AcMYB266, a key regulator of the red coloration in pineapple peel: a case of subfunctionalization in tandem duplicated genes

Abstract Red fruit peel is an attractive target for pineapple breeding. Various pineapple accessions with distinct red coloration patterns exist; however, the precise molecular mechanism accounting for these differences remains unknown, which hinders the pineapple breeding process from combining high fruit quality with red peel. In this study, we characterized a transcription factor, AcMYB266, which is preferentially expressed in pineapple peel and positively regulates anthocyanin accumulation. Transgenic pineapple, Arabidopsis, and tobacco plants overexpressing AcMYB266 exhibited significant anthocyanin accumulation. Conversely, transient silencing of this gene led to decreased anthocyanin accumulation in pineapple red bracts. In-depth analysis indicated that variations of AcMYB266 sequences in the promoter instead of the protein-coding region seem to contribute to different red coloration patterns in peels of three representative pineapple varieties. In addition, we found that AcMYB266 was located in a cluster of four MYB genes exclusive to and conserved in Ananas species. Of this cluster, each was proved to regulate anthocyanin synthesis in different pineapple tissues, illustrating an interesting case of gene subfunctionalization after tandem duplication. In summary, we have characterized AcMYB266 as a key regulator of pineapple red fruit peel and identified an MYB cluster whose members were subfunctionalized to specifically regulate the red coloration of different pineapple tissues. The present study will assist in establishing a theoretical mechanism for pineapple breeding for red fruit peel and provide an interesting case for the investigation of gene subfunctionalization in plants.


Introduction
Ananas comprises approximately eight species, classified into two groups based on their fruit usage: edible (pineapple) and ornamental [1,2].The ripe fruits of most edible pineapples exhibit a relatively monotone coloration, with yellow fruit peels, green leaves, and red inf lorescences.In contrast, some ornamental groups are rich in anthocyanins, resulting in bright red inf lorescences, peels, and even the whole plant [2,3].Ananas fruit is a conglomerate fruit developed from bracts, calyx, ovary, receptacle, and total pedicel, and its peel is composed of bracts and persistent calyx (BC) (Supplementary Data Fig.S1).In most pineapple varieties, the accumulation of anthocyanins occurs exclusively in inf lorescences and heart leaves during f lower bud differentiation to f lowering [4,5].A major aim of pineapple breeding programs has been to integrate the red-skinned characteristics of ornamental pineapple species into edible varieties through hybridization, seeking red-skinned edible pineapple varieties with exceptional fresh food quality [6].However, it was frequently observed that the fresh food quality of these red-skinned hybrid offspring fell short of that exhibited by their parent edible varieties [6].Hence, exploring the molecular mechanism regulating anthocyanin accumulation in pineapple peel will aid in the creation of high-quality edible varieties with red skin and increase their commercial and nutritional value.
Members of the R2R3-MYB transcription factor family are thought to be key regulators of the anthocyanin biosynthesis pathway, and bind directly to the promoters of structural genes [11,21,22].In dicotyledonous plants, AtMYB75 in Arabidopsis thaliana regulates anthocyanin accumulation in the hypocotyls and cotyledons of seedlings [23].Similar findings have been reported in vegetables and fruit trees.StMYBA1, StMYB113, and StAN1 in potatoes (Solanum tuberosum) promote anthocyanin accumulation in leaves or tubers [24,25], and MdMYB10 in apple (Malus domestica) is responsible for regulating fruit color [26].Regarding monocotyledonous plants, OsC1 in rice (Oryza sativa) and TaPpm1 in wheat (Triticum aestivum) were confirmed to be MYB transcription factors involved in anthocyanin synthesis [27,28].In maize (Zea mays), ZmC1 specifically regulates anthocyanin synthesis in the aleurone layers, while ZmP1 contributes to anthocyanin regulation in other maize tissues [29,30].Ananas are monocotyledonous plants with abundant germplasm resources.The process of anthocyanin accumulation in the leaves, inf lorescences, and peels of different accessions shows obvious differences, providing a variety of materials for analyzing the molecular mechanism of anthocyanin accumulation.Although a group of genes related to anthocyanin synthesis were identified through RNA-seq analyses, few transcription factors have been confirmed to regulate anthocyanin synthesis in pineapple [4,31,32].
In our previous studies, we obtained a spatiotemporal transcriptome profile of pineapple cultivar SW (Ananas comosus cv.'Shen Wan') and characterized 94 R2R3-MYB family members [31,32].We found that a potential MYB transcription factor, AcMYB266 (Aco023266; XP_020110371.1),seems to be the master regulator of the red coloration of pineapple peels.In the present study, we integrated physiological, cytological, and molecular evidence to uncover its regulatory mechanism for specifically promoting the accumulation of anthocyanins in pineapple peel and its effect on pineapple peel fading.Additionally, we examined the impact of the gene cluster that encompasses AcMYB266 on the accumulation of anthocyanins in pineapple, as well as its evolutionary variation within and across various species.

Identification of AcMYB266 as a potential major regulator of red coloration of pineapple fruit peels
Previously, we characterized 89 R2R3-MYB transcription factors in pineapple [31].In-depth transcriptomic exploration in 14 pineapple tissues led to the identification of AcMYB266 (Fig. 1A).It was preferentially expressed in fruit peel (BC), the same as the tissue-specific accumulation pattern of anthocyanin (Fig. 1C, Supplementary Data Fig.S3).To verify these findings, we employed RT-qPCR and assessed expression levels of AcMYB266 in 10 tissues, including BC, discs, ovules, stamens, pistils, cores, petals, stems, leaves, and roots.All results are in line with those in the transcriptomic data (Fig. 1B and C).To better characterize the function of AcMYB266, we cloned its complete coding sequence (CDS) which was found to be 648 nucleotides in length, encoding a 215 amino acid protein that contains an R2R3 signature domain, amino acid residues bound by bHLH, and a conserved C-terminal motif.Through a phylogenetic analysis of MYB transcription factors that regulate anthocyanin synthesis across various species, we found that AcMYB266 clusters with OsC1 and ZmC1, which regulate anthocyanin synthesis in black rice and the aleurone layers of maize (Supplementary Data Fig.S4).Localization of pineapple mesophyll protoplasts showed that AcMYB266 is a nuclear protein localized in the nucleus, conforming with the expected localization of transcription factors in cells (Supplementary Data Fig.S4).

AcMYB266 positively regulates anthocyanin accumulation in pineapple peel
To verify the association between AcMYB266 expression and anthocyanin content, we sampled BCs at various stages of SW inf lorescence development for further analysis.Samples were collected at four distinct stages (Fig. 2A): the inf lorescence appearance period (IAP, 42 days after ethylene-induced f lowering, following the elongation of the peduncle and independent emergence of a complete inf lorescence), the early f lowering period (EFP, 49 days after ethylene-induced f lowering, to before f loret bloom), the f lowering period (FP, 60 days after ethyleneinduced f lowering, when f lorets in the middle of the inf lorescence are in bloom), and the fruit set period (FSP, 70 days after ethyleneinduced f lowering, when all f lorets have withered).AcMYB266 expression decreased during inf lorescence development, with highest levels in the IAP (∼2.2 times higher than FSP).This correlated with the trend in anthocyanin content found in BC (Fig. 2B).Statistical analysis showed a significant association between the two variables (Supplementary Data Fig.S5).In addition, we analyzed the expression of pineapple f lavonoid synthesis genes (AcPAL, AcCHI, AcCHS, AcF3H, AcF3 H, AcDFR, AcANS, and AcUFGT) across four developmental stages.Their expression is also highest during the IAP, and their expression levels gradually decrease as the anthocyanins fade during peel development (Fig. 2C).The late biosynthetic genes AcDFR and AcANS showed higher expression, and their expression in IAP was ∼1.5-500 times higher than other genes (Fig. 2C).Whether AcMYB266 directly regulates these genes involved in the anthocyanin synthesis pathway and activates their expression would be the next question.We employed yeast one-hybrid (Y1H) experiments to confirm the effects of AcMYB266 on the early biosynthetic gene AcCHS, and the late biosynthetic genes AcDFR, AcANS, and AcUFGT of the anthocyanin synthesis pathway.After fusing the bait plasmid with the prey plasmid into Y1HGold-competent cells, we found that the co-transformed competent cells of all combinations except AcCHS grew normally on SD−Leu + AbA medium as well as the positive control (p41plus + pGAD-53) (Fig. 2D).Thus, AcMYB266 can bind to the AcDFR, AcANS, and AcUFGT promoters, but not with the AcCHS promoter.We also conducted a dual-luciferase reporter assay to confirm the effects of AcMYB266 on the promoter activity of AcCHS, AcANS, AcDFR, and AcUFGT (Fig. 2E).Our findings revealed that the expression of AcMYB266 can activate the promoters of AcANS, AcDFR, and AcUFGT, but not AcCHS, which is consistent with the results of Y1H.The activation effects were ∼2.5, 5.7, and 4.5 times greater than the control group.
We overexpressed AcMYB266 in Arabidopsis and tobacco.In three transgenic Arabidopsis lines (OE-AcMYB266-At1, OE-AcMYB266-At2, OE-AcMYB266-At3) with ectopic expression levels of AcMYB266, red pigmentation in hypocotyls and cotyledon petioles were observed after culturing the T 3 generation transgenic lines compared with that in the wild type (WT) Arabidopsis seeds on MS medium (Supplementary Data Fig.S6A).In transgenic tobacco lines overexpressing AcMYB266, a significant accumulation of red pigment in the stamens (anthers and filaments) of the transgenic tobacco lines was observed in the T 1 generation compared with WT (Supplementary Data Fig.S6B).
Semiquantitative PCR analysis and anthocyanin content determination showed that overexpression of AcMYB266 enhanced anthocyanin synthesis in the transgenic Arabidopsis seedlings, and the stamens and petals of transgenic tobacco plants (Supplementary Data Fig.S6C and D).
As in pineapple, virus-induced gene silencing (VIGS) and transient overexpression of AcMYB266 were conducted on red bracts and the leaf bases (which are white), respectively.Virusinduced suppression of AcMYB266 significantly reduced anthocyanin production in red bracts, and transient overexpression of AcMYB266 promoted the accumulation of anthocyanin in leaf bases (Fig. 3A and B).In addition, referring to our previous genetic transformation method [33], we managed to obtain transgenic pineapple plantlets overexpressing AcMYB266.Stable overexpression of AcMYB266 led to distinct red pigmentation in the adventitious shoots and callus material, in contrast to the WT samples (Fig. 3C).Expression levels of AcMYB266, AcCHS, AcF3H, AcDFR, AcANS, and AcUFGT in the above transgenic lines were also assessed using RT-qPCR, which showed results consistent with the phenotypes (Fig. 3D and E).
All results confirmed that AcMYB266 is involved in the regulation of anthocyanin accumulation, and is a key activator of the pineapple anthocyanin synthesis structural genes AcDFR, AcANS, and AcUFGT in pineapple peels.

Variations in the promoter region of AcMYB266 attributed to the distinct coloration pattern of pineapple peels
As in pineapple, different materials possess different red coloration patterns on fruit peels.We selected three representative varieties, KH (A. comosus cv.'Ka Hong') (stable accumulation during the fruiting stage), BTH (A. comosus cv.'Red Sugar') (unstable accumulation during the fruiting stage), and SW (accumulation during the f lowering stage only) to survey how AcMYB266 is involved in this biological process (Fig. 4A-C).We cloned their full-length CDSs, which were found to be identical (Supplementary Data Fig.S7).In contrast, three types of AcMYB266 promoter were obtained, and which are named Promoter I (from BTH), II (from KH), and III (from SW) (Fig. 4D).
Several SNPs and indels were found among these sequences and thus we hypothesize these differences in the promoter region contributed to variant activation ability.We constructed GUS expression vectors driven by three types of AcMYB266 promoter and transiently infected pineapple fruit peel (BC).GUS staining showed that the negative control (injected with Agrobacterium without vector) was not stained, and the BC of II::GUS was stained the deepest.The others, transiently transformed BCs by I::GUS and III::GUS, were stained similarly.GUS gene expression and enzyme activity determination also confirmed the histochemical staining results (Fig. 4E and F), which showed that the type II promoter has the highest activity, and the activities of type I and type III are similar.

Identification of gene cluster containing AcMYB266 that regulates anthocyanin accumulation in various tissues of pineapple
Tandem duplication is a major driver for gene family expansion, and always results in gene members with almost identical sequences and probably redundant functions.In the present study, we happened to find that AcMYB266 was located in an MYB gene cluster, adjacent to AcMYB267, AcMYB262, and AcMYB263 on pineapple chromosome 2 from 1 126 000 to 11 440 000 bp (Fig. 5A).Amino acid sequence and collinear analysis showed that this cluster evolved from a tandem duplication coupled with an inverted duplication.The tandem paired genes AcMYB267/AcMYB266 and AcMYB263/AcMYB262 are separated by 87 kb, showing a mirrored distribution (Fig. 5A and B).These results indicate that they might have similar molecular functions.But do they take part in the same/overlapped biological process, especially the red coloration of pineapple peel that is the focus of this study?If not, what potential process they might be involved in?
To answer these questions, in addition to using the transcriptome data of 14 tissues of pineapple in our previous work [32] we performed an RNA-seq experiment on pineapple red and green leaves [which is a rare trait featured only in a few pineapple varieties, e.g.LY (A. lucidus var.LY), which has high anthocyanin accumulation in leaves] to more accurately describe the expression patterns of gene cluster members (Fig. 5C).Interestingly, although AcMYB266 and AcMYB263 are similar in sequence (Fig. 5A), AcMYB266 is specifically highly transcribed in pineapple BC (Fig. 5C) while AcMYB263 is relatively highly transcribed in the red part of 'Sanse' leaves [4].AcMYB262 is specifically highly transcribed in red leaves (Fig. 5C and D).AcMYB267, which has a similar sequence to AcMYB262, is also highly transcribed in red leaves; however, it is also highly expressed in petals (Fig. 5C and D).RT-qPCR further verified the expression patterns of AcMYB262 and AcMYB267 in all anthocyanin-accumulating tissues of Ananas (LY), and the results were consistent with the transcriptome data (Fig. 5D).
Taking the results together, we found that: (i) AcMYB266 specifically regulates the accumulation of anthocyanins in the peel of pineapple; (ii) AcMYB263 was identified as a key transcription factor gene regulating leaf anthocyanin biosynthesis in 'Sanse' [4]; (iii) AcMYB267 promotes anthocyanin accumulation in pineapple leaves and petals (Feng JT et al., data not shown; Zhang W et al., data not shown); (iv) AcMYB262 is highly expressed in the red leaves of LY, and its expression levels in F 1 leaves of LY (red leaves) and XG (A. comosus cv.'Xi Gua') (green leaves) are consistent with their leaf color phenotype (Supplementary Data Fig.S8).Each member of this MYB cluster seems to implement its function in a specific tissue/stage.
To explore the origin of the AARM cluster, we conducted local collinearity analysis and found that the collinear block containing AARM is conserved only in Ananas (Fig. 6D-G).While there are four MYB family members in the associated region of the basal angiosperm Amborella trichopoda, there is no collinear relationship with pineapple F153 in the adjacent region, and the MYBs are also differently arranged.In both rice and banana, only one MYB gene is present, and no tandem duplication has occurred.In the association region of PY, which belongs to the same family (Bromeliaceae) but a different genus, only one pair of tandemly arranged MYBs (PY_026205T1/PY026206T1) is present, without any mirror image MYBs resulting from a similar duplication and inversion (Fig. 6G).In pineapple CB5, two pairs of tandemly arranged MYB transcription factors (CB5.17G0008030/CB5.17G0008050and CB5.17G0008090/CB5.17G0008100)exist with opposite transcriptional directions, which is completely consistent with the distribution of AARM cluster members in F153, and they are distributed in a mirror-symmetric manner at a distance of 9 kb.The sequence identity between CB5.17G0008030 and CB5.17G0008100, CB5.17G0008050, and CB5.17G0008090 reached 96% (Fig. 6G, Supplementary Data Fig.S9).Taking these results together, the AARM cluster is supposed to have originated from a common ancestor of Ananas and is also exclusively conserved in Ananas.

AcMYB266 is a key regulator of the red coloration of pineapple peel
Pineapple is an important horticultural plant, and the level of anthocyanin in the peel directly affects its commercial potential.Our recent research, which employed a multi-omics strategy, has revealed that anthocyanin plays a critical role in the development of red coloration in the peel of pineapple [41].Until now, the molecular regulatory mechanism of anthocyanin accumulation in pineapple has been largely unknown.The present study has identified an R2R3-type transcription factor gene, AcMYB266, which exhibits specific expression in the peel of pineapple that accumulates anthocyanins (Fig. 1).RT-qPCR, Y1H experiments, and stable overexpression of AcMYB266 in pineapple, Arabidopsis, and tobacco, as well as transient expression and silencing experiments in pineapple, collectively demonstrated its positive Four genes are symmetrically distributed on Ananas chromosome 2. B The AAMR cluster regulates the synthesis of anthocyanin in specific tissues of Ananas: AcMYB267 regulates the accumulation of anthocyanin specifically in Ananas petals and LY leaves; AcMYB266 regulates the accumulation of anthocyanin specifically in Ananas peel (BC); AcMYB262 regulates the accumulation of anthocyanin specifically in LY leaves; AcMYB263 regulates the accumulation of anthocyanin specifically in 'Sanse' leaves.C Regulatory mechanism by which AcMYB266 promotes anthocyanin synthesis in pineapple peel.AcMYB266 targets the promoter sequences of pineapple anthocyanin synthesis late genes (AcDFR, AcANS, and AcUFGT) and activates their expression.The width of the arrows corresponds to activation intensity.EBGS, early biosynthetic gene; LBGS, late biosynthetic gene.D-F Evolutionary analysis of MYB transcription factors in Arabidopsis, Amborella trichopoda, banana, rice, CB5, F153, MD-2, and PY.G Species trees were generated for Amborella trichopoda, banana, rice, CB5, F153, and PY, and collinearity analysis was performed on MYB transcription factors that are homologous to the members of the AARM cluster in each of these regions.Green and yellow rectangles represent genes on the forward and reversed chromosome strands, respectively.regulation of anthocyanin accumulation in pineapple (Fig. 3, Supplementary Data Fig.S6).This result indicated that the function of AcMYB266 in promoting anthocyanin accumulation is conserved in monocots and dicots.Three types of promoter of AcMYB266 were obtained from representative accessions, and their activities were significantly different (Fig. 4D-F).This finding leads us to speculate about the close association between the difference in promoter sequence defined the expression levels of AcMYB266 and thus affected the timing of anthocyanin accumulation of anthocyanin accumulation in pineapple peel.

The AARM cluster in Ananas is an interesting case of subfunctionalization in tandem duplicated genes
We identified four transcription factors that individually regulate anthocyanin accumulation in different tissues of pineapple.These four genes occur as two pairs of tandem MYBs, which are gathered together in a small gene cluster (the AARM cluster) located on the LG2 chromosome of pineapple (F153) (Fig. 6A).Regional collinearity analysis among species found that the MYB genes in the AARM cluster were acquired or lost during evolution.There are MYB clusters in both Amborella trichopoda and bromeliads, but the tandemly replicated MYB genes in rice and banana may be lost (Fig. 6G).
Gene duplication has made a major contribution to the evolution of organisms, and these genes may be lost, subfunctionalized, or neofunctionalized following natural selection [42,43].A similar phenomenon exists in the AARM cluster of this study.The four transcription factors AcMYB266, AcMYB263, AcMYB267, and AcMYB262 with highly homologous sequences were specifically expressed in different tissues of Ananas plants (Fig. 5).The inverted duplication of genes in Ananas gave rise to a gene cluster containing the four MYB transcription factors, and each MYB transcription factor underwent subfunctionalization. Similar findings were also found in citrus: a gene, Ruby2, with high homology was found near a known gene, Ruby1, that regulates anthocyanin accumulation, forming a cluster with Ruby1.They exerted different anthocyanin regulation abilities in different accessions of citrus [44].Thus, it is plausible to assume that the AARM cluster's four MYB transcription factors should have a common ancestor gene, AcMYB, which possesses the potential to regulate anthocyanin accumulation in all organs of Ananas.
There is also an interesting finding of MYB homologies closely related to the AARM cluster members in other species.In different species, MYB transcription factors within the same clade as the AARM cluster display a divergence in their functions and regulatory locations in tissue.The members found in rice (LOC_Os03g29614.1.MSUv7.0,ATR0598G349.1)were previously reported to regulate the accumulation of anthocyanins in the seed coat of rice [45].However, the member in the banana (Macma4_03_g30340.1) is a TT2-like transcription factor, which has been confirmed to synthesize proanthocyanidins in Arabidopsis [46].In the present study, the MYB transcription factor in the AARM cluster has been demonstrated to promote the production of anthocyanins in pineapple, whereas its expression in seed coat tissue is comparatively low (Supplementary Data Fig.S10).This suggests that the function and regulated tissue of action of the AARM cluster homologs have diverged during evolution.Subfuntionalization happens in gene orthologs within and across species.The four members of the AARM cluster regulate anthocyanin accumulation in different tissues of pineapple, which is possibly attributable to gene subfunctionalization after duplication in Ananas.

Conclusions
In pineapple, limited research has been reported on the regulation of fruit peel color.Through a systematic study, we characterized AcMYB266 to be specifically expressed in peels and confirmed its role in regulating the accumulation of anthocyanins.The variable promoter sequence of AcMYB266 corresponds to different gene activation abilities, and thus to the distinct red color patterns of three representative pineapple varieties.In addition, we found a cluster of four AcMYB genes in pineapple, whose members were connected in pairs and distributed in mirror images on chromosome 2.They seem to have undergone subfunctionalization and regulated the accumulation of anthocyanins in different tissues of pineapple.These findings will provide a basis for further exploration of the molecular mechanism of the red coloration of pineapple fruit peel, and also serve as an interesting case for the study of gene duplication and subfunctionalization in plants.

Plant materials
Tobacco (Nicotiana tabacum cv.K326) and A. thaliana Columbia-0 were used as recipients for heterologous stable genetic transformation.The callus of Ananas cultivar SW (A. comosus cv.'Shen Wan') was employed as the recipient for Agrobacterium-mediated stable genetic transformation, and the leaf tissue served as the transient overexpression material.The large-bracted A. bracteatus variety 'Sanse' was employed for VIGS transformation experiments.Tobacco (Nicotiana benthamiana) was used as a material for the dual-luciferase assay.The Ananas cultivars SW, KH (A. comosus cv.'Ka Hong'), and BTH (A. comosus cv.'Red Sugar') were selected as representative varieties demonstrating three distinct types of anthocyanin accumulation in Ananas peel.LY (A. lucidus var.LY) and XG (A. comosus cv.'Xi Gua') were used as parental plants to generate a total of 11 hybrid offspring.The aforementioned pineapple experimental materials were sourced from the Pineapple Germplasm Resource Garden, located at the Institute of Tropical Crops Variety Resources, Chinese Academy of Tropical Agricultural Sciences in Danzhou, Hainan province.

Determination of anthocyanin content
One hundred milligrams of sample was dissolved in 5 ml of methanol containing 0.1% HCl, and the sample was incubated at 4 • C in the dark for 24 h and oscillated three times.The supernatant was collected by centrifugation at 12 000 rpm and 4 • C for 15 min, and absorbance was obtained at 530 and 650 nm using a microplate reader (Fluoroskan Ascent FL).Total anthocyanin content was calculated according to the following formula: OD 600 = (A530 − 0.25 × A650) g −1 .All analyses and error bars were determined from at least three biological replicates.

Total RNA extraction and RT-qPCR analysis
Total RNA was extracted using Kangwei Century Bio-Company's extraction kit (CW2598S), and RT-qPCR was conducted with the assistance of Yeasen's Reverse Transcription Kit (11141ES60), quantitative reagent (11203ES08), and a Bio-Rad f luorescence quantitative PCR instrument (CFX Touch).The RT-qPCR results were normalized with reference genes (NtActin, AtActin, AcActin) and then the relative expression levels of genes were calculated using the 2 −ΔΔCT method (primers are listed in Supplementary Data Table S1).All analyses and error bars were determined from at least three biological replicates.

Gene cloning and expression vector construction
We designed sense and antisense primers (primers are listed in Supplementary Data Table S1) based on the Ananas genome in the NCBI database (NCBI Taxonomy ID: 4615).Leaf cDNA from SW was used as the PCR template.Amplified fragments were connected to the T-vector pCloon 007 and confirmed by Sanger sequencing before being used in subsequent experiments.Overexpression vectors were constructed by Gateway technology using the primers listed in Supplementary Data Table S1 [47].The target fragment of AcMYB266 was ligated into the entry vector pDONR221 in the BP recombination reaction.The recombination reaction followed kit instructions from Applied Biological Materials (ABM, Richmond, Canada).The recombinant plasmid pDONR-AcMYB266 was then subjected to the LR recombination reaction, resulting in the 35S promoter-driven overexpression recombinant plasmid pK7WG2D-AcMYB266.

Virus-induced gene silencing
VIGS was carried out as described previously [48].The CDS of AcMYB266 was inserted into the pTRV2 vector to generate the recombinant plasmid (pTRV2-AcMYB266).Agrobacterium tumefaciens (GV3101) colonies containing pTRV2-AcMYB266, pTRV2, and pTRV1 respectively were suspended in permeation buffer containing 10 mM MgCl 2 , 10 mM MES, and 150 μM acetosyringone until the OD 600 reached 0.8.The pTRV2-AcMYB266 and pTRV2 vectors were mixed with the pTRV1 suspension at a ratio of 1:1 and allowed to stand in the dark for 4-6 h.The mixture of pTRV1 + pTRV2-AcMYB266 or pTRV1 + pTRV2 was injected into the middle and upper part of the bracts using an Injex-30 syringe during fruit setting.Six inf lorescences were injected per injection, for a total of 240 bracts, each receiving 100 μl of the mixture.The treated inf lorescences were subjected to 12 h of dark incubation, followed by cultivation under normal light conditions.The phenotype was observed after 5 days.

Transient overexpression in pineapple leaves
The vector pK7WG2D-AcMYB266 was transferred into A. tumefaciens GV3101 by the freeze-thaw method.Positive colonies were selected and suspended in permeation buffer containing 10 mM MgCl 2 , 10 mM MES, and 150 μM acetosyringone solution with an OD 600 of ∼0.6.We used an Injex-30 type syringe to administer the pK7WG2D-AcMYB266 and pK7WG2D solutions into the leaf base of SW.Each leaf was injected with 50 μl of the mixture.The leaves were treated in the dark for 12 h after injection before being cultured under normal light for 5 days; the phenotype was then observed and RNA was sampled.

Generation of transgenic lines of pineapple, tobacco, and Arabidopsis overexpressing AcMYB266
The overexpression vector pK7WG2D-AcMYB266 driven by the 35S promoter was mediated by A. tumefaciens GV3101 to transform pineapple, tobacco, and Arabidopsis plants through previous research methods [33,49,50].Carbenicillin (200 mg/l) and kanamycin (50 mg/l) were used for selecting the transgenic tobacco and Arabidopsis lines.After rooting and acclimation, transgenic plants were transferred to the greenhouse and grown until f lowering.Resistant plants were verified by RT-qPCR.

AcMYB266 promoter cloning and expression vector construction
DNA was extracted from leaves of KH, BTH, and SW.Based on Ananas genome data, the nucleotide sequence ∼1000 bp upstream of the AcMYB266 coding region was selected for promoter cloning (Supplementary Data Table S1).The obtained AcMYB266 promoter (AcMYB266 Promoter I, AcMYB266 Promoter II, AcMYB266 Promoter III) was connected to the expression vector pBI121 instead of the 35S promoter.We obtained three expression vectors driven by AcMYB266 promoters (I::GUS, II::GUS, III::GUS).

Identification, evolution, and collinearity analysis of MYBs
To obtain the MYB members in each species, we obtained the hidden Markov models of the MYB DNA-binding domain (PF00249) from the Pfam database (http://pfam.xfam.org/).We used HMMER3.3(http://hmmer.org/) to query each species' protein sequence for the MYB domain, after which the NCBI Batch CDD server was used to further check for the MYB core sequence.Complete MYB protein sequences were used to study their evolutionary relationships.MAFFT with default parameters was used for multiple sequence alignment of MYB amino acid sequences, and FastTree was used to construct the maximum likelihood tree.Species trees were manually drawn according to previous research [53].We performed local collinearity analysis between species based on the species tree using TBtools-II [54].

Statistical analysis
All experiments were performed with at least three biological replicates, and the results were expressed as error bars.Differences were analyzed by t-test and were considered statistically significant when P < 0.05.

FFigure 1 .
Figure 1.AcMYB266 is preferentially expressed in pineapple peel.A Expression pattern of 89 R2R3-MYB transcription factors in different pineapple tissues.FPKM values of genes are scaled individually from 0 to 1. Red color denotes high expression levels.B RT-qPCR verification of AcMYB266 expression levels in different tissues.Error bars indicate the standard deviation from three biological replicates per group.Significant differences (P < 0.05, t-test) are indicated by different letters above the columns.C Bubble plot visualizing expression levels of AcMYB266 in various tissues of 'SW'.Larger circle size denotes higher expression levels while cross lines in the circle denote undetectable expression levels.

Figure 2 .
Figure 2. Anthocyanin content and relative expression level of AcMYB266 during pineapple peel development and identification of AcMYB266 target genes.A The inf lorescence development process of SW.From left to right: IAP, EFP, FP, and FSP.The first row shows the inf lorescence development process, and the second and third rows show the bracts and calyces of the four developmental stages, respectively.B Anthocyanin content and relative expression levels of AcMYB266 in BC at four developmental stages.Error bars indicate standard deviation from three biological replicates per group.C Simplified diagram of anthocyanin synthesis pathway and heat map visualization of RT-qPCR analysis of relative expression levels of anthocyanin synthesis structural genes in pineapple in BC at four developmental stages.D Growth of co-transformants of AcMYB266 and promoters of four structural genes in SD−Leu(+AbA) medium.N.Control, negative control; P.Control, positive control.E Dual-luciferase experiment demonstrates the activation ability of AcMYB266 on the structural gene of pineapple anthocyanin synthesis.CK, empty-effector plasmids.Error bars indicate the standard deviation from three biological replicates per group.Significant differences in B and E (P < 0.05, t-test) are indicated by different letters above the columns.

Figure 3 .
Figure 3. Stable or transient overexpression and silencing of AcMYB266 affect the accumulation of anthocyanins in Ananas.A Phenotypes produced by transient silencing of AcMYB266 in 'Sanse' (A.bracteatus var.Sanse) bracts (TRV2-AcMYB266).B Phenotypes produced by transient overexpression of AcMYB266 in SW leaves (pK7WG2D-AcMYB266); C Phenotypes produced by pineapple callus transgenic lines stably overexpressing AcMYB266 (OE-AcMYB266-1, OE-AcMYB266-2, OE-AcMYB266-3).D Relative expression levels of AcMYB266 in Ananas materials with phenotypes shown in A, B, and C. Error bars indicate standard deviation from three biological replicates per group.E Relative expression levels of structural genes of the anthocyanin synthesis pathway in Ananas materials with phenotypes shown in A, B, and C. Error bars indicate standard deviation from three biological replicates per group.Significant differences in D and E (P < 0.05, t-test) are indicated by different letters above the columns.

Figure 4 .
Figure 4. Anthocyanin accumulation types in fruit peel of representative pineapple varieties and the corresponding AcMYB266 promoter activation ability.A Comparison of the inf lorescence and fruit development of three types of pineapple at four stages.The stages, from left to right, are FP (f lowering period), FSP (fruit set period), FGP (fruit growth period), and MP (fruit maturity period).The types, from top to bottom, are KH germplasm, a stable accumulation type during fruiting; BTH, an unstable accumulation type during fruiting; and SW, an accumulation type during f lowering.B Determination of the anthocyanin content in three types of pineapple at four developmental stages by measuring absorbance with a photometer.The green dashed lines in B represent the mean BC anthocyanin content during four developmental periods for each cultivar.Error bars indicate standard deviation from three biological replicates per group.C Comparison of the relative gene expression levels of AcMYB266 in FGP and MP of three types of pineapple.The green dashed lines in C represent the mean expression level of AcMYB266 during two developmental periods for each cultivar.Error bars indicate standard deviation from three biological replicates per group.D Three types of AcMYB266 promoter isolated from three accessions in A. E, F Activity analysis of three types of AcMYB266 promoter.E is the transient GUS staining of pineapple BC driven by three types of promoter of AcMYB266, and F is the analysis of GUS activity and relative expression levels in pineapple BC driven by three types of promoter of AcMYB266.Error bars indicate the standard deviation from three biological replicates per group.Significant differences in B, C, and F (P < 0.05, t-test) are indicated by different letters.

Figure 5 .
Figure 5.The AcMYB gene cluster and tissue-specific expression patterns of its members.A AcMYB gene cluster (AcMYB267\AcMYB266\AcMYB263\AcMYB262) location and member sequence analysis.B Collinearity analysis between AcMYB gene cluster segments.C Heat map visualizing the expression pattern of each gene cluster member in various tissue parts of pineapple based on RNA-seq data.D RT-qPCR further analyzed the expression patterns of AcMYB267 and AcMYB262 in Ananas anthocyanin accumulation tissues.Error bars indicate the standard deviation from three biological replicates per group.Significant differences (P < 0.05, t-test) are indicated by different letters above the columns

Figure 6 .
Figure6.Summary of tissue-specific regulation of anthocyanin synthesis of the AARM and its exclusive conservation in Ananas.Distribution of the AARM cluster in Ananas chromosomes.Four genes are symmetrically distributed on Ananas chromosome 2. B The AAMR cluster regulates the synthesis of anthocyanin in specific tissues of Ananas: AcMYB267 regulates the accumulation of anthocyanin specifically in Ananas petals and LY leaves; AcMYB266 regulates the accumulation of anthocyanin specifically in Ananas peel (BC); AcMYB262 regulates the accumulation of anthocyanin specifically in LY leaves; AcMYB263 regulates the accumulation of anthocyanin specifically in 'Sanse' leaves.C Regulatory mechanism by which AcMYB266 promotes anthocyanin synthesis in pineapple peel.AcMYB266 targets the promoter sequences of pineapple anthocyanin synthesis late genes (AcDFR, AcANS, and AcUFGT) and activates their expression.The width of the arrows corresponds to activation intensity.EBGS, early biosynthetic gene; LBGS, late biosynthetic gene.D-F Evolutionary analysis of MYB transcription factors in Arabidopsis, Amborella trichopoda, banana, rice, CB5, F153, MD-2, and PY.G Species trees were generated for Amborella trichopoda, banana, rice, CB5, F153, and PY, and collinearity analysis was performed on MYB transcription factors that are homologous to the members of the AARM cluster in each of these regions.Green and yellow rectangles represent genes on the forward and reversed chromosome strands, respectively.