Characteristics of Pineapple Flower
After the inflorescence of pineapple emerges, its inflorescence comes out from among leaf clumps and looks like a pine cone. The entire inflorescence development to bloom is approximately 1 month. The capitulum of pineapple is composed of 50 to 200 florets. The blossoming order of the capitulum is from the bottom to the top (Fig. 1A) [22]. The pineapple flowers are hermaphrodite and consist of three sepals, three petals, five stamens, and one pistil (Fig. 1B). The edible part of pineapple involves the fleshy axis of inflorescence and the ovary of florets.
Identification and Classification of MADS-box Genes in Pineapple
The MADS-box protein sequences were used to Hidden Markov Model (HMM) search, and 54 candidate genes were originally obtained. The sequence analysis indicated that ten candidate MADS-box genes containing incomplete MADS-box domains were removed. Forty-four MADS-box genes were selected and annotated in pineapple (Supplementary Table S1). Two maximum likelihood trees (ML) were further constructed on the basis of the full-length sequence alignment of all AcMADS genes together with grape and Arabidopsis to provide a reference for the evolutionary relationship of the MADS-box family in pineapple (Fig. 2). The 44 AcMADS genes of the pineapple can be divided into two categories, namely, type I (16) and type II (28). In the first ML tree, type I AcMADS genes were further classified into three subclasses: Mα, Mβ, and Mγ (Fig. 2A). One MIKC*-type and 27 MIKCC-type genes were showed in the second ML tree, and the MIKCC -type genes were further classed into 11 major groups (Fig. 2B). These groups were named as follows: SVP (SHORT VEGETATIVE PHASE), AGL12, SEP, AGL6, AP1, FLC (FLOWERING LOCUS C), SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1), AG, PI/AP3, TT16 (TRANSPARENT TESTA16), and ANR.
Chromosomal Location and Duplication Analysis of Pineapple MADS-box Genes
Forty-four MADS-box genes were unevenly mapped to the 18 linkage groups (LGs) and five scaffolds and named from AcMADS1 to AcMADS44 according to their order on the LGs. The largest number of six genes (13.64%) was found in LG01, and the other LGs contained less than three AcMADS genes. Among the 44 AcMADS genes, 16 genes of type I were distributed on 10 chromosomes and three scaffolds, and 28 genes of type II were mapped to 16 chromosomes and two scaffolds. LG04 and LG13 only contained type I genes, while LG05, LG06, LG10, LG16, LG20, LG21, LG22, and LG24 only had type II genes (Fig. 3). Tandem duplication event refers to a region of chromosomes within 200 kb containing two or more genes [23]. The gene replication events of the MADS-box family found that one pair of genes (AcMADS17/AcMADS18) underwent tandem repeat events within the AcMADS gene family on LG7 (Fig. 3). These results showed randomness and nonuniform distribution of the AcMADS family in pineapple.
Syntenic Analysis of Pineapple AcMADS Genes
The segmental and tandem duplication events of the AcMADS genes family were identified to test the duplication effect in pineapple. In addition to the above-mentioned two tandem duplication events, twenty-two AcMADS genes were clustered into 15 segmental duplication events by BLASTP and MCScanX methods (Fig. 4, Supplementary Table S2). We found many copies of the segmental duplicated gene pairs from the same group, such as AcMADS08/15.AcMADS08/34 and AcMADS14/27 were from the ANR and SVP groups, respectively. AcMADS3/12 and AcMADS3/35 were from the AG group, AcMADS1/16 were from the AP1 group, and AcMADS38/39 were from SOC1. These AcMADS genes showed highly orthologous relationship, thereby indicating that they were obtained by gene duplication and may be from a common ancestor.
The comparative syntenic maps between pineapple with other five representative species (Arabidopsis, grape, banana, rice, and maize) were performed to further derive the origin and evolutionary mechanisms of pineapple MADS family (Fig. 5). Twenty-four AcMADS genes displayed syntenic relationship with those in banana, followed by maize (23, 52%), rice (22, 50%), grape (10, 32%), and Arabidopsis (6, 13%) through the whole genome-wide comparative analysis. The numbers of orthologous pairs between pineapple and other five species (banana, rice, maize, grape, and Arabidopsis) were 42, 42, 40, 14, and 12, respectively (Supplementary Table S3). Many collinear gene pairs were only found in monocots but not in dicots, thereby suggesting evolutionary difference between dicotyledonous and monocotyledonous plants. The mutual collinear pairs involving three AcMADS genes were identified between pineapple and all five other species. This finding indicates that these orthologous pairs may be derived from the same ancestor, and duplication occurred before species divergence. These pairs may participate in the evolution of MADS family.
Tissue-specific Expression Patterns of AcMADS Genes in Pineapple
MADS-box genes were reported to participate in plant organ development, especially floral organ specification [2]. The expression patterns of 44 pineapple AcMADS genes in different tissues were obtained from the transcriptome data. The results showed that the transcriptional abundance of MADS genes in pineapple greatly varied in all detected samples (Fig. 6). The genes with high expression were mainly concentrated in type II subfamily. Twenty-six AcMADS genes (59%) were highly expressed in the flower of pineapple. Meanwhile, 14 AcMADS genes (32%) were highly expressed in the fruit of pineapple. Sixteen genes (36%) were expressed at low levels or not expressed in pineapple root, bud, leaf, flower, and fruit (Supplementary Table S4). Multiple of AcMADS genes were specifically expressed in flower and fruit. For example, five AcMADS genes (AcMADS5/10/16/21/24) showed high transcript abundance in flower and fruit, two genes (AcMADS06/13) only demonstrated high transcript abundance in flowers, and two genes (AcMADS36/4) only presented high expression in the roots (Fig. 6A). These implied MADS-box genes might participate in the flower development of pineapple, which was in line with previous reports [24].
The expressions of all 44 AcMADS genes in different floral organs were investigated to further verify the potential functions of the AcMADS genes in the formation of floral organs (Fig. 6B). Thirty-eight AcMADS genes exhibited relatively high expression in one or more flower organs of pineapple, and fifteen of them were highly expressed. Most AcMADS genes with high expression in floral organs originated from the type II subfamily, thereby suggesting that AcMADS genes of type II may play crucial roles in the development of floral organs of pineapple contrast with type I (Fig. 6B). Twenty-eight type II AcMADS genes were further performed by qRT-PCR to validate the RNA-seq results (Fig. 7). The qRT-PCR results showed that the expression profiles of most AcMADS genes were consistent with RNA-Seq. Many AcMADS genes showed obvious tissue specificity. For example, AcMADS12 only expressed in pistil, AcMADS03, AcMADS31, AcMADS33, and AcMADS35 had specifically high expression in the ovary, and AcMADS08 only expressed in the stamen (Fig. 8).
Co-expression network of key AcMADS genes
Plant flowering is a complex physiological process that requires multiple genes to work together. Studying gene co-expression networks contributes to the exploration of the potential functions of genes. The qRT-PCR and RNA-seq results indicated that six AcMADS genes with specific expressions in the floral organs of pineapple were selected to construct an interaction network through the String Protein Interaction Database (https://string-db.org/). A total of 129 protein pairs with interactions were detected. These interaction proteins were mainly involved in the floral organ developmental genes and floral induction, including AP1, CO, WUS, SEU, AP2, UFO, and LFY (Fig. 9). In the co-expression network diagram, AcMADS3 interacted with 10 known proteins with the largest number of interacting proteins. AcMADS35, AcMADS12, AcMADS31, and AcMADS33 interacted with 2, 4, 6, and 8 known proteins, respectively. AcMADS8 only interacted with one known protein. These results will be beneficial to future research and verify its biological function on the basis of relevant experiments.