Genome-Wide Identification and Expression Profiling of ATP-Binding Cassette (ABC) Transporter Gene Family in Pineapple (Ananas comosus (L.) Merr.) Reveal the Role of AcABCG38 in Pollen Development.

Pineapple (Ananas comosus L.) cultivation commonly relies on asexual reproduction which is easily impeded by many factors in agriculture production. Sexual reproduction might be a novel approach to improve the pineapple planting. However, genes controlling pineapple sexual reproduction are still remain elusive. In different organisms a conserved superfamily proteins known as ATP binding cassette (ABC) participate in various biological processes. Whereas, till today the ABC gene family has not been identified in pineapple. Here 100 ABC genes were identified in the pineapple genome and grouped into eight subfamilies (5 ABCAs, 20 ABCBs, 16 ABCCs, 2 ABCDs, one ABCEs, 5 ABCFs, 42 ABCGs and 9 ABCIs). Gene expression profiling revealed the dynamic expression pattern of ABC gene family in various tissues and different developmental stages. AcABCA5, AcABCB6, AcABCC4, AcABCC7, AcABCC9, AcABCG26, AcABCG38 and AcABCG42 exhibited preferential expression in ovule and stamen. Over-expression of AcABCG38 in the Arabidopsis double mutant abcg1-2abcg16-2 partially restored its pollen abortion defects, indicating that AcABCG38 plays important roles in pollen development. Our study on ABC gene family in pineapple provides useful information for developing sexual pineapple plantation which could be utilized to improve pineapple agricultural production.

Introduction

The ATP-binding cassette (ABC) gene family is one of the largest expressed gene subfamilies (Campa et al., 2008). Most ABC transporter subfamilies had highly conservative amino acid sequence domains: the nucleotide-binding domain (NBDs) and the transmembrane domains (TMDs) including five or six helices (Higgins and Linton, 2004). The NBD provides energy by hydrolyzing ATP, and the TMDs determines the substrate specificity (Schneider and Hunke, 1998). NBDs contains three exceptional motifs: the signature motif, the Walker A motif and the Walker B motif. The signature motif is unique to ABC proteins while the Walker A and Walker B motif are responsible for nucleotide binding (Davidson et al., 2008). ABC transporter proteins have been classified into two categories based on the length of structural domain: full-sized transporters (two NBDs and two TMDs) and half-sized transporters (one NBD) (Davidson et al., 2008). Protein structures were closely associated with function. The ABCE and ABCF subfamilies lack of TMDs domain and do not function as transporter. For example, the first human and mammalian ABC protein ABC50 which does not contain TMDs, were endowed with ribosome assembly (Richard et al., 1998; Tyzack et al., 2000). ABCE (Y39E4B.1) had been reported for regulating transcription and translation in eukaryotes (Zhao et al., 2004).

The majority of ABC family members are involved in transporting a variety of compounds through biological membranes, such as lipids, suberin, steroids, irons, amino acids and other metabolic substances (Klein et al., 1999). Disruption of ABC transporter proteins has huge impact on bacterial physiology such as toxic effect (Henderson and Payne, 1994). The ABC transporters also participated in plant development. In Arabidopsis, auxin regulate almost every biological processes from early embryonic development to leaf senescence (Paponov et al., 2005). AtPGP1 and AtPGP19, two of Arabidopsis multidrug-resistance-like ABC transporters control polar auxin transport and the double mutant atpgp1-1atpgp19-1 exhibits epinastic growth and small inflorescence size (Geisler et al., 2003). It was recently shown that AtABCG1 and AtABCG16 implicated in the integrity of exine and nexine of pollen wall and the formation of pollen-derived intine layer (Yadav and Reed, 2014). The microspores of double mutants abcg1abcg16 exhibit defects in pollen mitosis in postmeiotic stages of male gametophyte development as well as lack of intact nexine and intine pollen layers in Arabidopsis (Yim et al., 2016).

Pineapple (Ananas comosus L.), a perennial monocot from the Bromeliaceae family, is one of the famous tropical and edible flavorful fruits. Pineapple is usually diploid (2n = 50) and the haploid genome size is estimated to be 526 Mb (Arumuganathan and Earle, 1991). In agriculture production, pineapple planting is impeded by abiotic and/or biotic factors, such as cold, fusarium wilt, and other diseases (Pujol and Kado, 2000; Peckham et al., 2010; Wang et al., 2014; Santos et al., 2016). In addition, pineapple is one of the self-incompatible species, and cultivated mostly by vegetative propagation. The pineapple varieties resulted by vegetative propagation greatly reduces fruit quality and heterogeneity (Fassinou Hotegni et al., 2015). These disadvantages of asexual cultivation could be effectively improved by sexual propagation. However, sexual propagation through clearing harmful mutations could improve the fruit quality of generations, which was more adaptive to effective evolutionary responses to natural selection (Otto, 2009; Neiman and Schwander, 2011; Levitis et al., 2017). Recent study shows that pollen development is crucial for sexual reproduction in plants and the quantity and quality of pollen received determines pollen limitation (Aizen and Harder, 2007; Rodger and Ellis, 2016). Overall, there is a serous limitation in pineapple vegetative propagation and sexual reproduction could become novel approach to overcome these problems. In view of the widely function were involved in reproduction development for ABC transporter proteins but how AcABC proteins function in pineapple reproduction is unknown, genome-wide identification, characterization and function study of the ABC transporter gene family in pineapple would become very meaningful.

Recently, sequencing and genome assembly of the entire genome of pineapple has been completed (Ming et al., 2015), which made it possible to systematically study of the ABC subfamily in pineapple. In this study, we performed the analyses of gene phylogeny, gene structure, and expression profiles of ABC proteins in different reproductive organs and different developmental stages in pineapple. We provided comprehensive information of ABC proteins in pineapple and determined the critical role of the stamen enriched gene AcABCG38 in pollen development. Present work may contribute to improve pineapple production though sexual reproduction.

Materials and Methods

Identification of ABC Transporter Genes in Pineapple Genome

The AcABC protein and genome sequence were downloaded from Phytozome v12.1[1]. To identify the AcABC genes, the HMMER3.0[2] was used with default parameters settings to search the proteins sequences containing PFAM ABC domain (PF00005) (Verrier et al., 2008). To achieve accuracy in our analysis, we further used NCBI-CDD with E-value threshold 0.01[3] to analyze the conservative sequences and to remove any sequences which lack ABC annotation (Zhang et al., 2017). Isoelectric point (PI) and molecular weight (MW) of the AcABC family member proteins were obtained using ExPAsy website[4] (Table ).

Phylogenetic Analysis

To understand the phylogenetic relationship of ABC proteins between pineapple and Arabidopsis, all the identified ABC amino acid sequences of pineapple and Arabidopsis were used to construct the phylogenetic tree. AcABC and AtABC protein sequences were aligned using MAFFT with default parameters[5]. Then, FastTree software was used to establish phylogenetic tree using 1,000 resamples, and constructed by maximum-likelihood using the JTT + CAT model. FastTree provides local support values by the Shimodaira-Hasegawa (SH) test, the resulting support values were closely correlated with the traditional bootstrap (r = 0.975) (Price et al., 2010; Chen et al., 2017). The local support values below 60 are hidden in the constructed tree. All amino acid sequences were used for phylogenetic and alignments analysis in the study were supplied in Supplementary Datasets , .

Gene Structure Analysis and Conserved Motif Identification

The exon–intron characteristics of the ABC genes family were exhibited using the Gene Structure Display Server[6] (Guo et al., 2007). Through a comparison with the full-length predicted coding sequence (CDS). The motifs of the AcABCG proteins were determined with the appropriate number of motifs using the MEME program[7]. The lower E-value (the most statistically significant), the more accurate of expected motifs.

Plant Materials and Growth Conditions

The wild-type Arabidopsis background Col-0 (CS60000) and the T-DNA insertions of AtABCG1 (abcg1-1: SALK_061511; abcg1-2: SALK_055389) and AtABCG16 (abcg16-1: SALK_087501; abcg16-2: SALK_119868C) were obtained from Arabidopsis Biological Resource Center (ABRC)[8]. Pineapple (Ananas comosus) variety MD2 was collected by Qin Lab[9]. Pineapple were grown in plastic pots with soil mix [peat moss: perlite = 2:1 (v/v)] and placed in greenhouse at about 30°C with light availability of 60–70 mMolL-1 photons m-2s-1 under 70% humidity with 16 h light/8 h dark photoperiod. Arabidopsis was grown with the conditions as described by Cai et al. (2017).

RNA-Seq and qRT-PCR

Tissue samples of MD2 were collected from different developmental stages of ovule, petal, sepal and stamen. The criterion of different stage samples was referenced to Su et al. (2017), including seven stages of ovule (Ov1-Ov7), three stages of petal (Pe1–Pe3), five stages of stamen (St1–St5) and four stages of sepal (Se1–Se4). Collected samples were quickly stored in liquid nitrogen until total RNA extraction. The RNA was extracted following manufacturer’s protocol using RNA extraction Kit (Omega Bio-Tek, Shanghai, China). Total RNA was diluted with nuclease-free water and then mRNA was isolated, following by fragmentation, and the first and second strand cDNA synthesized. Double-stranded cDNA was then purified using 1.8× Agencourt AMPure XP Beads. Performing End Repair/dA-tail of cDNA Library followed by adaptor ligation using Blunt/TA Ligase Master Mix and diluted NEB Next Adaptor. Purifying the ligation reaction and approximate insert size was kept 25–400 bp with final library was set to 300–500 bp. Performing PCR Library construction followed by purity of the PCR reaction using Agencourt AMPure XP Beads and assessed library quality on a Bioanalyzer® (Agilent high sensitivity chip) and send to company for sequencing (NEB next Ultra RNA Library Prep Kit for Illumina Biolabs). The RNA-seq data of root, leaf, flower and developing fruit were download from NCBI database[10], and all of RNA-seq data were analyzed following Trapnell et al. (2012). Raw reads were filtered by TRIMMOMATIC v0.3 to remove the adapter sequence, the clean reads were then aligned using the Tophat software with default parameters. Alignment results were processed using Cufflinks, and FPKM values were calculated by using Cuffdiff (FC ≥ 2, FDR ≤ 0.05) for following Dai et al. (2017) analysis. R software was employed to construct the heat-map using FPKM values of RNA-seq for each gene. RNA-seq data were further validated by qRT-PCR analysis in different tissues (i.e., ovule Ov3, stamen St2, sepal Se3 and petal Pe3). qRT-PCR was carried out using the SYBR Premix Ex Taq II (TaKaRa, China) on a Bio-Rad Real-time PCR system (Foster, United States), and the program was: 95°C for 30 s; 40 cycles of 95°C for 5 s and 60°C for 34 s; 95°C for 15 s. Three technical replicates and at least three independent biological replicates were performed in each condition.

Vector Construction

The full-length of coding sequence of AcABCG38 were amplified using primers listed in Supplementary Table . The PCR fragment was cloned into the pENTR/D-TOPO vector (Invitrogen) and sequenced. The positive clone was recombined into the destination vector pGWB505 by LR reaction. The Agrobacterium tumefaciens (GV3101) with AcABCG38 was used to transform into the abcg1-2abcg16-2 double mutant and WT using a floral dip procedure (Clough and Bent, 1998). The multiple independent T1 plants were analyzed.

Alexander Red Staining

Arabidopsis were fixed in FAA for 24 h, and then picked the anthers on slide, heated until the pollens were dyed in red color. The stained anthers were taken picture by microscope (Carl-Zeiss, Germany).

Results

Identification and Characterization of the Pineapple ABC Transporters

To identify the ABC gene family in pineapple, the HMMER3.0 (Eddy, 2011) was used to get ABC protein sequences containing PFAM ABC domain (PF00005) in pineapple genome database downloaded from the Phytozome v12.1. To obtain accurate AcABC members, the conserved sequences of AcABC were analyzed using NCBI and sequences that lacked ABC annotation were removed. Finally, a total of 100 ABC genes were identified in the pineapple genome by BLAST and phylogenetic analysis with Arabidopsis ABC proteins. Those genes chromosomes distribution was showed in Supplementary Figure . The 100 pineapple proteins were grouped into eight subfamilies, including 5 ABCAs, 20 ABCBs 16 ABCCs, 2 ABCDs, 1 ABCEs, 5 ABCFs, 42 ABCGs and 9 ABCIs (Table ). Although the ABCH subfamily were ubiquitously existed in insects, fishes, echinodermata and myxomycetes (Shao et al., 2013), and it was not identified in the pineapple genomes, this result is similar to the finding of other plant species (Pang et al., 2013). The pineapple ABC genes were named and further classified according to the sequence similarity of Arabidopsis ABC genes. AcABCAs were subdivided into two types: ATH (ABC1 homologous) and AOH (ABC1 homologous), and AcABCBs included three subgroups: MDR (multidrug resistance protein), TAP (transporter associated with antigen processing) and ATM (ABC transporter of the mitochondria); AcABCGs were also classified into two categories: WBC (white-brown complex homologue) and PDR (pleiotropic drug resistance) (Sánchezfernández et al., 2001). The amino acids numbers ranged from 88 aa (AcABCG14) to 2068 aa (AcABCC6) with the corresponding molecular weight varied from 9.32 to 228.97 Kd. The series of information about AcABCs including subgroup names, gene transcript ID, numbers of amino acid residues, proteins size, protein isoelectric point and main expression location were showed in Table .

Phylogenetic Analysis of ABC Family in Pineapple

To further understand the relationship between the AcABCs and AtABCs, full-length protein sequences of pineapple and Arabidopsis ABCs were aligned using MAFFT, and a combined phylogenetic tree was constructed using FastTree. The result showed that the ABC genes of these two species can be divided into eight subfamilies (Figure ). Among those subfamilies, ABCG had the largest members with 42 pineapple genes and 43 Arabidopsis genes. ABCB was the second largest subgroup, containing eighteen pineapple genes and twenty-eight Arabidopsis genes. The smallest subgroup was ABCD and ABCE, both contain only four members: two AcABCD genes and two AtABCD genes, and one AcABCEs and three AtABCEs. According to functional characteristic, the ABCA proteins were further classified into in two distinct types with two members in ATH and 22 in AOH. Similarly, the ABCBs subfamily were also divided into three groups (34 MDRs, 8 TAPs and 4 ATMs) and ABCGs were divided into two types (56 WBCs and 29 PDRs). While ABCI subfamily was divided into three clusters in phylogenetic tree (Figure ). One of which contained only two Arabidopsis members (AtABCI15 and AtABCI16) without any pineapple members, indicating that the ABCI family could had undergone evolutionary divergence between dicotyledonous and monocotyledonous plants.

Phylogenetic relationship of ABC gene among Pineapple (Ac) and Arabidopsis (At). All the AcABC protein sequences were aligned by using MAFFT and phylogenetic tree was constructed using FastTree. Different colors represented individual subfamily. Every subfamily had another classified names: ATH (ABC1 homologous), AOH (ABC1 homologous), MDR (multidrug resistance protein), TAP (transporter associated with antigen processing), ATM (ABC transporter of the mitochondria), WBC (white-brown complex homolog), PDR (pleiotropic drug resistance). AcABCG38, AtABCG1 and AtABCG16 were indicated by asterisk.

To investigate the structure diversity, we analyzed the predicted CDS of individual pineapple ABC gene family using Gene Structure Display Server (Guo et al., 2007). The result showed that the exons number of AcABC genes varied from 1 to 41. AcABCB15 had the maximum exons numbers, while seven AcABC genes (AcABCG4, AcABCG10, AcABCG14, AcABCG23, AcABCG32, AcABCG33 and AcABCG38) have only one exon with no UTR (Figure ). To reveal the diversification of ABCG subfamily in pineapple, the putative motifs were predicted and identified by MEME with the motif numbers setting from 1 to 15 (Bailey and Elkan, 1994). The ABCG subfamily had two types: PDR and WBC. The numbers of motifs in each AcPDRs were approximately twice as many as that of AcWBCs (Figure ). AcABCG14 belonging to AcWBCs had only one motif; while most of AcPDRs contained 15 motifs except for AcABCG1, AcABCG19, and AcABCG42.

Intron–exon structure of AcABC genes in pineapple genome. Yellow bars indicates exon (CDS), Blue bars indicated UTR while plain lines showing introns. black lines represented introns. On the left panel is the phylogenetic trees of ABC transporter proteins in pineapple which was constructed by Maximum likelihood method. The numbers on the right indicate the genomic length of the corresponding genes. bp, base pair.

The motif analysis of AcABCG proteins in pineapple. Motifs with specific colors can be find on respective AcABCG genes. The combined phylogenetic trees of AcABCG subfamily on the left panel. The motifs of corresponding proteins are shown on the right panel with specific colors on behalf of different motifs using the MEME motif search tool (http://meme-suite.org/tools/meme). The order of the motifs corresponds to their position within individual protein sequences.

Expression Profiles of Pineapple ABC Genes in Different Tissues

To analyze the expression profiles, the expression level of 100 AcABC genes were analyzed based on their FPKM values from RNA-seq data of different tissues. On basis of the average log values of each gene in given tissue, a hierarchical cluster and expression patterns of these genes were generated (Figure ). According to the expression specificity, those AcABC genes could be classified into five types. In type I, the genes expressed ubiquitously in most or all tissues such as AcABCB1, AcABCC2, AcABCE1, AcABCF1 and AcABCG31. Conversely, in type II, eleven genes demonstrated comparatively low expression levels in almost all tissues, especially, AcABCB3, AcABCG14 and AcABCG24 had extremely low expression. Sixty-three genes were moderately and evenly expressed in all organs in type III. The eleven genes in type IV had remarkably high expression levels in particular vegetative and reproductive organs (i.e., AcABCA5 and AcABCC4 in ovule and stamen, AcABCC7, AcABCC9, and AcABCG29 in ovule). The ten genes were highly and specifically expressed in one or two development stages of organs in type V (AcABCA4 in root, AcABCB5, AcABCB17 and AcABCAI2 in petal Pe3, AcABCC14 and AcABCG13 in flower and leaf, AcABCG20 in stamen St1, St2, and St5, AcABCG42 in stamen St1–St4 as well as AcABCG26 and AcABCG38 in stamen St3 and St4) (Figure ). We found some genes, which belong to the same group, had analogical expression profiles. For example, the majority of the AcABCC genes were relatively highly expressed in all of tissues, and nearly all of the AcABCI members had similar expression levels. To further verify the reliability from RNA-seq data, the expression levels of four genes (AcABCA5, AcABCG38, AcABCD2 and AcABCI2) in four different tissues were selected for qRT-PCR validation. The results revealed that expression patterns of qRT-PCR were consistent with that from RNA-seq analysis (Figure ).

Tissue-specific expression profiles of AcABC genes in pineapple. Heat-map of tissue-specific expression profiles of AcABC genes in pineapple. RNA-seq expression level can be understood using the given scale and roman numbers on right-side shows hierarchical clusters based on gene expression. The combined phylogenetic trees of pineapple ABC family on the left panel. Red color indicates high levels of transcript abundance, and green indicates low transcript abundance. The color scale is shown at the bottom. Samples are mentioned at the top of each lane: ovule S1–S7, sepal S1–S4, stamen S1–S5, petal S1–S3, root, leaf, flower, fruit S1–S7. “S” is abbreviation of word “stage.”

Validation of AcABC genes RNA-seq by qRT-PCR analysis of four genes’ expression at five different tissues. Line graphs are constructed from relative gene expression (Left side Y-Axis) in different tissues (qRT-PCR) data and FPKM values (RNA-seq) data (Right side Y-Axis) for these tissues.

AcABCG38 Might Play Crucial Roles in Pollen and Anther Development

As showed in Figure , the expression profiling revealed that AcABC38 expressed highly in stamen St3 and St4, implying that this gene may be involved in stamen development. As previous reports, two ABC genes (AtABCG1 and AtABCG16) play significant roles in pollen development (Yadav and Reed, 2014; Yim et al., 2016). Base on phylogenetic analysis, our results showed that AtABCG1, AtABCG16 and AcABCG38 clustered the same subclade. Those results further indicated AcABCG38 functions in pollen development. Thereby, we cloned AcABCG38 gene and transformed into the Arabidopsis double mutant abcg1-2abcg16-2 to check it whether functions or not.

As showed in Figure , the abcg1-2abcg16-2 double mutant has short siliques, lower seed set (Figures ) and collapsed pollens (Figures ). Reciprocal crosses between abcg1-2abcg16-2 double mutant and wild type showed that the reduced fertility in abcg1-2abcg16-2 was due to defective male gametophyte function (Supplementary Table ). Moreover, the transmission of abcg1 allele through pollen but not female reproductive organ was significantly reduced in abcg16-2/abcg16-2 background (p < 0.05) (Supplementary Table ). While the transmission of abcg16 alleles through both male and female was not affected in abcg1-2/abcg1-2 background (Supplementary Table ). These results suggested that AtABCG1 plays dominant role in controlling male gametophytic function.

Over-expressing of AcABCG38 can partly recover the fertility and pollen development defects of double mutant abcg1-2abcg16-2. (A) Alexander red staining of WT anther. (B) Alexander red staining of p35S:AcABCG38 anther. (C) Alexander red staining of double mutant of abcg1-2abcg16-2 anther. (D) Alexander red staining of the abcg1-2abcg16-2 p35S:AcABCG38 line 8# anther. (E) Main branch of the plants with genotype as indicated (left: col-0, mid: abcg1-2abcg16-2, right: abcg1-2abcg16-2 p35S:AcABCG3 line 2#). (F) Siliques of plants with genotype as indicated (left: col-0, mid: abcg1-2abcg16-2, right: transgenic plant 8# in abcg1-2abcg6-2 background). (G) Mature pollens of col-0. (H) Mature pollens of abcg1-2abcg16-2 plant. (I) Mature pollens of the abcg1-2abcg16-2 p35S:AcABCG38 8#. (J) Seed set phenotype of the 25 siliques from bottom to top in main branch of the abcg1-2abcg16-2 plants. Red dots represent the short siliques with reduced seed set; green dots represent the normal siliques with full seed set. (K) Seed set phenotype of the 25 siliques from bottom to top in main branch of the individual abcg1-2abcg16-2 p35S:AcABCG38 lines.

In the T1 generation of the p35S:AcABCG38 transgenic plants, we selected nine individual lines that showed elongated siliques (Figures ) and less aborted pollens (Figures ) compared with the double mutant abcg1-2abcg16-2 (Figures and Table ), however, they had slightly shorter siliques and higher percentage of aborted pollen compared to WT plants (Figures and Table ). These results suggesting that overexpression of AcABCG38 can partially rescue the reduced fertility and pollen abortion phenotype in abcg1-2abcg16-2. The transcript level of AcABCG38 was also evaluated in individual transgenic lines using qRT-PCR to confirm the complemented phenotype with the increased AcABCG38 expression. The results showed that complemented line 8# exhibited the highest transcriptional level of AcABCG38 (Figure , Table , and Supplementary Figure ). Five independent AcABCG38-overexpressing transgenic plants in WT background showed comparable vegetative growth to WT plant and full seed set with normal pollens development as indicated by Alexander red staining (Supplementary Figure ). Taken together, these results indicated that AcABCG38 plays vital roles in pineapple pollen development.

Discussion

Diversity of ABC Transporters in Plants

It is reported that the ABC proteins are relatively conserved across different species but distinct between plants and animals (Xie et al., 2012; Pang et al., 2013). Compared with animal ABC transporters, plant ABC proteins exist numerous and diverse. A number of ABC genes are found in different plant species, e.g., the model plant Arabidopsis thaliana contains 129 ABC genes, maize 130 ABC transporters and rice 128 transporters (Ponte-Sucre, 2007; Pang et al., 2013). In this study, 100 AcABC proteins have been identified genome-wide. Additionally, those ABC proteins can be divided into as many as eight subfamilies, each subfamily can further be divided into a quantity of subsets (Shao et al., 2013). We also identified 18 AcABCB genes, 16 AcABCC genes and 42 AcABCG genes in pineapple genome, accounting for 76% of all AcABC members. Among those subfamilies, AcABCB genes includes 13 AcMDRs, 4 AcTAPs and 1 AcATMs in this study. Previous studies reported that MDRs are responsible for transporting varieties of substrates, such as lipid proteins, bactericin, peptides, cell surface components, auxin transport and so on (Noh et al., 2001; Sasaki et al., 2002; Moons, 2008). ATMs play a vital role in resistance to heavy metals toxicity in plants (Kim et al., 2006). In Arabidopsis, two proteins AtMRP4 and AtMRP5 particularly expressed in guard cells and control stomata conductance (Gaedeke et al., 2001; Frelet, 2003). Maize MRP (ZmABCC5), involved in resistance system, also plays role in anthocyanin transport (Goodman et al., 2004). As showed in Figure , the majority of AcABCC proteins were expressed in all organs in our study, suggesting their potential role in pineapple growth and development. ABCG subfamily participates in transporting lipid precursors of cutin and wax (Pighin et al., 2004). AcABCG contains 42 members and their expression profiles suggests that either they are highly expressed or specific to tissue. Those data clearly indicate that plants require numerous ABC proteins for growth and development. The reason maybe that plants develop a complicated transporter system to survival in diversity environment through a rapid divergence across a long process of natural evolution and selection.

AcABC Gene Expression Profiles Analysis

Recently, the release of pineapple genome sequence makes it available for researchers to explore pineapple specific agronomic traits (Ming et al., 2015). To explore the function of ABC transporters in pineapple, we downloaded the pineapple different tissues RNA-seq data, and analyzed the expression levels of 100 AcABC genes based on their FPKM values. As showed in Figure , a hierarchical cluster and expression patterns of these genes were generated. Those AcABC genes could express specifically. For example, AcABCB6 expressed in floral organs, AcABCB5 and AcABCB17 in Pe3 shows tissue-specific expression. AtABCB1 localized in the plasma membrane and participated in IAA-induced plant development (Sasaki et al., 2002). AcABCB1, clustered with AtABCB1 in same clades by phylogenetic analysis, expressed broadly in all pineapple tissues, suggesting that AcABCB1 could also be involved in auxin induced pineapple development.

Interesting, some AcABCs expressed remarkably in reproductive tissues, e.g., AcABCA5 and AcABCC4 in ovule and stamen; AcABCC7, AcABCC9 and AcABCG29 in ovule; and AcABCG31 was specific to floral organs; indicating their potential roles in pineapple flower development. AtABCG1 and AtABCG16 and redundantly in controlling Arabidopsis pollen development (Yadav and Reed, 2014; Yim et al., 2016) and suggests that AcABCG38 may also be involved in pollen development. The expression pattern of AcABCs together with the reported functions of corresponsive Arabidopsis orthologs could provide clue to understand the function of AcABCs in pineapple.

AcABC38 Regulates Pineapple Reproductive Development

Sexual propagation could be an effective and potential way to improve the pineapple agricultural production (Kondrashov, 1993). However, the function of genes in pineapple development are barely reported. Our study aims to identify ABC genes that are crucial for pineapple propagation. For instance, AcABCG26 and AcABCG38 were highly expressed in different stages of stamen (Figure ). The expression profiles of these genes in reproductive organs at different developmental stages could determine its functional role. To validate functions of AcABCs, AcABCG38 was selected for functional study as it showed high expression in stamens at St3 and St4. Also Arabidopsis homologs of AcABCG38, AtABCG1 and AtABCG16 are known to work redundantly in pollen development (Yadav and Reed, 2014; Yim et al., 2016).

Over-expression of AcABCG38 in Arabidopsis double mutant abcg1-2abcg16-2 was able to partly rescue the defective pollen development phenotype, indicating that AcABCG38 might play important roles for pollen development in pineapple. The mechanism underlying the regulation of AcABCG38 on pineapple pollen development is still unknown and further studies could shed light on it. Present study of pineapple AcABC genes provide useful information on gene function and may act as foundation for future pineapple research.

Conclusion

The ABC transporter gene families are ubiquitous and important in all kind of life events for all life organism. Whereas the information about pineapple ABC gene family is not available. Here we identified 100 ABC genes in the pineapple genome and grouped them into eight subfamilies. Gene expression profiling revealed many tissue specific genes particularly in reproductive organs. Furthermore, over-expression of AcABCG38 in Arabidopsis double mutant abcg1-2abcg16-2 partly rescued the defective pollen development phenotype, indicating that AcABCG38 might have important function for pollen development in pineapple. Overall, the characterization and expression profile study of pineapple AcABC genes provide useful information for gene functional study as well as insights into future pineapple researches for improving pineapple sexual plant reproduction.

Author Contributions

Created and designed the experiments: PC, YiL, and YQ. Carried out the experiments: PC, LZ, and ZH. The analysis of data: YiL. Contributed reagents/materials/analysis tools: PC, LZ, ZH, MY, BH, YaL, SA, ZZ, ZR, and LL. Wrote the paper: PC and YQ.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Table 1

The ABC gene family in pineapple.

Group	Transcipt ID	Gene name	Topology	Ch	Length(aa)	MW(Kd)	pI	Main loca.
Subfamily A, 5 members
AOH	Aco007263.1	AcABCA1	(TMD-NBD)2	LG23	1885	209.74	7.28	fruit.S7
ATH	Aco028137.1	AcABCA2	TMD-NBD	scaffold_1582	967	107.06	7.81	Flower
	Aco006844.1	AcABCA3	TMD-NBD	LG01	961	106.43	6.95	Stamen.S5
	Aco006845.1	AcABCA4	TMD-NBD	LG01	890	99.17	8.62	Ovule.S7
	Aco014363.1	AcABCA5	TMD-NBD	LG05	947	106.5	8.76	Flower
Subfamily B, 18 members
MDR	Aco010196.1	AcABCB1	(TMD-NBD)2	LG25	1252	144.98	9.32	Ovule.S1
	Aco012952.1	AcABCB2	(TMD-NBD)2	LG03	1216	139.29	9.18	Petal.S3
	Aco027643.1	AcABCB3	TMD-NBD-TMD	scaffold_777	992	107.44	8.91	Root
	Aco016500.1	AcABCB4	(TMD-NBD)2	LG11	1613	108.46	9.03	Stamen.S4
	Aco022110.1	AcABCB5	(TMD-NBD)2	LG04	1294	140.81	7.89	fruit.S4
	Aco001135.1	AcABCB6	(TMD-NBD)2	LG02	1239	136.8	8.75	fruit.S4
	Aco018951.1	AcABCB7	(TMD-NBD)2	LG12	1258	140.34	8.97	Stamen.S2
	Aco006827.1	AcABCB8	(TMD-NBD)2	LG01	1295	135.1	9.03	Stamen.S3
	Aco019592.1	AcABCB9	(TMD-NBD)2	LG10	978	138.8	8.68	Flower
	Aco013278.1	AcABCB10	(TMD-NBD)2	LG24	1395	139.04	8.69	Ovule.S6
	Aco016496.1	AcABCB11	(TMD-NBD)2-NBD	LG11	1407	174.98	6.21	Root
	Aco022325.1	AcABCB12	(TMD-NBD)2	LG19	1263	135.39	8.81	fruit.S1
	Aco003987.1	AcABCB19	(TMD-NBD)2	LG15	645	151.04	6.27	Stamen.S3
	Aco001486.1	AcABCB20	(TMD-NBD)2	LG18	1270	155.88	6.4	Stamen.S1
	Aco008863.1	AcABCB13	(TMD-NBD)2	LG09	1340	132.98	8.9	Stamen.S2
TAP	Aco007808.1	AcABCB14	TMD-NBD	LG21	1653	70.41	7.98	Stamen.S3
	Aco017569.1	AcABCB15	TMD-(NBD)2-TMD-NBD	LG09	756	179.38	6.29	fruit.S4
	Aco015743.1	AcABCB16	TMD2-NBD	LG09	741	82.03	9.05	Ovule.S4
	Aco017326.1	AcABCB17	TMD-NBD	LG01	755	82.29	8.86	Ovule.S2
ATM	Aco011166.1	AcABCB18	TMD-NBD	LG04	309	82.67	9.32	fruit.S7
Subfamily C, 16 members
	Aco009750.1	AcABCC1	(TMD-NBD)2	LG10	1545	170.6	7.71	Flower
	Aco014828.1	AcABCC2	(TMD-NBD)2	LG14	1543	173.73	6.83	Ovule.S1
	Aco027927.1	AcABCC3	(TMD-NBD)2	scaffold_1613	1544	170.76	8.16	Stamen.S5
	Aco019200.1	AcABCC4	(TMD-NBD)2	LG13	1404	154.1	6.18	Stamen.S4
	Aco010163.1	AcABCC5	(TMD-NBD)2	LG25	1522	168.42	8.27	Flower
	Aco019507.1	AcABCC6	(TMD-NBD)2	LG07	2068	228.97	7.1	Ovule.S3
	Aco009944.1	AcABCC7	(TMD-NBD)2	LG10	1463	163.87	8.43	Stamen.S4
	Aco015193.1	AcABCC8	(TMD-NBD)2	LG05	1463	163.62	8.82	fruit.S3
	Aco000353.1	AcABCC9	(TMD-NBD)2	LG12	1491	166.33	7.94	Stamen.S5
	Aco005539.1	AcABCC10	(TMD-NBD)2	LG11	1485	164.82	8.42	Stamen.S1
	Aco006625.1	AcABCC11	(TMD-NBD)2	LG01	1583	177.22	8.71	Ovule.S2
	Aco025745.1	AcABCC12	NBD	LG01	348	39.08	5.29	Ovule.S1
	Aco016941.1	AcABCC13	(TMD-NBD)2	LG02	1285	143.55	6.29	Petal.S3
	Aco017858.1	AcABCC14	(TMD-NBD)2	LG23	1261	140.25	6.04	Sepal.S2
	Aco012910.1	AcABCC15	(TMD-NBD)2	LG03	1514	169.94	8.6	Leaf
	Aco016196.1	AcABCC16	TMD-NBD	LG17	309	33.65	9.69	Ovule.S4
Subfamily D, 2 members
	Aco023353.1	AcABCD1	(TMD-NBD)2	LG18	1342	149.88	9.16	Stamen.S5
	Aco010685.1	AcABCD2	TND-NBD	LG10	750	83.8	8.48	Stamen.S3
Subfamily E, 1 members
	Aco010811.1	AcABCE1	NBD2	LG10	605	68.22	8.04	Stamen.S5
Subfamily F, 5 members
	Aco010200.1	AcABCF1	NBD3	LG25	601	67.25	6.07	Petal.S3
	Aco006898.1	AcABCF2	NBD3	LG22	603	67.03	6.37	Leaf
	Aco009295.1	AcABCF3	NBD3	LG22	719	79.99	5.92	Stamen.S1
	Aco018812.1	AcABCF4	NBD2	LG13	742	81.44	5.87	Stamen.S4
	Aco000176.1	AcABCF5	NBD3	LG12	717	79.54	6.07	fruit.S2
Subfamily G,42 members
PDR	Aco005449.1	AcABCG1	TMD-NBD-TMD2	LG11	1234	140.02	7.35	Sepal.S2
	Aco005451.1	AcABCG2	(NBD-TMD)2	LG11	1393	157.13	8.66	Stamen.S1
	Aco027149.1	AcABCG6	(NBD-TMD)2	scaffold_1231	1419	159.96	8.93	Sepal.S3
	Aco009786.1	AcABCG8	(NBD-TMD)2	LG10	1347	151.89	8.83	Flower
	Aco006783.1	AcABCG12	(NBD-TMD)2	LG01	1456	164.5	8.47	fruit.S5
	Aco006142.1	AcABCG13	(NBD-TMD)2	LG16	1476	166.35	6.59	Sepal.S1
	Aco023493.1	AcABCG19	(NBD-TMD)2	LG01	1416	160.42	7.85	Stamen.S4
	Aco015369.1	AcABCG20	(NBD-TMD)2	LG23	1449	161.94	8.62	Stamen.S5
	Aco021666.1	AcABCG21	(NBD-TMD)2	LG07	1282	145.07	8.65	fruit.S1
	Aco009791.1	AcABCG31	(NBD-TMD)2	LG10	1452	165.01	8.77	Ovule.S7
	Aco006143.1	AcABCG36	(NBD-TMD)2	LG16	1410	158.85	6.51	Ovule.S4
	Aco014323.1	AcABCG39	(NBD-TMD)2	LG05	1450	164.01	7.54	Sepal.S2
	Aco013658.1	AcABCG41	(NBD-TMD)2	LG13	1452	164.64	7.72	Stamen.S3
	Aco015172.1	AcABCG42	NBD2-TMD-NBD-TMD	LG05	1584	177.88	8.91	Stamen.S4
WBC	Aco010405.1	AcABCG3	NBD-TMD	LG03	727	80.78	9.09	Root
	Aco030876.1	AcABCG4	NBD-TMD	scaffold_1304	612	65.76	8.91	Sepal.S3
	Aco011308.1	AcABCG5	NBD-TMD	LG01	610	66.69	9.39	fruit.S5
	Aco021246.1	AcABCG7	NBD-TMD	LG10	691	75.26	8.96	fruit.S1
	Aco009154.1	AcABCG9	NBD-TMD	LG09	642	70.19	8.61	Stamen.S3
	Aco006105.1	AcABCG10	NBD-TMD	LG16	591	64.95	9.14	Root
	Aco010632.1	AcABCG11	NBD-TMD	LG07	723	80.1	9.18	Stamen.S2
	Aco023881.1	AcABCG14	NBD	LG20	88	9.32	10.37	/
	Aco007271.1	AcABCG15	NBD-TMD	LG23	677	75.25	9.59	Ovule.S1
	Aco020513.1	AcABCG16	NBD	LG01	283	31.43	11.09	Leaf
	Aco010077.1	AcABCG17	NBD-TMD	LG25	714	78.57	8.54	Ovule.S4
	Aco001046.1	AcABCG18	Arf-NBD-TMD	LG02	896	100.18	8.27	fruit.S7
	Aco021024.1	AcABCG22	NBD-TMD	LG15	743	81.87	9.15	Sepal.S1
	Aco002328.1	AcABCG23	NBD-TMD	LG04	612	68.93	6.81	Root
	Aco023879.1	AcABCG24	NBD	LG20	201	21.67	9.49	Root
	Aco003255.1	AcABCG25	NBD-TMD	LG17	605	65.4	9.38	Ovule.S7
	Aco023619.1	AcABCG26	NBD-TMD	LG19	641	71.96	9.36	Stamen.S4
	Aco013155.1	AcABCG27	NBD-TMD	LG24	601	66.02	9.42	Stamen.S3
	Aco006887.1	AcABCG28	NBD	LG22	1089	120.53	9.12	Ovule.S5
	Aco007658.1	AcABCG29	NBD-TMD	LG08	721	79.81	8.84	Flower
	Aco001237.1	AcABCG30	NBD-TMD	LG02	728	80.13	7.59	Flower
	Aco031846.1	AcABCG32	NBD-TMD	scaffold_3635	372	39.56	8.97	Stamen.S1
	Aco004405.1	AcABCG33	NBD-TMD	LG05	605	64.96	8.82	Sepal.S4
	Aco000542.1	AcABCG34	NBD-TMD	LG12	642	70.76	10.47	Leaf
	Aco010996.1	AcABCG35	NBD-TMD	LG04	707	77.84	8.43	Sepal.S2
	Aco005255.1	AcABCG37	NBD-TMD	LG07	720	79.28	8.97	Petal.S3
	Aco001503.1	AcABCG38	NBD-TMD	LG18	794	87.24	9.1	Stamen.S3
	Aco019952.1	AcABCG40	NBD	LG08	861	96.25	9.2	Stamen.S4
Subfamily I, 9 members
	Aco008459.1	AcABCI1	NBD	LG19	224	24.87	9.69	Petal.S3
	Aco028846.1	AcABCI2	NBD	scaffold_627	301	32.04	8.48	Stamen.S1
	Aco018474.1	AcABCI3	NBD	LG21	431	48.82	7.17	Stamen.S1
	Aco005118.1	AcABCI4	NBD	LG07	283	30.25	8.56	Sepal.S1
	Aco001741.1	AcABCI5	NBD	LG18	307	33.24	8.68	Stamen.S2
	Aco007380.1	AcABCI6	NBD	LG23	326	34.26	6.62	Stamen.S1
	Aco006975.1	AcABCI7	NBD	LG22	291	32.74	5.48	fruit.S4
	Aco030323.1	AcABCI8	NBD	scaffold_1361	312	32.85	6.4	Petal.S2
	Aco004516.1	AcABCI9	NBD	LG05	350	38.23	8.2	fruit.S3

Table 2

The abcg1-2abcg6-2 p35S:AcABCG38 plants have higher percentage of normal pollen than p35S:AcABCG38 plants.

	WT	abcg1-2-/- abcg16-2-/-	2#	3#	5#	6#	7#	8#	15#	18#	19#
Normal	1331	275	965	656	591	979	478	1044	1418	657	784
Abnormal	3	1015	434	449	476	551	353	275	633	448	393
Total	1334	1290	1399	1105	1066	1530	831	1319	2051	1105	1177
Normal (%)	99.48	21.3	69.0	59.4	55.4	64.0	57.3	79.2	69.1	59.5	66.6