Proteogenomic Gene Structure Validation in the Pineapple Genome.

MD2 pineapple (Ananas comosus) is the second most important tropical crop that preserves crassulacean acid metabolism (CAM), which has high water-use efficiency and is fast becoming the most consumed fresh fruit worldwide. Despite the significance of environmental efficiency and popularity, until very recently, its genome sequence has not been determined and a high-quality annotated proteome has not been available. Here, we have undertaken a pilot proteogenomic study, analyzing the proteome of MD2 pineapple leaves using liquid chromatography-mass spectrometry (LC-MS/MS), which validates 1781 predicted proteins in the annotated F153 (V3) genome. In addition, a further 603 peptide identifications are found that map exclusively to an independent MD2 transcriptome-derived database but are not found in the standard F153 (V3) annotated proteome. Peptide identifications derived from these MD2 transcripts are also cross-referenced to a more recent and complete MD2 genome annotation, resulting in 402 nonoverlapping peptides, which in turn support 30 high-quality gene candidates novel to both pineapple genomes. Many of the validated F153 (V3) genes are also supported by an independent proteomics data set collected for an ornamental pineapple variety. The contigs and peptides have been mapped to the current F153 genome build and are available as bed files to display a custom gene track on the Ensembl Plants region viewer. These analyses add to the knowledge of experimentally validated pineapple genes and demonstrate the utility of transcript-derived proteomics to discover both novel genes and genetic structure in a plant genome, adding value to its annotation.

Integrative Analysis of Metabolome and Transcriptome Provides Insights into the Mechanism of Flower Induction in Pineapple (Ananas comosus (L.) Merr.) by Ethephon.

Exogenous ethylene is commonly utilized to initiate flower induction in pineapple (Ananas comosus (L.) Merr.). However, the molecular mechanisms and metabolic changes involved are not well understood. In this study, we explored the genetic network and metabolic shifts in the 'Comte de Paris' pineapple variety during ethylene-induced flowering. This was achieved through an integrative analysis of metabolome and transcriptome profiles at vegetative shoot apexes (0 d after ethephon treatment named BL_0d), the stage of bract primordia (8 d after ethephon treatment named BL_8d), stage of flower primordia (18 d after ethephon treatment named BL_18d), and the stage of stopped floret differentiation (34 d after ethephon treatment named BL_34d). We isolated and identified 804 metabolites in the pineapple shoot apex and inflorescence, categorized into 24 classes. Notably, 29, 31, and 46 metabolites showed significant changes from BL_0d to BL_8d, BL_8d to BL_18d, and BL_18d to BL_34d, respectively. A marked decrease in indole was observed, suggesting its role as a characteristic metabolite during flower induction. Transcriptomic analysis revealed 956, 1768, and 4483 differentially expressed genes (DEGs) for BL_0d vs. BL_8d, BL_8d vs. BL_18d, and BL_18d vs. BL_34d, respectively. These DEGs were significantly enriched in carbohydrate metabolism and hormone signaling pathways, indicating their potential involvement in flower induction. Integrating metabolomic and transcriptomic data, we identified several candidate genes, such as Agamous-Like9 (AGL9), Ethylene Insensitive 3-like (ETIL3), Apetala2 (AP2), AP2-like ethylene-responsive transcription factor ANT (ANT), and Sucrose synthase 2 (SS2), that play potentially crucial roles in ethylene-induced flower induction in pineapple. We also established a regulatory network for pineapple flower induction, correlating metabolites and DEGs, based on the Arabidopsis thaliana pathway as a reference. Overall, our findings offer a deeper understanding of the metabolomic and molecular mechanisms driving pineapple flowering.

Comparative transcriptome analysis reveals candidate genes for cold stress response and early flowering in pineapple.

Pineapple originates from tropical regions in South America and is therefore significantly impacted by cold stress. Periodic cold events in the equatorial regions where pineapple is grown may induce early flowering, also known as precocious flowering, resulting in monetary losses due to small fruit size and the need to make multiple passes for harvesting a single field. Currently, pineapple is one of the most important tropical fruits in the world in terms of consumption, and production losses caused by weather can have major impacts on worldwide exportation potential and economics. To further our understanding of and identify mechanisms for low-temperature tolerance in pineapple, and to identify the relationship between low-temperature stress and flowering time, we report here a transcriptomic analysis of two pineapple genotypes in response to low-temperature stress. Using meristem tissue collected from precocious flowering-susceptible MD2 and precocious flowering-tolerant Dole-17, we performed pairwise comparisons and weighted gene co-expression network analysis (WGCNA) to identify cold stress, genotype, and floral organ development-specific modules. Dole-17 had a greater increase in expression of genes that confer cold tolerance. The results suggested that low temperature stress in Dole-17 plants induces transcriptional changes to adapt and maintain homeostasis. Comparative transcriptomic analysis revealed differences in cuticular wax biosynthesis, carbohydrate accumulation, and vernalization-related gene expression between genotypes. Cold stress induced changes in ethylene and abscisic acid-mediated pathways differentially between genotypes, suggesting that MD2 may be more susceptible to hormone-mediated early flowering. The differentially expressed genes and module hub genes identified in this study are potential candidates for engineering cold tolerance in pineapple to develop new varieties capable of maintaining normal reproduction cycles under cold stress. In addition, a total of 461 core genes involved in the development of reproductive tissues in pineapple were also identified in this study. This research provides an important genomic resource for understanding molecular networks underlying cold stress response and how cold stress affects flowering time in pineapple.

Identification of bromelain subfamily proteases encoded in the pineapple genome.

Papain (aka C1A) family proteases, including bromelain enzymes, are widespread across the plant kingdom and play critical regulatory functions in protein turnover during development. The proteolytic activity exhibited by papain family proteases has led to their increased usage for a wide range of cosmetic, therapeutic, and medicinal purposes. Bromelain enzymes, or bromelains in short, are members of the papain family that are specific to the bromeliad plant family. The only major commercial extraction source of bromelain is pineapple. The importance of C1A family and bromelain subfamily proteases in pineapple development and their increasing economic importance led several researchers to utilize available genomic resources to identify protease-encoding genes in the pineapple genome. To date, studies are lacking in screening bromelain genes for targeted use in applied science studies. In addition, the bromelain genes coding for the enzymes present in commercially available bromelain products have not been identified and their evolutionary origin has remained unclear. Here, using the newly developed MD2 v2 pineapple genome, we aimed to identify bromelain-encoding genes and elucidate their evolutionary origin. Orthologous and phylogenetic analyses of all papain-family proteases encoded in the pineapple genome revealed a single orthogroup (189) and phylogenetic clade (XIII) containing the bromelain subfamily. Duplication mode and synteny analyses provided insight into the origin and expansion of the bromelain subfamily in pineapple. Proteomic analysis identified four bromelain enzymes present in two commercially available bromelain products derived from pineapple stem, corresponding to products of four putative bromelain genes. Gene expression analysis using publicly available transcriptome data showed that 31 papain-family genes identified in this study were up-regulated in specific tissues, including stem, fruit, and floral tissues. Some of these genes had higher expression in earlier developmental stages of different tissues. Similar expression patterns were identified by RT-qPCR analysis with leaf, stem, and fruit. Our results provide a strong foundation for future applicable studies on bromelain, such as transgenic approaches to increase bromelain content in pineapple, development of bromelain-producing bioreactors, and studies that aim to determine the medicinal and/or therapeutic viability of individual bromelain enzymes.

Integrated Metabolome and Transcriptome Analysis Reveals a Potential Mechanism for Water Accumulation Mediated Translucency in Pineapple (Ananas comosus (L.) Merr.) Fruit.

A physiological disease of the pineapple fruit called pineapple translucency causes the pulp to become water-soaked, which affects the fruit's taste, flavor, shelf life, and integrity. In the present study, we analyzed seven pineapple varieties, of which three were watery and four were non-watery. There were no apparent macronutritional (K, P, or N) differences in their pulp, but the non-watery pineapple varieties had higher dry matter and soluble sugar content. The metabolomic analysis found 641 metabolites and revealed differential expression of alkaloids, phenolic acids, nucleotide derivatives, lipids, and other metabolites among the seven species. Transcriptome analysis and further KEGG enrichment showed downregulation of 'flavonoid biosynthesis' pathways, differential expression of metabolic pathways, secondary metabolites biosynthesis, plant-pathogen interaction, and plant hormone signal transduction. We believe this study will provide critical molecular data supporting a deeper understanding of pineapple translucency formation and greatly benefit future research on this commercially important crop.

Unveiling the molecular mechanism involving anthocyanins in pineapple peel discoloration during fruit maturation.

Peel color is a key factor that affects the fruit's aesthetic and economic values. Limited knowledge is available on the regulation of pineapple peel discoloration. Here, we report that a decrease in anthocyanin biosynthesis, particularly cyanidin, is predominantly associated with the pineapple peel color change during maturation. The findings suggest that the changes in the expression of key structural genes (early and late biosynthetic genes) of the anthocyanin (cyanidin) biosynthesis pathway are responsible for peel discoloration. Based on a gene co-expression analysis and a transient expression, two transcription factors i.e., AcHOX21 and AcMYB12, were identified, whose' downregulation leads to reduced anthocyanin accumulation with fruit maturation. The endogenous levels of jasmonic acid, gibberellic acid, and auxins are also involved in anthocyanin-content-led peel discoloration. Overall, the discovery of genes regulating anthocyanin biosynthesis in pineapple peel provides a theoretical basis for improving the fruit's aesthetic value through genetic engineering.

Genetic and phenotype recovery of Ananas comosus var. MD2 in response to ionizing radiation.

Due to their sessile nature, plants are exposed to various environmental stressors such as exposure to high levels of harmful ultraviolet (UV), ionizing, and non-ionizing radiations. This exposure may result in various damages, ranging from DNA and chromosomal aberrations to phenotypic abnormalities. As an adaptation, plants have evolved efficient DNA repair mechanisms to detect and repair any damage caused by exposure to these harmful stressors to ensure their survival. In this study, the effects of gamma radiation (as a source of ionizing radiation) on clonal Ananas comosus var. MD2 was evaluated. The morphology and physiology of the clonal plantlets before and after exposure to gamma radiation were monitored at specific time intervals. The degree of genetic variation between the samples pre- and post-irradiation was also analyzed by using inter-simple sequence repeat (ISSR) markers. The resulting data revealed that the heights of the irradiated plantlets were significantly reduced (compared to control), but improved with the recovery period. Irradiated samples also exhibited relatively good photosynthetic efficiency that further improved as the plantlets recover. These observations were supported by the ISSR analysis, where the genetic dissimilarities between the irradiated samples and control were reduced by 0.1017, after 4 weeks of recovery. Overall, our findings suggested that the phenotype recovery of the clonal A. comosus var. MD2 plantlets was contributed by their ability to detect and repair the DNA lesions (as exemplified by the reduction in genetic dissimilarity after 4 weeks) and hence allow the plantlets to undergo phenotype reversion to normal plant stature.

Genome-Wide Identification and Expression Patterns of AcSWEET Family in Pineapple and AcSWEET11 Mediated Sugar Accumulation.

Pineapple (Ananas comosus L.) is an important fruit crop in tropical regions, and it requires efficient sugar allocation during fruit development. Sugars Will Eventually be Exported Transporters (SWEETs) are a group of novel sugar transporters which play critical roles in seed and fruit development. However, the function of AcSWEETs remains unknown in the sugar accumulation. Herein, 17 AcSWEETs were isolated and unevenly located in 11 chromosomes. Analysis of a phylogenetic tree indicated that 17 genes were classified into four clades, and the majority of AcSWEETs in each clade shared similar conserved motifs and gene structures. Tissue-specific gene expression showed that expression profiles of AcSWEETs displayed differences in different tissues and five AcSWEETs were strongly expressed during fruit development. AcSWEET11 was highly expressed in the stage of mature fruits in 'Tainong16' and 'Comte de paris', which indicates that AcSWEET11 was important to fruit development. Subcellular localization analysis showed that AcSWEET11 was located in the cell membrane. Notably, overexpression of AcSWEET11 could improve sugar accumulation in pineapple callus and transgenic tomato, which suggests that AcSWEET11 might positively contribute to sugar accumulation in pineapple fruit development. These results may provide insights to enhance sugar accumulation in fruit, thus improving pineapple quality in the future.

MicroRNA expression profile of cholesterol metabolism analysis mediated by Thelenota ananas desulfated holothurin A saponin in RAW264.7 macrophage-foam cells.

Saponins from the sea cucumber, Thelenota ananas, have been reported to modulate cholesterol metabolism and may be useful in treating atherosclerosis and related diseases, but the mechanism remains unclear. This study is designed to investigate the effect of the saponin desulfated holothurin A (desHA), prepared from Thelenota ananas, on cholesterol and lipid metabolism in oxidized low-density lipoprotein (oxLDL)-induced RAW264.7 macrophage-foam cells. The detection and prediction of miRNAs were conducted by high-throughput sequencing and associated bioinformatics analysis. We found that desHA could play an essential role in the species and expression of miRNAs. There were more abundant miRNAs in the desHA-treated groups. miR-365-2-5p, -125b-1-3p, -744-5p, -330-3p, -125a-3p and -3057-5p were extremely suppressed by desHA under the oxLDL treatment, while miR-212-3p and miR-132-3p were significantly upregulated by desHA. The clusters of orthologous groups (COG) analysis indicated that the detected miRNAs participated in lipid transport and metabolism (I) and secondary metabolite biosynthesis, transport, and catabolism (Q), which were closely linked with cholesterol metabolism. The network of the nine miRNAs with higher degree of differential expression (miR-125a-3p, -23b-5p, -3057-5p, -330-3p, -365-2-5p and -744-5p, -345-5p -212-3p and -132-3p) and their target genes further showed relationships with inflammatory response, cell proliferation and cholesterol metabolism. Due to miR-125a-3p and miR-365-2-5p being involved in the regulation of more genes, which are mostly related to the functions involved in cholesterol metabolism, they may be potential targets for desHA to prevent or treat atherosclerosis.

Heterologous Expression and Catalytic Properties of Codon-Optimized Small-Sized Bromelain from MD2 Pineapple.

Bromelain is a unique enzyme-based bioactive complex containing a mixture of cysteine proteases specifically found in the stems and fruits of pineapple (Ananas comosus) with a wide range of applications. MD2 pineapple harbors a gene encoding a small bromelain cysteine protease with the size of about 19 kDa, which might possess unique properties compared to the other cysteine protease bromelain. This study aims to determine the expressibility and catalytic properties of small-sized (19 kDa) bromelain from MD2 pineapple (MD2-SBro). Accordingly, the gene encoding MD2-SBro was firstly optimized in its codon profile, synthesized, and inserted into the pGS-21a vector. The insolubly expressed MD2-SBro was then resolubilized and refolded using urea treatment, followed by purification by glutathione S-transferase (GST) affinity chromatography, yielding 14 mg of pure MD2-SBro from 1 L of culture. The specific activity and catalytic efficiency (kcat/Km) of MD2-SBro were 3.56 ± 0.08 U mg-1 and 4.75 ± 0.23 × 10-3 µM-1 s-1, respectively, where optimally active at 50 °C and pH 8.0, and modulated by divalent ions. The MD2-SBro also exhibited the ability to scavenge the 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) with an IC50 of 0.022 mg mL-1. Altogether, this study provides the production feasibility of active and functional MD2-Bro as a bioactive compound.

High-temperature ethanol fermentation from pineapple waste hydrolysate and gene expression analysis of thermotolerant yeast Saccharomyces cerevisiae.

High-temperature ethanol fermentation by thermotolerant yeast is considered a promising technology for ethanol production, especially in tropical and subtropical regions. In this study, optimization conditions for high-temperature ethanol fermentation of pineapple waste hydrolysate (PWH) using a newly isolated thermotolerant yeast, Saccharomyces cerevisiae HG1.1, and the expression of genes during ethanol fermentation at 40 °C were carried out. Three independent variables, including cell concentration, pH, and yeast extract, positively affected ethanol production from PWH at 40 °C. The optimum levels of these significant factors evaluated using response surface methodology (RSM) based on central composite design (CCD) were a cell concentration of 8.0 × 107 cells/mL, a pH of 5.5, and a yeast extract concentration of 4.95 g/L, yielding a maximum ethanol concentration of 36.85 g/L and productivity of 3.07 g/L. Gene expression analysis during high-temperature ethanol fermentation using RT-qPCR revealed that the acquisition of thermotolerance ability and ethanol fermentation efficiency of S. cerevisiae HG1.1 are associated with genes responsible for growth and ethanol stress, oxidative stress, acetic acid stress, DNA repair, the pyruvate-to-tricarboxylic acid (TCA) pathway, and the pyruvate-to-ethanol pathway.

Ectopic Overexpression of Pineapple Transcription Factor AcWRKY31 Reduces Drought and Salt Tolerance in Rice and Arabidopsis.

Pineapple (Ananas comosus (L.) Merr.) is an important tropical fruit with high economic value, and its growth and development are affected by the external environment. Drought and salt stresses are common adverse conditions that can affect crop quality and yield. WRKY transcription factors (TFs) have been demonstrated to play critical roles in plant stress response, but the function of pineapple WRKY TFs in drought and salt stress tolerance is largely unknown. In this study, a pineapple AcWRKY31 gene was cloned and characterized. AcWRKY31 is a nucleus-localized protein that has transcriptional activation activity. We observed that the panicle length and seed number of AcWRKY31 overexpression transgenic rice plants were significantly reduced compared with that in wild-type plant ZH11. RNA-seq technology was used to identify the differentially expressed genes (DEGs) between wild-type ZH11 and AcWRKY31 overexpression transgenic rice plants. In addition, ectopic overexpression of AcWRKY31 in rice and Arabidopsis resulted in plant oversensitivity to drought and salt stress. qRT-PCR analysis showed that the expression levels of abiotic stress-responsive genes were significantly decreased in the transgenic plants compared with those in the wild-type plants under drought and salt stress conditions. In summary, these results showed that ectopic overexpression of AcWRKY31 reduced drought and salt tolerance in rice and Arabidopsis and provided a candidate gene for crop variety improvement.

Genome-wide identification and characterization of the BBX gene family in pineapple reveals that candidate genes are involved in floral induction and flowering.

B-box zinc finger proteins contain one or two B-box domains, and sometimes, a CCT domain, which are involved in many biological processes, such as photomorphogenesis, flowering, anthocyanin synthesis and abiotic stress resistance. But the BBX gene family in pineapple has not been systematically studied. Nineteen BBX genes were detected in pineapple genome and divided into five groups according to phylogenetic analysis. The results of transcriptome analysis and RT-qPCR showed that most of AcBBX members were highly expressed during the flowering process, indicating that AcBBX gene may be involved in flower bud differentiation and morphogenesis. Transcriptional activation analysis showed that AcBBX6 and AcBBX18 had transcriptional activity and were located in the nucleus. Overexpression of AcBBX18 promoted flowering in Arabidopsis thaliana. These results provided a basis for further study functions and regulatory mechanism of BBX members in pineapple floral induction and flower development.

Gene prediction for leaf margin phenotype and fruit flesh color in pineapple (Ananas comosus) using haplotype-resolved genome sequencing.

Pineapple (Ananas comosus (L.) Merr.) is one of the most economically important tropical fruit species. The major aim of the breeding programs in several countries, including Japan, is quality improvement, mainly for the fresh market. 'Yugafu', a Japanese cultivar with distinctive pipe-type leaf margin phenotype and white flesh color, is popular for fresh consumption. Therefore, genome sequencing of 'Yugafu' is expected to assist pineapple breeding. Here, we developed a haplotype-resolved assembly for the heterozygous genome of 'Yugafu' using long-read sequencing technology and obtained a pair of 25 pseudomolecule sequences inherited from the parental accessions 'Cream pineapple' and 'HI101'. The causative genes for leaf margin and fruit flesh color were identified. Fine mapping revealed a 162-kb region on CLG23 for the leaf margin phenotype. In this region, 20 kb of inverted repeat was specifically observed in the 'Cream pineapple' derived allele, and the WUSCHEL-related homeobox 3 (AcWOX3) gene was predicted as the key gene for leaf margin morphogenesis. Dominantly repressed AcWOX3 via RNAi was suggested to be the cause of the pipe-type leaf margin phenotype. Quantitative trait locus (QTL) analysis revealed that the terminal region of CLG08 contributed to white flesh and low carotenoid content. Carotenoid cleaved dioxygenase 4 (AcCCD4), a key gene for carotenoid degradation underlying this QTL, was predicted as the key gene for white flesh color through expression analysis. These findings could assist in modern pineapple breeding and facilitate marker-assisted selection for important traits.

The highly continuous reference genome of a leaf-chimeric red pineapple (Ananas comosus var. bracteatus f. tricolor) provides insights into elaboration of leaf color.

Ananas comosus var. bracteatus f. tricolor (GL1) is a red pineapple accession whose mostly green leaves with chimeric white leaf margins turn red in spring and autumn and during flowering. It is an important ornamental plant and ideal plant research model for anthocyanin metabolism, chimeric leaf development, and photosynthesis. Here, we generated a highly contiguous chromosome-scale genome assembly for GL1 and compared it with other 3 published pineapple assemblies (var. comosus accessions MD2 and F153, and var. bracteatus accession CB5). The GL1 assembly has a total size of ∼461 Mb, with a contig N50 of ∼2.97 Mb and Benchmarking Universal Single-Copy Ortholog score of 97.3%. More than 99% of the contigs are anchored to 25 pseudochromosomes. Compared with the other 3 published pineapple assemblies, the GL1 assembly was confirmed to be more continuous. Our evolutionary analysis showed that the Bromeliaceae and Poaceae diverged from their nearest common ancestor ∼82.36 million years ago (MYA). Population structure analysis showed that while GL1 has not undergone admixture, bracteatus accession CB5 has resulted from admixture of 3 species of Ananas. Through classification of orthogroups, analysis of genes under positive selection, and analysis of presence/absence variants, we identified a series of genes related to anthocyanin metabolism and development of chimeric leaves. The structure and evolution of these genes were compared among the published pineapple assemblies with reveal candidate genes for these traits. The GL1 genome assembly and its comparisons with other 3 pineapple genome assemblies provide a valuable resource for the genetic improvement of pineapple and serve as a model for understanding the genomic basis of important traits in different pineapple varieties and other pan-cereal crops.

Integrative transcriptomic and metabolomic analysis of D-leaf of seven pineapple varieties differing in N-P-K% contents.

BACKGROUND: Pineapple (Ananas comosus L. Merr.) is the third most important tropical fruit in China. In other crops, farmers can easily judge the nutritional requirements from leaf color. However, concerning pineapple, it is difficult due to the variation in leaf color of the cultivated pineapple varieties. A detailed understanding of the mechanisms of nutrient transport, accumulation, and assimilation was targeted in this study. We explored the D-leaf nitrogen (N), phosphorus (P), and potassium (K) contents, transcriptome, and metabolome of seven pineapple varieties. RESULTS: Significantly higher N, P, and K% contents were observed in Bali, Caine, and Golden pineapple. The transcriptome sequencing of 21 libraries resulted in the identification of 14,310 differentially expressed genes in the D-leaves of seven pineapple varieties. Genes associated with N transport and assimilation in D-leaves of pineapple was possibly regulated by nitrate and ammonium transporters, and glutamate dehydrogenases play roles in N assimilation in arginine biosynthesis pathways. Photosynthesis and photosynthesis-antenna proteins pathways were also significantly regulated between the studied genotypes. Phosphate transporters and mitochondrial phosphate transporters were differentially regulated regarding inorganic P transport. WRKY, MYB, and bHLH transcription factors were possibly regulating the phosphate transporters. The observed varying contents of K% in the D-leaves was associated to the regulation of K+ transporters and channels under the influence of Ca2+ signaling. The UPLC-MS/MS analysis detected 873 metabolites which were mainly classified as flavonoids, lipids, and phenolic acids. CONCLUSIONS: These findings provide a detailed insight into the N, P, K% contents in pineapple D-leaf and their transcriptomic and metabolomic signatures.

Recent amplification of microsatellite-associated miniature inverted-repeat transposable elements in the pineapple genome.

BACKGROUND: Miniature inverted-repeat transposable elements (MITEs) are non-autonomous DNA transposable elements that play important roles in genome organization and evolution. Genome-wide identification and characterization of MITEs provide essential information for understanding genome structure and evolution. RESULTS: We performed genome-wide identification and characterization of MITEs in the pineapple genome. The top two MITE families, accounting for 29.39% of the total MITEs and 3.86% of the pineapple genome, have insertion preference in (TA) n dinucleotide microsatellite regions. We therefore named these MITEs A. comosus microsatellite-associated MITEs (Ac-mMITEs). The two Ac-mMITE families, Ac-mMITE-1 and Ac-mMITE-2, shared sequence similarity in the terminal inverted repeat (TIR) regions, suggesting that these two Ac-mMITE families might be derived from a common or closely related autonomous elements. The Ac-mMITEs are frequently clustered via adjacent insertions. Among the 21,994 full-length Ac-mMITEs, 46.1% of them were present in clusters. By analyzing the Ac-mMITEs without (TA) n microsatellite flanking sequences, we found that Ac-mMITEs were likely derived from Mutator-like DNA transposon. Ac-MITEs showed highly polymorphic insertion sites between cultivated pineapples and their wild relatives. To better understand the evolutionary history of Ac-mMITEs, we filtered and performed comparative analysis on the two distinct groups of Ac-mMITEs, microsatellite-targeting MITEs (mt-MITEs) that are flanked by dinucleotide microsatellites on both sides and mutator-like MITEs (ml-MITEs) that contain 9/10 bp TSDs. Epigenetic analysis revealed a lower level of host-induced silencing on the mt-MITEs in comparison to the ml-MITEs, which partially explained the significantly higher abundance of mt-MITEs in pineapple genome. The mt-MITEs and ml-MITEs exhibited differential insertion preference to gene-related regions and RNA-seq analysis revealed their differential influences on expression regulation of nearby genes. CONCLUSIONS: Ac-mMITEs are the most abundant MITEs in the pineapple genome and they were likely derived from Mutator-like DNA transposon. Preferential insertion in (TA) n microsatellite regions of Ac-mMITEs occurred recently and is likely the result of damage-limiting strategy adapted by Ac-mMITEs during co-evolution with their host. Insertion in (TA) n microsatellite regions might also have promoted the amplification of mt-MITEs. In addition, mt-MITEs showed no or negligible impact on nearby gene expression, which may help them escape genome control and lead to their amplification.

Comparative transcriptome analysis of a fan-shaped inflorescence in pineapple using RNA-seq.

Pineapple plant usually has a capitulum. However, a fan-shaped inflorescence was exceptionally evolved in pineapple, having multiple crown buds. In order to reveal the molecular mechanisms of the formation of the fan-shaped inflorescence, fruit traits and the transcriptional differences between the fan-shaped inflorescence and the wild-shaped inflorescence pineapples were analyzed in three tissues, i.e., the flower stem apex, the base of the inflorescence, and the inflorescence axis. The weight (i.e., individual yield) of fan-shaped fruit is 4.5 times that of wild-shaped fruit；and non-significant difference in soluble solids, soluble sugar, titratable acid, and Vitamin C was found. Between the fan-shaped inflorescence and wild-shaped inflorescence, a total of 5370 differentially expressed genes were identified across the three tissues. Of these genes, there were 489 overlapping differentially expressed genes in all three tissue comparisons. Between the two pineapples, functional analysis indicated that 444 transcription factors and 206 inflorescence development-related genes were differentially expressed in at least one tissue comparison, while 45 transcription factors and 21 inflorescence development-related genes were overlapped across three tissues. Among the 489 overlapping differentially expressed genes in the three tissue comparisons, excluding the inflorescence development-related genes and transcription factors, 80 of them revealed a higher percentage of involvement in the biological processes relating to response to auxin, and reproductive processes. RNA-seq value and real-time quantitative PCR analysis exhibited the similar gene expression patterns in the three tissues. Our result provided novel cues for understanding the molecular mechanisms of the formation of the fan-shaped inflorescence in pineapple, making a valuable resource for the study of plant breeding and the speciation of pineapple.

Genome-wide characterization and expression profiling of B3 superfamily during ethylene-induced flowering in pineapple (Ananas comosus L.).

BACKGROUND: The B3 superfamily (B3s) represents a class of large plant-specific transcription factors, which play diverse roles in plant growth and development process including flowering induction. However, identification and functional surveys of B3 superfamily have not been reported in ethylene-induced pineapple flowering (Ananas comosus). RESULTS: 57 B3 genes containing B3 domain were identified and phylogenetically classified into five subfamilies. Chromosomal localization analysis revealed that 54 of 57 AcB3s were located on 21 Linkage Groups (LG). Collinearity analysis demonstrated that the segmental duplication was the main event in the evolution of B3 gene superfamily, and most of them were under purifying selection. The analysis of cis-element composition suggested that most of these genes may have function in response to abscisic acid, ethylene, MeJA, light, and abiotic stress. qRT-PCR analysis of 40 AcB3s containing ethylene responsive elements exhibited that the expression levels of 35 genes were up-regulated within 1 d after ethephon treatment and some were highly expressed in flower bud differentiation period in stem apex, such as Aco012003, Aco019552 and Aco014401. CONCLUSION: This study provides a basic information of AcB3s and clues for involvement of some AcB3s in ethylene-induced flowering in pineapple.

A novel ampelovirus associated with mealybug wilt of pineapple (Ananas comosus).

Mealybug wilt of pineapple (MWP) is the most important and complex viral disease affecting pineapple worldwide. High-throughput sequencing was conducted to characterize a new virus identified only in symptomatic pineapple plants and tentatively named pineapple mealybug wilt-associated virus 6 (PMWaV-6). Data analyses revealed a genome of 17,854 nucleotides with an organization resembling members of the genus Ampelovirus, family Closteroviridae. Encoded proteins shared sequence identity with the corresponding proteins of grapevine leafroll-associated virus 3, blackberry vein banding-associated virus, and PMWaV-2. The present study reports the discovery of PMWaV-6, a putative and distinct new member of the genus Ampelovirus, subgroup I, its potential involvement in MWP, and the development of PMWaV-6-specific RT-PCR assays to detect and monitor this virus in field samples.