Genome sequence and RNA-seq analysis reveal genetic basis of flower coloration in the giant water lily Victoria cruziana

Victoria cruziana is well known for its huge floating leaves covered with sharp spines and its night blooming. Reports indicate that white flowers open during the first night and turn pinkish during the following day and the second night. Here, we set out to unravel the molecular basis and ecological function of the flower color change in V. cruziana. A high quality genome sequence with a N50 of 14.3 Mbp and a total assembly size of 3.54 Gbp was generated as the genetic basis for this study. Comparative transcriptomics revealed the genes required for anthocyanin biosynthesis genes and their transcriptional regulators as differentially expressed between the white and the pinkish stage of a flower. Structural genes with expression differences between white and pinkish flower stages include VcrF3’H, VcrF3’5’H, VcrDFR, VcrANS, and VcrarGST. The expression pattern of the corresponding transcription factors VcrMYBSG5, VcrMYBSG6, VcrTT8, and VcrTTG1 also showed differences that aligned with the flower color.


Introduction
Waterlilies of the genus Victoria are best known for their giant floating leaves that can endure a substantial weight if equally distributed across the leaf.The genus Victoria belongs to the Nymphaeaceae and harbors three robust species V. amazonica, V. cruziana and V. boliviana that were recently proposed (Smith et al., 2022).These aquatic species are night bloomers, considered as short-lived perennial plants and naturally grow under tropical conditions.Distinctive shared characteristics are the sharp spines covering most plant parts, massive leaves, and a grand flower.The whole development from seeds to a mature giant water lily takes less than five months (Smith et al., 2022).Victoria spp.also have an economic relevance, as the large seeds and rhizomes can be used as a food source (Bortolotto et al., 2015).
The flower, a special feature of Victoria spp., has been reported to undergo a color change from white to pinkish (Figure 1) (Wu et al., 2018).Development of the Victoria flower bud starts underwater and the bud only opens after reaching the water surface.Interestingly, Victoria plants bloom at night.To the best of our knowledge, one individual plant exhibits only a single blooming flower at any given time.Each flower undergoes a flowering period of two consecutive nights, during which the flower color changes from white to pinkish.In addition, anthesis also occurs during these consecutive nights, starting with the female reception, followed by staminate maturation (male phase) (Seymour & Matthews, 2006;Jiang et al., 2021).The ecological reason and molecular basis of the flower color change are currently unknown, but multiple hypotheses exist that could explain it: (1) flower color might change following a visitation/pollination event, (2) light could trigger the accumulation of pigments once flowers emerge from the water, or (3) a developmental program could lead to pigmentation in an age-dependent manner.In the following sections, we will provide context for each of these hypotheses.
Previous studies described flower color changes following pollination events for other plant species (Weiss, 1991;Ruxton & Schaefer, 2016).Although these events are rare, floral color change can serve as a signal between plant and insect to transmit the information that pollination, or at least visitation, already occurred (Weiss, 1991;Ruxton & Schaefer, 2016;Garcia et al., 2022).Flowers of V. cruziana appear white in the first night, combined with a fruity smell, secreted by the carpellary appendages which could suggest a function in pollinator attraction (Zini et al., 2019).A previously reported pollinator of Victoria spp.are Cyclocephalini, a tribe of scarab beetles that are pantropical distributed (Moore & Jameson, 2013).Willmer et al. described the floral color alterations from lilac to white in Desmodium setigerum after receiving visitors (Willmer et al., 2009).
Light-induced accumulation of red anthocyanins has been described before in many plant species like Arabidopsis thaliana (Maier et al., 2013;Li et al., 2016), Malus spec.(Merzlyak & Chivkunova, 2000;Takos et al., 2006), or Pyrus spec.(Dussi et al., 1995).The molecular basis of this light response has been extensively studied and involves an up-regulation of the transcription factors (TFs) controlling the anthocyanin biosynthesis genes (Takos et al., 2006;Li et al., 2016).Anthocyanins can serve as sunscreen thus protecting plants against excessive light irradiation (Gould, 2004) and are strong antioxidants that can scavenge reactive oxygen species (Wang et al., 1997).
Anthocyanin formation might also take place as part of a developmental program, thus depending on the age of the flower and not directly connected to environmental conditions.For example, a study in Fuchsia excorticata demonstrated that the flower color change from green to red is age-dependent and not caused by pollination (Delph & Lively, 1989).An investigation in Pulmonaria collina also noted an age-dependent color change from red to blue and explains this as a mechanism to direct pollinators towards the young flowers thus increasing the pollination efficiency (Oberrath & Böhning-Gaese, 1999).Similar findings have been reported for Pedicularis monbeigiana which shows a color change from white to purple with increasing flower age (Sun et al., 2005).The pigments responsible for flower coloration are well studied and often belong to the anthocyanins, carotenoids, or betalains (Tanaka et al., 2008).Anthocyanins and carotenoids are taxonomically widespread and responsible for most flower colors (Tanaka et al., 2008), while betalains are restricted to the Caryophyllales (Timoneda et al., 2019).Anthocyanins are derived from phenylalanine involving the general phenylpropanoid pathway and the flavonoid biosynthesis (Figure 2).Structural genes required for the anthocyanin biosynthesis include phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL) as part of the general phenylpropanoid pathway followed by chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), anthocyanin-related Glutathione S-transferase (arGST), and UDP-glucose: flavonoid-3-O-glucosyltransferase (UFGT) as one branch of the flavonoid biosynthesis (Winkel-Shirley, 2001;Eichenberger et al., 2023).Activity of these structural genes is orchestrated by an ensemble of transcription factors.The two largest transcription factor families in plants, MYBs and bHLHs, contribute substantially to the regulation of different branches of the flavonoid biosynthesis.MYB transcription factors are able to bind DNA through a highly conserved DNA-binding repeat domain (Prouse & Campbell, 2012), while bHLHs can bind DNA with a stretch of basic amino acids (Voronova & Baltimore, 1990).MYBs can be divided into different groups based on the number of characteristic repeats with the R2R3-MYBs playing a predominant role in the control of the flavonoid biosynthesis (Baudry et al., 2004;Stracke et al., 2007;Gonzalez et al., 2008;Li, 2014;Marin & Pucker, 2023).Specific subgroups of these R2R3-MYBs have different and even opposing functions.Subgroup 4, including AtMYB4, AtMYB32, AtMYB7, AtMYB3, is known for encoding transcription factors with repressive functions (Jin et al., 2000;LaFountain & Yuan, 2021).AtMYB123 and AtMYB5 belong to subgroup 5 and regulate proanthocyanidin biosynthesis in Arabidopsis thaliana (Baudry et al., 2004;Xu et al., 2014).MYBs assigned to subgroup 6 (AtMYB113, AtMYB114, AtMYB90, AtMYB75) are involved in controlling the anthocyanin biosynthesis in A. thaliana (Borevitz et al., 2000;Marin & Pucker, 2023) whereas AtMYB111, AtMYB12 and AtMYB11 (subgroup 7) are regulating the production of flavonols (Stracke et al., 2007;Dubos et al., 2010).Over the last years, various orthologous of the well characterized MYBs in A. thaliana have been identified across plant species which enables a broader understanding of regulatory mechanisms in the plant kingdom.For example, AtMYB123 orthologs have been reported to control the anthocyanin biosynthesis in addition to the proanthocyanidin biosynthesis (Martínez-Rivas et al., 2023) and orthologs of the AtMYB5 have been reported to activate the anthocyanin biosynthesis in strawberry (Jiang et al., 2023).This suggests that the generally well conserved regulation of the anthocyanin biosynthesis might be subject to lineage-specific differences.Given that A. thaliana does not have colorful fruits and flowers, it is likely that many secrets of the anthocyanin regulation can only be discovered by exploring non-model species.Previous studies investigated the pigment composition of Victoria cruziana and identified four anthocyanins (two cyanidin and two delphinidin derivatives) in the flower (Wu et al., 2018).An increase in anthocyanin content across the blooming days correlates with the intensifying pigmentation and supports the role of anthocyanins as the predominant flower colorants (Wu et al., 2018).
To identify the genes involved in the anthocyanin biosynthesis and its regulation in V. cruziana, we performed long-read sequencing of the genome and transcriptome.Here, we provide a genome sequence of V. cruziana and a corresponding annotation supported by RNA-seq and direct RNA sequencing that serve as the basis for metabolic pathway explorations.Additionally, we conducted RNA-seq experiments to identify differentially expressed genes between white and pinkish flower stages of V. cruziana.Specifically, we aimed to unravel the genetic network underlying the flower color transition from white to pinkish.

Material and Methods
The genome of V. cruziana was sequenced with Oxford Nanopore Technologies (ONT) long reads to generate a high quality genome sequence that served as basis for the transcriptomics investigation based on direct RNA sequencing and RNA-seq (Figure 3).

Plant cultivation, DNA extraction and RNA extraction
For genomic DNA extraction, a V. cruziana plant (XX-0-BRAUN-7477852) was cultivated at 28 °C in a 500 L container to facilitate repeated sampling of young leaves and immediate processing of material.Light was provided for 16 hours per day with two LEDs (Niello ® , 300W) placed 1 m above the water surface.High molecular weight DNA extraction and quality assessment were performed based on a previously developed CTAB protocol optimized for high molecular weight DNA extraction from plants (Siadjeu et al., 2020;Wolff et al., 2023).In brief, fresh leaf material was ground in liquid nitrogen and the fine powder subjected to the CTAB-based DNA extraction procedure.RNase treatment in the CTAB-TE buffer was performed overnight at room temperature.After initial quality assessment via NanoDrop and agarose gel, short DNA fragments were depleted with the Short Read Eliminator kit.DNA quantification prior to library preparation for the nanopore sequencing was performed via Qubit.
For transcriptome analysis, young V. cruziana petals at the white and pink coloring stage were collected.Sampling was conducted in two consecutive nights during July 2023 in a dedicated Victoria glasshouse located in the Botanical Garden of TU Braunschweig (52.27043343004546, 10.5336707275785).Inner petals of the same flower at both color stages were carefully detached, immediately frozen in liquid nitrogen, and stored at -70 °C prior to RNA extraction.The initial sampling occurred on July 19th, 2023, at 10:50 PM.Four samples were taken from the not yet fully opened white flower.The weather on this day was cloudy.The subsequent day, July 20th 2023, had cloudy and sunny conditions, and five samples were collected from the pink blooming flower.The ambient temperature in the greenhouse was 24 °C during the day and 22 °C at night.The ventilation temperature remained constant at 34 °C, while humidity fluctuated between 80 % and 100 %.The plants did not receive any artificial light exposure.For irrigation, tap water with a temperature of 27 °C was used.The plants were cultivated in a pool with a depth of 1.07 meters and a width of 8.8 m x 5.5 m, with a substrate ratio of 1:1 (compost to peat).Each liter of water received 6 grams of Osmocote Pro and 6 grams of lime as fertilizer.
Total RNA was extracted with the RNA plant and fungi kit (Macherey & Nagel), together with a DNase treatment according to the manufacturer's instructions.Cell homogenization was performed using the Bertin Ribolyser at 6000 RPM with three cycles of 30 seconds each, with a 30 second break between cycles, using 3 mm steel beads.The extracted RNA quality and quantity was determined by a NanoDrop measurement and an agarose gel.The RNA was stored at -70 °C until it was sent for RNA-seq.Paired-end RNA-seq (2 x 150 nt) was conducted using Illumina NovaSeq 6000 (BMKGene).

Genome sequencing
Per library, 1 µg of high molecular weight DNA was utilized following ONT's SQK-LSK109 protocol.Priming of R9.4.1 flow cells and washing steps between sequencing runs were performed according to ONT's protocols (EXP-FLP002, EXP-WSH004).Sequencing was performed on a MinION Mk1B.FAST5 files were collected and subjected to basecalling using Guppy v6.4.6+ae70e8f with the dna_r9.4.1_450bps_hac model and a minimum quality score of 9.
Direct RNA sequencing RNA for direct RNA sequencing was extracted from one white petal with the Monarch Total RNA miniprep Kit (New England BioLabs) combined with the manufacturer specific DNase treatment.Homogenization was performed using the Bertin Ribolyser at 6500 RPM with two cycles of 30 seconds each, with a 10 second break between cycles.The extracted RNA quality and quantity was determined by NanoDrop measurement and additional Qubit measurement before conducting sequencing.Direct RNA sequencing was performed on a MinION Mk1B using an R9.4.1 flow cell and ONT's direct RNA sequencing kit SQK-RNA002 following the suppliers' protocol.Sequencing data were collected in FAST5 files and subjected to basecalling using Guppy v6.4.6+ae70e8f with the rna_r9.4.1_70bps_hac model and a minimum quality score of 7. Reads generated by direct RNA sequencing were aligned to the V. cruziana genome sequence (VB02) with minimap2 (v2.17 r941) using the following parameters: -ax splice -uf -k14 (Li, 2018).Sorting of the resulting SAM file and conversion into BAM was conducted with samtools v1.10 (Li et al., 2009).Mappings to the genome sequence were visualized in the context of the structural annotation with Integrative Genomics Viewer (IGV) v2.17.4 (Robinson et al., 2011).
Direct RNA sequencing data was utilized to validate the structure of genes of interest through manual inspection of the long read alignments.

Structural annotation
The prediction of gene models was conducted with AUGUSTUS v3.3 (Stanke et al., 2006;Keller et al., 2011) for the assembly VB01 with parameters previously optimized for the detection of non-canonical splice sites (Pucker et al., 2017).For the assembly VB02, BRAKER3 v3.0.8 (Gabriel et al., 2023) was applied with protein hints derived from Viridiplantae.fa (Kuznetsov et al., 2023) and RNA-seq hints.The RNA-seq hints were derived from a mapping of paired-end RNA-seq data generated with HISAT2 v2.2.1 (Kim et al., 2019) with default parameters against the assembly VB02.Samtools v1.10 (using htslib 1.10.2-3ubuntu0.1)(Li et al., 2009) was applied to generate a sorted BAM file which was then passed to BRAKER3.The final structural annotation was largely based on the BRAKER3 results produced for VB02 only complemented with the coding sequence of an anthocyanin synthase (ANS) gene model lifted from the VB01 annotation.The derived polypeptide sequence of ANS was compared against the VB02 assembly via tBLASTn v2.15.0+ (Gertz et al., 2006) to locate this gene.

Functional annotation and candidate gene identification
Genes associated with the anthocyanin metabolism were identified with three dedicated tools.To identify the structural genes of the flavonoid biosynthesis pathway, an analysis with KIPEs v3.2.4 (Rempel et al., 2023) and the flavonoid baits data set v.3.1.7 was conducted (Additional File 2).Flavonoid biosynthesis controlling MYB transcription factors were annotated using the MYB_annotator v1.0.1 (Pucker, 2022) with parameters described in Additional File 3. The bHLH transcription factors were identified with the bHLH_annotator v1.04 (Thoben & Pucker, 2023) with parameters described in Additional File 4. A general annotation was produced using construct_anno.pyand the functional annotation available for A. thaliana (Pucker & Iorizzo, 2023).

Assembly results
A completeness assessment revealed 92.4% and 93.6% complete BUSCO genes for the VB01 (Shasta) and VB02 (NextDenovo2) assembly, respectively.Due to a higher completeness, VB02 was selected as the representative assembly.VB02 has a size of 3.54 Gbp comprising 837 contigs with an N50 length of 14.3 Mbp.In total, 49,301 protein coding genes were annotated in VB02.The assembled genome sequence and the corresponding annotation are available via LeoPARD (https://doi.org/10.24355/dbbs.084-202406040628-0).

Structural genes of the anthocyanin biosynthesis are differentially expressed between white and pink petals
A principal component analysis (PCA) of the transcriptomic data revealed distinct clusters of the replicates derived from white flower and pink flower samples, respectively, supporting clear differences between the two groups and homogeneity within each group (Additional File 5).To understand the molecular basis of the flower color variations in V. cruziana, structural genes required for anthocyanin biosynthesis were identified.Expression profiles of the best candidates were analyzed with respect to the white and pink flower color (Figure 4).The majority of genes required for anthocyanin biosynthesis (F3'H, F3'5'H, DFR, ANS, arGST, and UFGT) exhibited higher expression levels in anthocyanin-pigmented pink petals compared to white petals (Additional File 6).Genes encoding enzymes of the general phenylpropanoid pathway (PAL, C4H and 4CL) displayed increased expression levels in the pink petals.The expression pattern of CHS and CHI matches better to the flavonol biosynthesis gene FLS than to the anthocyanin biosynthesis gene DFR, i.e., CHS and CHI exhibited a higher expression within the white petals.To convert flavanones into dihydroflavonols, F3H is required and showed increased expression levels in the samples taken from pinkish petals.Branching reactions towards delphindin or cyanidin are catalyzed by F3'5'H and F3'H, respectively.Both encoding genes are present in V. cruziana and exhibited a higher activity in pinkish petals.Dihydroflavonols are converted to the precursors of anthocyanidins, the leucoanthocyanidins, by DFR.Higher expression levels of DFR were observed in pinkish petals compared to white petals.Afterwards, the production of the anthocyanidins requires ANS and arGST.These enzymes catalyze the conversion to the anthocyanidins delphinidin and cyanidin in V. cruziana and both corresponding genes were more active in the pink petals.

Expression of anthocyanin biosynthesis regulators differs between white and pinkish flowers
Anthocyanin biosynthesis is regulated by various transcription factors, specifically the MYB family, the bHLH family, and specific WD40 proteins.These TFs are capable of forming complexes, thus activating anthocyanin production in plants.After identification of the required structural genes, best candidates for potential transcriptional regulators were analyzed.As one transcription factor with activatory properties for proanthocyanidin production, we identified a subgroup 5 MYB (VcrMYBSG5) which exhibited higher expression levels in pink compared to white petals.Further, we annotated a candidate belonging to MYB subgroup 6 (VcrMYBSG6), described for the positive regulation of anthocyanin biosynthesis.This VcrMYBSG6 was only expressed in pink petals in V. cruziana.Additionally we identified VcrMYBSG4, which could be an orthologue of the anthocyanin biosynthesis repressing regulator AtMYB004.Expression levels increased within the white petals thus showing a pattern opposite to anthocyanin accumulation.Additionally, VcrTT8 belonging to the bHLHs and VcrTTG1 representing the WD40 proteins, were predicted in the functional annotation process.Both corresponding genes indicated higher expression levels in pink petals compared to white (Figure 5).To explore other MYB transcription factors that have been reported in the context of anthocyanin biosynthesis in species closely related to V.cruiziana, a search for the ortholog of the recently published KcMYB1 from Kadsura coccinea was conducted.KcMYB1 belongs to the MYB subgroup 6 and overexpression in Nicotiana benthamiana resulted in anthocyanin accumulation (Huang et al., 2022).Using functional annotation and a phylogenetic analysis, g3771 was classified as a subgroup 5 R2R3-MYB, while KcMYB1 was located in the subgroup 6 clade close to the V. cruziana genes g3252-g3257 (Additional file 7).However, only g3253 (VcrMYBSG6) was substantially supported by aligned RNA-seq reads, indicating a promising SG6 MYB in V. cruziana.In contrast, genes without RNA-seq support might be incomplete gene models and have not been picked up as differentially expressed between white and pinkish petal samples (Additional file 7, Additional file 8).

Discussion
Differential expression of flavonoid biosynthesis genes explains anthocyanin production in pink petals of V. cruziana The continuous genome sequence of V. cruziana and the corresponding annotation enabled the identification of genes encoding relevant proteins and transcription factors required for the anthocyanin biosynthesis (Figure 6).The differential expression patterns compared between white and pink petals align with the active anthocyanin production in the pink flower.Besides higher expression levels of genes belonging to the phenylpropanoid pathway within pink petals, the first committed genes of the flavonoid biosynthesis (CHS, CHI) were higher expressed in the white flower.Additionally, FLS and LAR expression patterns did not match the expression level patterns of the other genes.The FLS pattern can be due to its activity in flavonol biosynthesis which is active in the white petals, supported by Wu et al., 2018 who identified flavonols in white V. cruziana flowers.The ANR expression pattern suggested some ongoing proanthocyanidin biosynthesis in white petals.Future metabolic studies are needed to confirm the presence of proanthocyanidins.Furthermore, F3'5'H as the branching point towards delphinidin production exhibited increased expression levels in pink petals.This finding is surprising as delphinidin is one anthocyanidin required for blue flower colors which has not been observed in Victoria spp.flowers.However, delphinidin derivatives were identified besides cyanidin derivatives as the major anthocyanins in the V. cruziana and V. amazonica (Wu et al., 2018).Perhaps blue colors are lacking due to overlay with cyanidin derivatives.As ANS and arGST encode major enzymes in anthocyanin biosynthesis, their expression pattern matches the anthocyanin accumulation in pink petals.Interestingly the arGST expression is significantly higher in pink petals compared to the corresponding ANS expression.The substantially higher expression of arGST compared to ANS might ensure the channeling of metabolites towards anthocyanins.
VcrMYBSG5 and VcrMYBSG6 may interact with VcrTT8 and VcrTTG1 to activate the anthocyanin biosynthesis in pink flowers of V. cruziana Transcription factors fulfill a crucial role when it comes to orchestrating gene expression.Essential for anthocyanin production in plants are MYB proteins, regulating various branches of the pathway.To understand why anthocyanin pigments are accumulating in flowers, the responsible transcription factor needs to be identified.VcrMYBSG5, most likely an AtMYB123 orthologue, was identified in V. cruziana.VcrMYBSG6 was identified as an potential orthologue of prominent anthocyanin-regulating MYBs like MYB75/MYB90/MYB113/MYB114 in A. thaliana.Furthermore, four candidates were identified as potential members of the flavonol-regulating MYB subgroup 7. It appears likely that VcrMYBSG6 and potentially VcrMYBSG5 are the main anthocyanin-regulating MYBs in V. cruziana.Other gene candidates proposed for subgroup 6 are not supported by petal-derived RNA-seq data which could indicate that they might be active in other plant parts.Alternatively, no further active subgroup 6 MYBs might exist in V. cruziana.Previous studies reported a role of subgroup 5 MYB (MYB5, MYB123) in activating the anthocyanin biosynthesis (Martínez-Rivas et al., 2023;Jiang et al., 2023) thus corroborating the putative role of VcrMYBSG5 in this context.The additional sequence alignment of KcMYB1 TF (JHA, Additional file 7) from K. coccinea further supports the assumption that VcrMYBSG6 encodes an anthocyanin activating MYB transcription factor as K. coccinea belongs to the Schisandraceae (Huang et al., 2022), a basal angiosperm lineage like the Nymphaceaee.
Further the VcrMYBSG4, an potential orthologue of the repressor AtMYB004, showed higher expression levels in the white compared to the pink flower, suggesting repressive function on anthocyanin production in the white outer petals.This can be further supported by a study, where they identified another AtMYB004 orthologue in Musa spp., MaMYB4, with repressive function on the anthocyanin biosynthesis genes (Deng et al., 2021).Li et al. observed decreased transcript levels in CsMYB4a transgenic plants suggesting a repressive function on the flavonoid biosynthesis pathway (Li et al., 2017).Furthermore, one of the two candidates for the VcrMYBSG4 might represent another repressor as AtMYB007 (Fornalé et al., 2014) is homologue to AtMYB004 (Jin et al., 2000;Stracke et al., 2001).Based on a continuous genome sequence, we successfully identified all structural genes required for anthocyanidin production in Victoria cruziana.However, there is potential to improve the structural annotation as 93.6% complete BUSCOs have been discovered.The inclusion of additional RNA-seq hints derived from different plant parts might contribute to further improvements of the structural gene prediction and subsequently result in an improved functional annotation.Further, we identified VcrMYBSG5 and VcrMYBSG6 as potential transcriptional activators of anthocyanin and proanthocyanidin biosynthesis based on co-expression with anthocyanin accumulation.To confirm the anthocyanin biosynthesis activation properties, the genes could be introduced into A. thaliana myb75 mutants to conduct a complementation experiment.Besides, expression levels of the structural genes as well as transcription factors of V. cruziana could be analyzed in leaves and other parts of the plant to further investigate potential TFs activating structural genes in the flavonoid biosynthesis.To reveal developmental and environmental factors triggering the coloration of V. cruziana flowers, future experiments need to control light exposure and pollinator access to the flowers.Since V. cruziana is only opening its flowers at night, light triggering the anthocyanin formation inside the flower would have to pass through the outer petals.However, if the signal to close the flower after the first night is the first sunlight of the next day, this could be enough light to induce anthocyanin biosynthesis.Further experiments are needed to fully understand the ecological relevance and environmental factors triggering anthocyanin formation in V. cruziana.

Figure 1 :
Figure 1: Flower of Victoria cruziana.Comparison of a flower between the first bloom (white) at night (A) and the consecutive night (pink) (B).

Figure 3 :
Figure 3: Workflow for the RNA-seq and long read genome sequencing project of Victoria cruziana.The left side of the figure illustrates the direct RNA sequencing and RNA-seq workflow targeting V. cruziana flowers, while the right side depicts the conducted long read genome sequencing workflow.

Figure 5 :
Figure 5: (A) Expression pattern of transcription factor genes putatively required for anthocyanin biosynthesis activation in V. cruziana.VcrMYBSG5 and VcrMYBSG6 expression patterns align with the anthocyanin accumulation.VcrTT8 represents the associated bHLH transcription factor and VcrTTG1 edcodes a WD40 protein required for anthocyanin biosynthesis activation.(B) VcrMYBSG4-A and VcrMYBSG4-B probably possess anthocyanin biosynthesis repressing activities and show an expression pattern opposite to anthocyanin accumulation.(C) MYB, associated bHLH transcription factor and WD40 protein forming a transcription factor complex.The WD40 protein is believed to serve as a scaffolding protein connecting MYB and bHLH.Expression values are z-score normalized TPM values.Values

Figure 6 :
Figure 6: Graphical abstract summarizing the molecular mechanism involved in anthocyanin biosynthesis and potential factors triggering the flower color change of V.cruziana.VcrMYBSG4 is active in white petals whereas VcrMYBSG5 and VcrMYBSG6 are expressed in pink petals.Factors that could trigger anthocyanin biosynthesis, thereby activating TFs and structural genes, might be visitation/pollination, light exposure, or a general developmental program of the plant.