocyanin pigmentation than lowland populations, and more so in the highland site than in the lowland site. Macrohair density was largely non-plastic, and Mexican landraces and highland landraces were generally more pilose. Analysis of δ13C indicated that lowland populations may have lower WUE. Each population demonstrated garden-specific correlations between highland trait expression and fitness, with stronger positive correlations in the highland site. These results give substance to the long-held presumption of local adaptation of New World maize landraces to elevation and other environmental variables across North and South America.

The effects of climate change on floral anthocyanin polymorphisms

Pigmentation affords resistance to abiotic stressors, and thus can respond adaptively or plastically to drought and extreme temperatures associated with global change. Plants frequently display variability in flower coloration that is underlain by anthocyanin pigmentation. While anthocyanin polymorphisms impact plant–animal interactions, they also impact reproductive performance under abiotic stress. We used descriptions of flower colour from over 1900 herbarium records representing 12 North American species spanning 124 years to test whether anthocyanin-based flower colour has responded to global change. Based on demonstrated abiotic associations with performance of anthocyanin colour morphs, we predicted pigmentation would increase in species experiencing increased aridity, but decline in those experiencing larger increases in temperature. We found that the frequency of reports of pigmented morphs increased temporally in some taxa but displayed subtle declines in others. Pigmentation was negatively associated with temperature and positively associated with vapour pressure deficit (a metric of aridity) across taxa. Species experiencing larger temperature increases over time displayed reductions in pigmentation, while those experiencing increases in aridity displayed increases in pigmentation. Change in anthocyanin-based floral colour was thus linked with climatic change. Altered flower coloration has the strong potential to impact plant–animal interactions and overall plant reproductive performance.