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In the present study, both acidic and alkaline hydrolysate of pineapple waste was utilised for the production of biohydrogen using locally isolated bacterial strains. The bacteria were isolated from different wastewater sources and were identified as Proteus mirabilis, Pseudomonas aeruginosa, Bacillus altitudinus, Bacillus subtilis, Paenibacillus alvei, and Lysinibacillus sphaericus . Experimental results showed that the highest biohydrogen yield of 836.33 ± 48.02 mL H 2 was produced from alkaline hydrolysate with Bacillus altitudinis during the 96 th hr of fermentation. Among the different bacterial strains, B. altitudinis showed higher H 2 production. Comparatively alkaline hydrolysates exhibited a higher yield of hydrogen than acidic hydrolysates. The final pH of the experiment was found to be in acidic range. The total VFA concentration ranged between 930 ± 207.85 mg/L to 3050 ± 476.97 mg/L. Both sugar degradation and COD reduction were more than 80% in the acidic and alkaline hydrolysates while the lowest sugar degradation and COD reduction were observed for the untreated biomass. The rationale behind this study was to convert the waste biomass into energy by utilising the potential of native bacterial communities.

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.

The gibberellic acid-stimulated Arabidopsis (GASA) gene family plays a crucial role in growth, development, and stress response, and it is specific to plants. This gene family has been extensively studied in various plant species, and its functional role in pineapple has yet to be characterized. In this study, 15 AcGASA genes were identified in pineapple through a genome-wide scan and categorized into three major branches based on a phylogenetic tree. All AcGASA proteins share a common structural domain with 12 cysteine residues, but they exhibit slight variations in their physicochemical properties and motif composition. Predictions regarding subcellular localization suggest that AcGASA proteins are present in the cell membrane, Golgi apparatus, nucleus, and cell wall. An analysis of gene synteny indicated that both tandem and segmental repeats have a significant impact on the expansion of the AcGASA gene family. Our findings demonstrate the differing regulatory effects of these hormones (GA, NAA, IAA, MeJA, and ABA) on the AcGASA genes. We analyzed the expression profiles of GASA genes in different pineapple tissue parts, and the results indicated that AcGASA genes exhibit diverse expression patterns during the development of different plant tissues, particularly in the regulation of floral organ development. This study provides a comprehensive understanding of GASA family genes in pineapple. It serves as a valuable reference for future studies on the functional characterization of GASA genes in other perennial herbaceous plants.

Pineapple color yellowing and quality promotion gradually manifest as pineapple fruit ripening progresses. To understand the molecular mechanism underlying yellowing in pineapples during ripening, coupled with alterations in fruit quality, comprehensive metabolome and transcriptome investigations were carried out. These investigations were conducted using pulp samples collected at three distinct stages of maturity: young fruit (YF), mature fruit (MF), and fully mature fruit (FMF). This study revealed a noteworthy increase in the levels of total phenols and flavones, coupled with a concurrent decline in lignin and total acid contents as the fruit transitioned from YF to FMF. Furthermore, the analysis yielded 167 differentially accumulated metabolites (DAMs) and 2194 differentially expressed genes (DEGs). Integration analysis based on DAMs and DEGs revealed that the biosynthesis of plant secondary metabolites, particularly the flavonol, flavonoid, and phenypropanoid pathways, plays a pivotal role in fruit yellowing. Additionally, RNA-seq analysis showed that structural genes, such as FLS , FNS , F3H , DFR , ANR , and GST , in the flavonoid biosynthetic pathway were upregulated, whereas the COMT , CCR , and CAD genes involved in lignin metabolism were downregulated as fruit ripening progressed. APX as well as PPO, and ACO genes related to the organic acid accumulations were upregulated and downregulated, respectively. Importantly, a comprehensive regulatory network encompassing genes that contribute to the metabolism of flavones, flavonols, lignin, and organic acids was proposed. This network sheds light on the intricate processes that underlie fruit yellowing and quality alterations. These findings enhance our understanding of the regulatory pathways governing pineapple ripening and offer valuable scientific insight into the molecular breeding of pineapples.

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., (© 2023. The Author(s).)

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., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Junhu He reports financial support was provided by Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Ananas comosus var. bracteatus ( Ac. bracteatus ) is a typical leaf-chimeric ornamental plant. The chimeric leaves are composed of central green photosynthetic tissue (GT) and marginal albino tissue (AT). The mosaic existence of GT and AT makes the chimeric leaves an ideal material for the study of the synergistic mechanism of photosynthesis and antioxidant metabolism. The daily changes in net photosynthetic rate (NPR) and stomatal conductance (SCT) of the leaves indicated the typical crassulacean acid metabolism (CAM) characteristic of Ac. bracteatus . Both the GT and AT of chimeric leaves fixed CO 2 during the night and released CO 2 from malic acid for photosynthesis during the daytime. The malic acid content and NADPH-ME activity of the AT during the night was significantly higher than that of GT, which suggests that the AT may work as a CO 2 pool to store CO 2 during the night and supply CO 2 for photosynthesis in the GT during the daytime. Furthermore, the soluble sugar content (SSC) in the AT was significantly lower than that of GT, while the starch content (SC) of the AT was apparently higher than that of GT, indicating that AT was inefficient in photosynthesis but may function as a photosynthate sink to help the GT maintain high photosynthesis activity. Additionally, the AT maintained peroxide balance by enhancing the non-enzymatic antioxidant system and antioxidant enzyme system to avoid antioxidant damage. The enzyme activities of reductive ascorbic acid (AsA) and the glutathione (GSH) cycle (except DHAR) and superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were enhanced, apparently to make the AT grow normally. This study indicates that, although the AT of the chimeric leaves was inefficient at photosynthesis because of the lack of chlorophyll, it can cooperate with the GT by working as a CO 2 supplier and photosynthate store to enhance the photosynthetic ability of GT to help chimeric plants grow well. Additionally, the AT can avoid peroxide damage caused by the lack of chlorophyll by enhancing the activity of the antioxidant system. The AT plays an active role in the normal growth of the chimeric leaves.

In plants, sexual reproduction relies on the proper development of floral organs that facilitate the successful development of fruits and seeds. Auxin responsive small auxin-up RNA (SAUR) genes play essential roles in floral organ formation and fruit development. However, little is known about the role of SAUR genes in pineapple floral organ formation and fruit development as well as stress responses. In this study, based on genome information and transcriptome datasets, 52 AcoSAUR genes were identified and grouped into 12 groups. The gene structure analysis revealed that most AcoSAUR genes did not have introns, although auxin-acting elements were abundant in the promoter region of AcoSAUR members. The expression analysis across the multiple flower and fruit development stages revealed differential expression of AcoSAUR genes, indicating a tissue and stage-specific function of AcoSAURs. Correlation analysis and pairwise comparisons between gene expression and tissue specificity identified stamen-, petal-, ovule-, and fruit-specific AcoSAURs involved in pineapple floral organs (AcoSAUR4/5/15/17/19) and fruit development (AcoSAUR6/11/36/50). RT-qPCR analysis revealed that AcoSAUR12/24/50 played positive roles in response to the salinity and drought treatment. This work provides an abundant genomic resource for functional analysis of AcoSAUR genes during the pineapple floral organs and fruit development stages. It also highlights the role of auxin signaling involved in pineapple reproductive organ growth., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

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.

To uncover the mechanism underlying membrane lipid metabolism of low temperature induced internal browning tolerance in pineapple, membrane phospholipid alterations of harvested 'Comte de Paris' winter pineapple fruit stored at either 10 °C or 25 °C was investigated. Fruit stored at 10 °C developed low levels of internal browning as compared to fruit stored at 25 °C and was associated with high contents of phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine, and low levels of phosphatidic acid. Storage at 10 °C down-regulated the expression levels of phospholipase As. Fruit stored at 10 °C also exhibited high ratio of unsaturated fatty acid to saturated fatty acid and index of unsaturated fatty acid level. These findings suggest that maintenance phospholipid abundance, reduction in phosphatidic acid accumulation and membrane lipid peroxidation may have contributed to the enhanced internal browning tolerance in 'Comte de Paris' winter pineapple fruit at low temperature storage., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kanghua Song reports was provided by No. Kanghua Song reports a relationship with No that includes: board membership, employment, and funding grants. Kanghua Song has patent pending to no. no., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Chlorophyll and heme are essential molecules for photosynthesis and respiration, which are competing branches of the porphyrin metabolism pathway. Chlorophyll and heme balance regulation is very important for the growth and development of plants. The chimeric leaves of Ananas comosus var. bracteatus were composed of central photosynthetic tissue (PT) and marginal albino tissue (AT), which were ideal materials for the study of porphyrin metabolism mechanisms. In this study, the regulatory function of ALA content on porphyrin metabolism (chlorophyll and heme balance) was revealed by comparing PT and AT, 5-Aminolevulinic Acid (ALA) exogenous supply, and interference of hemA expression. The AT remained similar in porphyrin metabolism flow level to the PT by keeping an equal ALA content in both tissues, which was very important for the normal growth of the chimeric leaves. As the chlorophyll biosynthesis in AT was significantly inhibited, the porphyrin metabolism flow was directed more toward the heme branch. Both tissues had similar Mg 2+ contents; however, Fe 2+ content was significantly increased in the AT. The chlorophyll biosynthesis inhibition in the white tissue was not due to a lack of Mg 2+ and ALA. A 1.5-fold increase in ALA content inhibited chlorophyll biosynthesis while promoting heme biosynthesis and hemA expression. The doubling of ALA content boosted chlorophyll biosynthesis while decreasing hemA expression and heme content. HemA expression interference resulted in a higher ALA content and a lower chlorophyll content, while the heme content remained at a relatively low and stable level. Conclusively, a certain amount of ALA was important for the stability of porphyrin metabolism and the normal growth of plants. The ALA content appears to be able to regulate chlorophyll and heme content by bidirectionally regulating porphyrin metabolism branch direction.

Protein phosphatase 2C (PP2C) is a negative regulator of serine/threonine residue protein phosphatase and plays an important role in abscisic acid (ABA) and abiotic-stress-mediated signaling pathways in plants. The genome complexity of woodland strawberry and pineapple strawberry is different due to the difference in chromosome ploidy. This study conducted a genome-wide investigation of the FvPP2C ( Fragaria vesca ) and FaPP2C ( Fragaria ananassa ) gene family. Fifty-six FvPP2C genes and 228 FaPP2C genes were identified from the woodland strawberry and pineapple strawberry genomes, respectively. FvPP2Cs were distributed on seven chromosomes, and FaPP2Cs were distributed on 28 chromosomes. The size of the FaPP2C gene family was significantly different from that of the FvPP2C gene family, but both FaPP2Cs and FvPP2Cs were localized in the nucleus, cytoplasm, and chloroplast. Phylogenetic analysis revealed that 56 FvPP2Cs and 228 FaPP2Cs could be divided into 11 subfamilies. Collinearity analysis showed that both FvPP2Cs and FaPP2Cs had fragment duplication, and the whole genome duplication was the main cause of PP2C gene abundance in pineapple strawberry. FvPP2Cs mainly underwent purification selection, and there were both purification selection and positive selection effects in the evolution of FaPP2Cs . Cis -acting element analysis found that the PP2C family genes of woodland and pineapple strawberries mainly contained light responsive elements, hormone responsive elements, defense and stress responsive elements, and growth and development-related elements. The results of quantitative real-time PCR (qRT-PCR) showed that the FvPP2C genes showed different expression patterns under ABA, salt, and drought treatment. The expression level of FvPP2C18 was upregulated after stress treatment, which may play a positive regulatory role in ABA signaling and abiotic stress response mechanisms. This study lays a foundation for further investigation on the function of the PP2C gene family.

The R2R3-MYB proteins comprise the largest class of MYB transcription factors, which play an essential role in regulating anthocyanin synthesis in various plant species. Ananas comosus var. bracteatus is an important colorful anthocyanins-rich garden plant. The spatio-temporal accumulation of anthocyanins in chimeric leaves, bracts, flowers, and peels makes it an important plant with a long ornamental period and highly improves its commercial value. We conducted a comprehensive bioinformatic analysis of the R2R3-MYB gene family based on genome data from A. comosus var. bracteatus . Phylogenetic analysis, gene structure and motif analysis, gene duplication, collinearity, and promoter analysis were used to analyze the characteristics of this gene family. In this work, a total of 99 R2R3-MYB genes were identified and classified into 33 subfamilies according to phylogenetic analysis, and most of them were localized in the nucleus. We found these genes were mapped to 25 chromosomes. Gene structure and protein motifs were conserved among AbR2R3-MYB genes, especially within the same subfamily. Collinearity analysis revealed four pairs of tandem duplicated genes and 32 segmental duplicates in AbR2R3-MYB genes, indicating that segmental duplication contributed to the amplification of the AbR2R3-MYB gene family. A total of 273 ABRE responsiveness, 66 TCA elements, 97 CGTCA motifs, and TGACG motifs were the main cis elements in the promoter region under response to ABA, SA, and MEJA. These results revealed the potential function of AbR2R3-MYB genes in response to hormone stress. Ten R2R3-MYBs were found to have high homology to MYB proteins reported to be involved in anthocyanin biosynthesis from other plants. RT-qPCR results revealed the 10 AbR2R3-MYB genes showed tissue-specific expression patterns, six of them expressed the highest in the flower, two genes in the bract, and two genes in the leaf. These results suggested that these genes may be the candidates that regulate anthocyanin biosynthesis of A . comosus var. bracteatus in the flower, leaf, and bract, respectively. In addition, the expressions of these 10 AbR2R3-MYB genes were differentially induced by ABA, MEJA, and SA, implying that these genes may play crucial roles in hormone-induced anthocyanin biosynthesis. Our study provided a comprehensive and systematic analysis of AbR2R3-MYB genes and identified the AbR2R3-MYB genes regulating the spatial-temporal anthocyanin biosynthesis in A. comosus var. bracteatus , which would be valuable for further study on the anthocyanin regulation mechanism of A. comosus var. bracteatus.

Asthma is a polygenic chronic inflammatory respiratory disease devastating the quality of life and state economies. Therefore, utilization of natural products as a therapeutic approach has attained wider consideration for development of novel drugs for asthma management. Bromelain, a mixture of natural bioactive cysteine proteases abundantly found in pineapple stem, has allured attention for its pharmacological activities. However, poor stability in gastric milieu, high dose and immunogenicity associated with prolonged use hinders its oral use. Therefore, need exists to explore alternative route of bromelain administration to achieve its plausible benefits. The present study investigated the preclinical prospects of nasal administration of bromelain on systemic bioavailability, tissue distribution and it's in vivo anti-histaminic, bronchodilator and anti-asthmatic activity in animal models. Pharmacokinetic studies revealed 1.43-fold higher relative bioavailability with faster absorption of bromelain on nasal administration at one-fourth oral dose. The enhanced cellular uptake and localization of bromelain in tissues of lung was observed significantly. Furthermore, faster onset and enhanced antihistaminic, bronchodilator and anti-asthmatic activity on bromelain's nasal administration signified faster absorption and higher in vivo stability of bromelain. Nasal administration significantly achieved decrease in level of oxidative and immunological markers along with restoration of antioxidant enzymes at considerably one-fourth dose administered orally. These findings distinctly manifested that nasal administration could be a substantial and effective route for bromelain delivery with enduring competency in asthma management., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Sharma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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.

Hypercholesterolaemia is a significant risk factor for developing vascular disease and fatty liver. Pineapple ( Ananas comosus ), a tropical fruit widely cultivated in Asia, is reported to exhibit antioxidant and cholesterol-lowering activity; however, the potential hypolipidaemic mechanisms of pineapple fruit remain unknown. Therefore, we aimed to identify the anti-hypercholesterolaemic mechanism of pineapple fruit and to study the effect of pineapple fruit intake on hypercholesterolaemia-induced vascular dysfunction and liver steatosis in a high-cholesterol diet (HCD)-fed rats. Male Sprague Dawley rats were fed with standard diet or HCD, and the pineapple fruit was orally administered to HCD-fed rats for 8 weeks. At the end of treatment, vascular reactivity and morphology of aortas, as well as serum nitrate/nitrite (NOx), were determined. Liver tissues were also examined for histology, lipid content, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) activity, and protein expression of cholesterol metabolism-related enzymes. Results showed that pineapple fruit reduced the levels of hepatic cholesterol and triglycerides, and improved histological characteristics of a fatty liver in HCD-fed rats. Pineapple fruit also increased serum NOx, restored endothelium-dependent vasorelaxation, and reduced structural alterations in aortas of rats fed the HCD. In addition, a reduction of HMGCR activity and the downregulation of hepatic expression of HMGCR and sterol-regulatory element-binding protein 2 (SREBP2), as well as the upregulation of hepatic expression of cholesterol 7α-hydroxylase (CYP7A1) and LDL receptor (LDLR) were found in pineapple fruit-treated hypercholesterolaemic rats. These results indicate that pineapple fruit consumption can restore fatty liver and protect vascular endothelium in diet-induced hypercholesterolaemia through an improvement of hepatic cholesterol metabolism.

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 × 10 7 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., (© 2022. The Author(s).)

Plant-specific calcineurin B-like proteins (CBLs) and their interacting kinases, CBL-interacting protein kinases (CIPKs) module, are essential for dealing with various biotic and abiotic stress. The kinases (CIPKs) of this module have been well studied in several plants; however, the information about pineapple CIPKs remains limited. To understand how CIPKs function against environmental cues in pineapple, the CIPK5 gene of pineapple was cloned and characterized. The phylogenetic analyses revealed that AcCIPK5 is homologous to the CIPK12 of Arabidopsis and other plant species. Quantitative real-time PCR (qRT-PCR) analysis revealed that AcCIPK5 responds to multiple stresses, including osmotic, salt stress, heat and cold. Under optimal conditions, AcCIPK5 gets localized to the cytoplasm and cell membrane. The ectopic expression of AcCIPK5 in Arabidopsis improved the germination under osmotic and salt stress. Furthermore, AcCIPK5 positively regulated osmotic, drought, salt and cold tolerance and negatively regulated heat and fungal stress in Arabidopsis. Besides, the expression of AcCIPK impacted ABA-related genes and ROS homeostasis. Overall, the present study demonstrates that AcCIPK5 contributes to multiple stress tolerance and has the potential to be utilized in the development of stress-tolerant crops., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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 AcWRKY 31 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.

Background: The increase in patients suffering from type I hypersensitivity, including hay fever and food allergy, is a serious public health issue around the world. Recent studies have focused on allergy prevention by food factors with fewer side effects. The purpose of this study was to evaluate the effect of dietary glucosylceramide from pineapples (P-GlcCer) on type I hypersensitivity and elucidate mechanisms., Results: Oral administration of P-GlcCer inhibited ear edema in passive cutaneous anaphylaxis reaction. In a Caco-2/RBL-2H3 co-culture system, P-GlcCer inhibited β-hexosaminidase release from RBL-2H3 cells. The direct treatment of P-GlcCer on RBL-2H3 did not affect β-hexosaminidase release, but sphingoid base moiety of P-GlcCer did. These results predicted that sphingoid base, a metabolite of P-GlcCer, through the intestine inhibited type I hypersensitivity by inhibiting mast cell degranulation. In addition, the inhibitory effects of P-GlcCer on ear edema and degranulation of RBL-2H3 cells were canceled by pretreatment of leukocyte mono-immunoglobulin-like receptor 3 (LMIR3)-Fc, which can block LMIR3-mediated inhibitory signals., Conclusion: It was demonstrated that a sphingoid base, one of the metabolites of P-GlcCer, may inhibit mast cell degranulation by binding to LMIR3. The oral administration of P-GlcCer is a novel and attractive food factor that acts directly on mast cells to suppress allergy. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

<b>Background and Objective:</b> Pineapple (<i>Ananas comosus</i>) is a popular fruit worldwide with natural antioxidant properties. This study examined how pineapple modified the expression of drug-metabolizing enzymes (CYP1A2, CYP2C9, CYP3A4, UGT1A6, NAT2 and SULT1A1) and a drug transporter (OATP1B1) in human hepatocarcinoma (HepG2) cells. <b>Materials and Methods:</b> HepG2 cells (2.5×10<sup>5</sup> cells/well in a 24-well plate) were incubated with pineapple juice extract (125-1,000 μg mL<sup>1</sup>) for 48 hrs in phenol red-free medium. Resazurin reduction, ROS, AST and ALT assays were performed. The mRNA expression of target genes was determined by RT/qPCR. <b>Results:</b> Pineapple juice slightly reduced HepG2 cell viability to 80% of the control, while ROS, AST and ALT levels were not changed. Pineapple juice did not alter the expression of CYP1A2, CYP2C9 and UGT1A6 mRNA. All tested concentrations of pineapple juice suppressed CYP3A4, NAT2 and OATP1B1 expression, while SULT1A1 expression was induced. <b>Conclusion:</b> Though pineapple juice slightly decreased the viability of HepG2 cells, cell morphology and cell function remained normal. Pineapple juice disturbed the expression of phase I (CYP3A4) and phase II (NAT2 and SULT1A1) metabolizing genes and the drug transporter OATP1B1. Therefore, the consumption of excessive amounts of pineapple juice poses a risk for drug interactions.

The main purpose of this work was to use pineapple crowns as substrate for optimizing laccase production by Trametes versicolor in lab-scale experiments. One-factor-at-the-time analysis and response surface methodology were used to optimize production. A single laccase with molecular weight of 45 kDa was the main protein produced. A maximal laccase activity of 60.73 ± 1.01 U/g was obtained in 7-day cultures, representing a 6.7-fold increase compared to non-optimized conditions. The optimized conditions were temperature: 28 °C; initial moisture: 90%; glucose: 8.38%; yeast extract: 2.86%. Combining activity and stability, the best conditions for using this laccase during the long periods required by large-scale processes are pH 4.0-5.0 and temperature of 40-50 °C. Under these conditions, the crude laccase was efficient in detoxifying the dye malachite green with a K M of 14.33 ± 1.94 µM and a V max of 0.482 ± 0.029 µM/min with 0.1 units/mL. It can be concluded that pineapple crown leaves can be effectively used as substrate by T. versicolor for producing laccase under solid-state culture conditions. Laccase is an industrially relevant enzyme and its production with concomitant valorization of pineapple crowns as substrate offers highly interesting perspectives.

There are about 4-6 slips on a fruit, and they are good materials for effective regeneration of Ananas comosus var. bracteatus . Adventitious root (AR) induction is essential for the propagation of Ananas comosus var. bracteatus slips. Growth regulator treatment, and culture medium are imperative factors that affect slip growth and rooting. In order to screen the optimal methods for slips rooting and reveal the anatomic procedure of slip rooting, this study induced slip rooting by different treatment of growth regulator, culture medium, observed the slip stem structure, AR origination and formation procedure through paraffin sections. The results showed that, slip cuttings treated with 100 mg/L of Aminobenzotriazole (ABT) for 6 hrs, cultured in river sand: coconut chaff: garden soil 2:2:1 medium is the optimal method for rooting. The proper supplementary of ABT can enhance the soluble sugar content, soluble protein content, polyphenol oxidase (PPO) activity and peroxidase (POD) enzyme activity, which resulted in the improvement of rooting. The slip stem structure is quite different from other monocots, which consists of epidermis, cortex, and stele with vascular tissues distributed in the cortex and stele. The AR primordia originates from the parenchyma cells located on the borderline between the cortex and stele. The vascular tissues in the AR develop and are connected with vascular tissue of the stem before the AR grew out the stem. The number of primary xylem poles in AR is about 30.

In the present study, protective effects of Ananas comosus i.e., pineapple pulp (PA) against cobalt chloride (CoCl 2 )‑induced hypoxia in Caco-2 cells were evaluated. PA reduces levels of lipid peroxidation, reactive oxygen and nitrogen species. It was proved to be cytoprotective and increased anti-oxidant activity against CoCl 2 -induced hypoxia. The inference drawn from this experiment was CoCl 2 -induced hypoxia that regulates hypoxia-inducible factor-1 (HIF-1), a transcription factor. It was also confirmed that PA pre-treatment inhibited the expression of HIF-1α, thereby downregulating the hypoxia-associated gene/protein expressions such as GLUT-1, VEGF, ANG and FGF. Finally, supplementation of PA could help in snow-balling the digestive hormones like leptin and CCK in hypoxic conditions. Therefore, this report provides substantial proof of reducing the hypoxia-induced loss of appetite at high-altitude environments. Graphical Abstract.

Toxic compounds in pineapple peel waste hydrolysate (PPWH), namely formic acid, 5-hydroxymethylfurfural (HMF), and furfural, are the major predicament in its utilization as a carbon source for bacterial cellulose (BC) fermentation. A rapid detoxification procedures using atmospheric cold plasma (ACP) technique were employed to reduce the toxic compounds. ACP treatment allows the breakdown of toxic compounds without causing excessive breakdown of sugars. Herein, the performance of two available laboratory ACP reactors for PPWH detoxification was being demonstrated. ACP-reactor-1 (R1) runs on plasma power of 80-200 W with argon (Ar) plasma source, while ACP-reactor-2 (R2) runs at 500-600 W with air plasma source. Treatment in R1, at 200 W for 15 min, results in 74.06%, 51.38%, and 21.81% reduction of furfural, HMF, and formic acid. Treatment in R2 at 600 W gives 45.05%, 32.59%, and 60.41% reductions of furfural, HMF, and formic acid. The BC yield from the fermentation of Komagateibacter xylinus in the R1-treated PPWH, R2-treated PPWH, and untreated-PPWH is 2.82, 3.82, and 2.97 g/L, respectively. The results show that ACP treatment provides a novel detoxified strategy in achieving agricultural waste hydrolysate reuse in fermentation. Furthermore, the results also imply that untreated PPWH can be an inexpensive and sustainable resource for fermentation media supplementation., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Unconventional parts of vegetables represent a rich source of health-promoting phytochemicals. The phenolic profile of cabbage-stalk flour (CSF), pineapple-crown flour (PCF), and their essential oils were characterized via UPLC-ESI-QTOF-MS E and GC-FID/MS. Antimicrobial activity was tested against five strains, and antioxidant activities were determined in free and bound extracts. Globally, 177 phenolics were tentatively identified in PCF (major p-coumaric acid, ferulic acid, and 4-hydroxybenzaldehyde) and 56 in CSF (major chlorogenicacid, quercetin 3-O-glucuronide, and p-coumaric acid). PCF exhibited a distinguished profile (lignans, stilbenes) and antioxidant capacity, especially in bound extracts (1.3 g GAE.100 g -1 ; 0.6 g catechin eq.100 g -1 ; DPPH: 244.7; ABTS: 467.8; FRAP: 762.6 µg TE.g -1 , ORAC: 40.9 mg TE.g -1 ). The main classes of volatile compounds were fatty acids, their esters, and terpenes in CSF (30) and PCF (41). A comprehensive metabolomic approach revealed CSF and PCF as a promising source of PC, showing great antioxidant and discrete antimicrobial activities., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Unlike the flower of the model monocot rice, which has diverged greatly from the ancestral monocot flower, the pineapple (Ananas comosus) flower is more typical of monocot flowers. Here, we identified 43 pineapple genes containing MADS-box domains, including 11 type I and 32 type II genes. RNA-seq expression data generated from five pineapple floral organs (sepals, petals, stamens, pistils, and ovules) and quantitative real-time PCR revealed tissue-specific expression patterns for some genes. We found that AcAGL6 and AcFUL1 were mainly expressed in sepals and petals, suggesting their involvement in the regulation of these floral organs. A pineapple 'ABCDE' model was proposed based on the phylogenetic analysis and expression patterns of MADS-box genes. Unlike rice and orchid with frequent species-specific gene duplication and subsequent expression divergence, the composition and expression of the ABCDE genes were conserved in pineapple. We also found that AcSEP1/3, AcAG, AcAGL11a/b/c, and AcFUL1 were highly expressed at different stages of fruit development and have similar expression profiles, implicating these genes' role in fruit development and ripening processes. We propose that the pineapple flower can be used as a model for studying the ancestral form of monocot flowers to investigate their development and evolutionary history.

<b>Background and Objective:</b> Obesity exerts negative influences on male reproductive capacity via changing the molecular and physical structure of male germ cells. This study was conducted to evaluate the mitigating effects of raw juice of pineapple on obesity-associated testicular impairment in male Wistar rats. <b>Materials and Methods:</b> Rats included the control group (G<sub>I</sub>, n = 6) who received a Normal Diet (ND) and the obese group (G<sub>II</sub>, n = 18) who received a High-Fat Diet (HFD). Obese rats (G<sub>II</sub>) were subdivided into 3 groups (6 rats each): G<sub>II</sub> represents the untreated obesity group that continued to receive HFD with plain Drinking Water (DW), G<sub>III</sub> received ND along with raw juice (15% v/v) in DW and G<sub>IV</sub> continued to receive HFD with raw juice (15% v/v) in DW. Rats were sacrificed at the end of the trial and testis was processed for histopathology and immunohistochemistry. <b>Results:</b> Testis from obese rats revealed a significant increment in spermatogenic cell degeneration, pro-inflammatory Nuclear factor of kappa B (NF-κB) and pro-apoptotic Caspase-3 immunoreactivities. Yet, Proliferating Cell Nuclear Antigen (PCNA) displayed poor immunoreactivity in obese rats' testis relative to controls. Administration of raw juice of pineapple to obese rats significantly reduced degeneration of spermatogenic cells, NF-κB and Caspase-3 immunoreactivities. Additionally, treatment with the juice significantly increased immunoreactivity to PCNA in obese rats. These ameliorating effects were more obvious in rats who received juice along with ND (G<sub>III</sub>) than in those who received it along with HFD (G<sub>IV</sub>). <b>Conclusion:</b> Treatment of obese rats with pineapple juice restored testicular homeostasis, indicating its potential validity to overcome obesity-induced male fertility disorders.

<b>Background and Objective:</b> Pineapple (<i>Ananas comosus</i> L.) has antioxidant and other pharmacological properties. This study examined how pineapple modified mitochondrial permeability transition and expression of drug-metabolizing enzymes, i.e., CYP1A2, CYP2C9, CYP3A4, UGT1A6, NAT2 and the drug transporter OATP1B1 in human colorectal adenocarcinoma (Caco-2) cells. <b>Materials and Methods:</b> Caco-2 cells (2.5×10<sup>5</sup> cells well<sup>1</sup> in 24-well plates) were incubated with pineapple (125 to 1,000 μg mL<sup>1</sup>) for 48 hrs in a phenol red-free medium. Mitochondrial permeability transition, resazurin cell viability and AST and ALT levels were investigated. The mRNA expression of target genes was determined by RT/qPCR. <b>Results:</b> Pineapple significantly reduced depolarized mitochondria, slightly decreased cell viability and did not change AST and ALT levels. Pineapple did not modify the mRNA expressions of CYP1A2, CYP2C9 and CYP3A4 but markedly induced UGT1A6 expression. The highest tested concentration of pineapple (1,000 μg mL<sup>1</sup>) significantly suppressed NAT2 and OATP1B1 expression. <b>Conclusion:</b> Although pineapple slightly decreased cell viability to ~80% of control, the morphology and functions of the cells were unaffected. Pineapple showed a beneficial effect to reduce depolarized mitochondria, which consequently decreased reactive oxygen species production. Pineapple did not modify the expression of CYPs, whilst it altered the expression of phase 2 metabolizing genes UGT1A6 and NAT2 and the transporter OATP1B1. Therefore, the consumption of large amounts of pineapple is of concern for the risk of drug interaction via alteration of UGT1A6, NAT2 and OATP1B1 expression.

Drought and salt stress are the main environmental cues affecting the survival, development, distribution, and yield of crops worldwide. MYB transcription factors play a crucial role in plants' biological processes, but the function of pineapple MYB genes is still obscure. In this study, one of the pineapple MYB transcription factors, AcoMYB4 , was isolated and characterized. The results showed that AcoMYB4 is localized in the cell nucleus, and its expression is induced by low temperature, drought, salt stress, and hormonal stimulation, especially by abscisic acid (ABA). Overexpression of AcoMYB4 in rice and Arabidopsis enhanced plant sensitivity to osmotic stress; it led to an increase in the number stomata on leaf surfaces and lower germination rate under salt and drought stress. Furthermore, in AcoMYB4 OE lines, the membrane oxidation index, free proline, and soluble sugar contents were decreased. In contrast, electrolyte leakage and malondialdehyde (MDA) content increased significantly due to membrane injury, indicating higher sensitivity to drought and salinity stresses. Besides the above, both the expression level and activities of several antioxidant enzymes were decreased, indicating lower antioxidant activity in AcoMYB4 transgenic plants. Moreover, under osmotic stress, overexpression of AcoMYB4 inhibited ABA biosynthesis through a decrease in the transcription of genes responsible for ABA synthesis ( ABA1 and ABA2 ) and ABA signal transduction factor ABI5 . These results suggest that AcoMYB4 negatively regulates osmotic stress by attenuating cellular ABA biosynthesis and signal transduction pathways.

Background: Lysine succinylation, an important protein posttranslational modification (PTM), is widespread and conservative. The regulatory functions of succinylation in leaf color has been reported. The chimeric leaves of Ananas comosus var. bracteatus are composed of normal green parts and albino white parts. However, the extent and function of lysine succinylation in chimeric leaves of Ananas comosus var. bracteatus has yet to be investigated., Results: Compared to the green (Gr) parts, the global succinylation level was increased in the white (Wh) parts of chimeric leaves according to the Western blot and immunohistochemistry analysis. Furthermore, we quantitated the change in the succinylation profiles between the Wh and Gr parts of chimeric leaves using label-free LFQ intensity. In total, 855 succinylated sites in 335 proteins were identified, and 593 succinylated sites in 237 proteins were quantified. Compared to the Gr parts, 232 (61.1%) sites in 128 proteins were quantified as upregulated targets, and 148 (38.9%) sites in 70 proteins were quantified as downregulated targets in the Wh parts of chimeric leaves using a 1.5-fold threshold (P < 0.05). These proteins with altered succinylation level were mainly involved in crassulacean acid metabolism (CAM) photosynthesis, photorespiration, glycolysis, the citric acid cycle (CAC) and pyruvate metabolism., Conclusions: Our results suggested that the changed succinylation level in proteins might function in the main energy metabolism pathways-photosynthesis and respiration. Succinylation might provide a significant effect in the growth of chimeric leaves and the relationship between the Wh and Gr parts of chimeric leaves. This study not only provided a basis for further characterization on the function of succinylated proteins in chimeric leaves of Ananas comosus var. bracteatus but also provided a new insight into molecular breeding for leaf color chimera.

Somatic embryogenesis (SE) is a more rapid and controllable method for plant propagation than traditional breeding methods. However, it often suffers from limited efficiency. SERK1 promotes SE in several plants, including pineapple ( Ananas comosus L.). We investigate the embryonic cell-specific transcriptional regulation of AcSERK1 by methylation analysis of CpG islands in AcSERK1 regulatory sequences. This revealed differences in the methylation status of CpG islands between embryonic callus and non-embryonic callus; the methylation inhibitor 5-azaC increased AcSERK1 expression and also accelerated SE. These findings indicate that the expression of AcSERK1 is regulated epigenetically. This study lays the foundation for further analysis of epigenetic regulatory mechanisms that may enhance the efficiency of SE in pineapple and other plants.

The plant-specific transcription factor gene family, YABBY, belongs to the subfamily of zinc finger protein superfamily and plays an essential regulatory role in lateral organ development. In this study, nine YABBY genes were identified in the pineapple genome. Seven of them were located on seven different chromosomes and the remaining two were located on scaffold 1235. Through protein structure prediction and protein multiple sequence alignment, we found that AcYABBY3 , AcYABBY5 and AcYABBY7 lack a C2 structure in their N-terminal C2C2 zinc finger protein structure. Analysis of the cis -acting element indicated that all the seven pineapple YABBY genes contain multiple MYB and MYC elements. Further, the expression patterns analysis using the RNA-seq data of different pineapple tissues indicated that different AcYABBYs are preferentially expressed in various tissues. RT-qPCR showed that the expression of AcYABBY2 , AcYABBY3 , AcYABBY6 and AcYABBY7 were highly sensitive to abiotic stresses. Subcellular localization in pineapple protoplasts, tobacco leaves and Arabidopsis roots showed that all the seven pineapple YABBY proteins were nucleus localized. Overexpression of AcYABBY4 in Arabidopsis resulted in short root under NaCl treatment, indicating a negative regulatory role of AcYABBY4 in plant resistance to salt stress. This study provides valuable information for the classification of pineapple AcYABBY genes and established a basis for further research on the functions of AcYABBY proteins in plant development and environmental stress response.

Quite a few studies have been conducted to improve the Agrobacterium -mediated transformation of pineapple, which is the second most important commercial tropical fruit crop worldwide. However, pineapple transformation remains challenging, due to technical difficulties, the lengthy regeneration process, and a high labor requirement. There have not been any studies specifically addressing the introduction of GFP-tagged genes into pineapples through Agrobacterium -mediated transformation, which would enable easy, non-destructive expression detection. It would also allow expression localization at the organelle level, which is not possible with GUS a reporter gene that encodes β-glucuronidase or a herbicide resistance reporter gene. Here, we report a method for the introduction of GFP-tagged genes into pineapples through Agrobacterium -mediated transformation. We used embryonic calli for transformation, and plants were regenerated through somatic embryogenesis. In this study, we optimized the incubation time for Agrobacterium infection, the co-cultivation time, the hygromycin concentration for selection, and the callus growth conditions after selection. Our strategy reduced the time required to obtain transgenic plants from 7.6 months to 6.1 months. The expression of GFP-tagged AcWRKY28 was observed in the nuclei of transgenic pineapple root cells. This method allows easy, non-destructive expression detection of transgenic constructs at the organelle level. These findings on pineapple transformation will help accelerate pineapple molecular breeding efforts to introduce new desirable traits.

Chromatin remodeling complex orchestrates numerous aspects of growth and development in eukaryotes. SWI2/SNF2-Related 1 chromatin remodeling complex (SWR1-C) is a member of the SWI/SNF ATPase-containing chromatin remodeling complex responsible for the exchange of H2A for H2A.Z. In plants, SWR1-C plays a crucial role by transcriptionally regulating numerous biological and developmental processes. However, SWR1-C activity remains obscure in pineapple. Here, we aim to identify the SWR1-C subunits in pineapple. By genome-wide identification, we found a total of 11 SWR1-C subunits in the pineapple. The identified SWR1-C subunits were named and classified based on the sequence similarity and phylogenetic analysis. RNA-Seq analysis showed that pineapple SWR1-C subunits are expressed differentially in different organs and at different stages. Additionally, the qRT-PCR of pineapple SWR1-C subunits during abiotic stress exposure showed significant changes in their expression. We further investigated the functions of pineapple SWR1 COMPLEX 6 (AcSWC6) by ectopically expressing it in Arabidopsis. Interestingly, transgenic plants ectopically expressing AcSWC6 showed susceptibility to fungal infection and enhanced resistance to salt and osmotic stress, revealing its involvement in biotic and abiotic stress. Moreover, the complementation of mutant Arabidopsis swc6 by pineapple SWC6 suggested the conserved function of SWC6 in plants., Competing Interests: 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.

Ca 2+ serves as a ubiquitous second messenger regulating several aspects of plant growth and development. A group of unique calcium sensor proteins, calcineurin B-like (CBL), interact with CBL-interacting protein kinases (CIPKs) to decode the Ca 2+ signature inside the cell. Although CBL-CIPK signaling toolkit has been shown to play significant roles in the responses to numerous stresses in different plants, the information about pineapple CBL-CIPK remains obscure. In the present study, a total of eight Ac CBL and 21 Ac CIPK genes were identified genome-wide in pineapple. The identified genes were renamed on the basis of gene ID in ascending order and phylogenetic analysis divided into five groups. Transcriptomic data analysis showed that Ac CBL and Ac CIPK genes were expressed differentially in different tissues. Further, the expression analysis of Ac CBL1 in different tissues showed significant changes under various abiotic stimuli. Additionally, the ectopic expression of Ac CBL1 in Arabidopsis resulted in enhanced tolerance to salinity, osmotic, and fungal stress. The present study revealed the crucial contribution of the CBL-CIPK gene in various biological and physiological processes in pineapple., Competing Interests: The authors declare that they have no competing interests.

Long noncoding RNAs (lncRNAs) have been identified in many mammals and plants and are known to play crucial roles in multiple biological processes. Pineapple is an important tropical fruit and a good model for studying the plant evolutionary adaptation to the dry environment and the crassulacean acid metabolism (CAM) photosynthesis strategy; however, the lncRNAs involved in CAM pathway remain poorly characterized. Here, we analyzed the available RNA-seq data sets derived from 26 pineapple leaf samples at 13 time points and identified 2,888 leaf lncRNAs, including 2,046 long intergenic noncoding RNAs (lincRNAs) and 842 long noncoding natural antisense transcripts (lncNATs). Pineapple leaf lncRNAs are expressed in a highly tissue-specific manner. Co-expression analysis of leaf lncRNA and mRNA revealed that leaf lncRNAs are preferentially associated with photosynthesis genes. We further identified leaf lncRNAs that potentially function as competing endogenous RNAs (ceRNAs) of two CAM photosynthesis pathway genes, PPCK and PEPC, and revealed their diurnal expression pattern in leaves. Moreover, we found that 48% of lncRNAs exhibit diurnal expression patterns in leaves, suggesting their important roles in CAM. This study conducted a comprehensive genome-wide analysis of leaf lncRNAs and identified their role in gene expression regulation of the CAM photosynthesis pathway in pineapple.

Antibacterial activity of cell-free supernatant from Escherichia coli E against selected pathogenic bacteria in food and aquaculture was the highest against Edwardsiella tarda 3, a significant aquaculture pathogen. Biochemical properties of the bacteriocins were studied and bacteriocin was found to be sensitive to proteinase K, demonstrating its proteinaceous nature. In addition, pH and temperature affected bacteriocin activity and stability. The bacteriocins were partially purified by ammonium sulfate precipitation. The antibacterial activity was only detected in 20% ammonium sulfate fraction and direct detection of its activity was performed by overlaying on the indicator strains. The inhibition zone associated with the antibacterial activity was detected in the sample overlaid by E. tarda 3 and Staphylococcus aureus DMST8840 with the relative molecular mass of about 27 kDa and 10 kDa, respectively. Bacteriocin showed no cytotoxic effect on NIH-3T3 cell line; however, two virulence genes, aer and sfa, were detected in the genome of E. coli E by PCR. The characteristics of bacteriocins produced by E. coli E exhibited the antibacterial activity against both Gram-positive and Gram-negative pathogenic bacteria and the safe use determined by cytotoxicity test which may have interesting biotechnological applications.

Pineapple (Ananas comosus (L.) Merr.) is a non-climacteric tropical fruit whose ripening could be accompanied by oxidative processes and the concurrent activation of enzymatic and non-enzymatic reactive oxygen species (ROS) scavenging systems. To better understand the variability of these processes among climatic environments or genotypes in pineapple, the temporal expression dynamics for genes encoding oxidative and antioxidative stress enzymes were analyzed by real-time RT-PCR during fruit development and ripening, among three cultivars: Queen Victoria, Flhoran 41 and MD-2 hybrid, and in two climatic areas. Pineapple development and ripening involved changes in the levels of transcripts encoding for polyphenol oxidase and transcripts involved in the first steps of the phenylpropanoid pathway and in the balance of ROS, especially those encoding for ascorbate peroxydase and metallothioneins, regardless of the cultivar. Our results confirm the same dynamic in gene expression from the two environmental crop areas, however climatic conditions influenced the level of the expression of the major transcripts studied that were linked to these oxidative and antioxidant metabolisms. MT3a and MT3b transcripts were not influenced by genetic factor. The genetic effect was not significant on the various transcripts linked to the first steps of the phenylpropanoid pathway and to phenol oxidation, except 4CL ones. In ripe pineapple, highly significant relationships were found between the contents in antioxidant metabolites, i.e., ascorbic acid and total phenolic compounds, and the transcript levels of genes involved in the enzymatic ROS-scavenging system and in the biosynthesis or regeneration of ROS-scavenging compounds, like phenylpropanoids, ascorbic acid, metallothioneins., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

A mannose binding jacalin-related lectin from Ananas comosus stem (AcmJRL) was purified and biochemically characterized. This lectin is homogeneous according to native, SDS-PAGE and N-terminal sequencing and the theoretical molecular mass was confirmed by ESI-Q-TOF-MS. AcmJRL was found homodimeric in solution by size-exclusion chromatography. Rat erythrocytes are agglutinated by AcmJRL while no agglutination activity is detected against rabbit and sheep erythrocytes. Hemagglutination activity was found more strongly inhibited by mannooligomannosides than by D-mannose. The carbohydrate-binding specificity of AcmJRL was determined in some detail by isothermal titration calorimetry. All sugars tested were found to bind with low affinity to AcmJRL, with K a values in the mM range. In agreement with hemagglutination assays, the affinity increased from D-mannose to di-, tri- and penta-mannooligosaccharides. Moreover, the X-ray crystal structure of AcmJRL was obtained in an apo form as well as in complex with D-mannose and methyl-α-D-mannopyranoside, revealing two carbohydrate-binding sites per monomer similar to the banana lectin BanLec. The absence of a wall separating the two binding sites, the conformation of β7β8 loop and the hemagglutinating activity are reminiscent of the BanLec His84Thr mutant, which presents a strong anti-HIV activity in absence of mitogenic activity.

A strain Leuconostoc citreum B-2 was isolated from homemade fermentation product of pineapple and its polysaccharide yield was 28.3g/L after cultivating the strain in Man-Rogosa-Sharpe (MRS) medium with 75g/L sucrose. The exopolysaccharide (EPS) was characterized by gas chromatography (GC), Fourier-transform infrared (FT-IR) spectra, high-performance size-exclusion chromatography (HPSEC), nuclear magnetic resonance (NMR) spectroscopy and scanning electron microscope (SEM) analysis in present study. The monosaccharide composition of EPS was glucose and molecular weight was 3.77×10 6 Da. FT-IR and NMR spectra revealed that the B-2 EPS was composed of 75% α-(1→6) linked d-glucopyranose units existing in the main chain with 19% α-(1→3) branching and only a few α-(1→2) branching. The SEM of the dried EPS appeared irregular sheets with glittering surface and compact structure. Water solubility index and water holding capacity of B-2 EPS were 80% and 450%, respectively. All the mentioned characteristics suggested that the EPS has a potential application in the food, cosmetic and pharmaceuticals industry., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Sugars will eventually be exported transporters (SWEETs) are a group of recently identified sugar transporters in plants that play important roles in diverse physiological processes. However, currently, limited information about this gene family is available in pineapple (Ananas comosus). The availability of the recently released pineapple genome sequence provides the opportunity to identify SWEET genes in a Bromeliaceae family member at the genome level. In this study, 39 pineapple SWEET genes were identified in two pineapple cultivars (18 AnfSWEET and 21 AnmSWEET) and further phylogenetically classified into five clades. A phylogenetic analysis revealed distinct evolutionary paths for the SWEET genes of the two pineapple cultivars. The MD2 cultivar might have experienced a different expansion than the F153 cultivar because two additional duplications exist, which separately gave rise to clades III and IV. A gene exon/intron structure analysis showed that the pineapple SWEET genes contained highly conserved exon/intron numbers. An analysis of public RNA-seq data and expression profiling showed that SWEET genes may be involved in fruit development and ripening processes. AnmSWEET5 and AnmSWEET11 were highly expressed in the early stages of pineapple fruit development and then decreased. The study increases the understanding of the roles of SWEET genes in pineapple., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Vinegar is widely used as a food additive, in food preparation and as a food supplement. This study compared the phenolic acid profiles and in vivo toxicities, and antioxidant and immunomodulatory effects of coconut, nipah and pineapple juice vinegars, which were respectively prepared via a two-step fermentation using Saccharomyces cerevisiae 7013 INRA and Acetobacter aceti vat Europeans., Results: Pineapple juice vinegar, which had the highest total phenolic acid content, also exhibited the greatest in vitro antioxidant capacity compared to coconut juice and nipah juice vinegars. Following acute and sub-chronic in vivo toxicity evaluation, no toxicity and mortality were evident and there were no significant differences in the serum biochemical profiles between mice administered the vinegars versus the control group. In the sub-chronic toxicity evaluation, the highest liver antioxidant levels were found in mice fed with pineapple juice vinegar, followed by coconut juice and nipah juice vinegars. However, compared to the pineapple juice and nipah juice vinegars, the mice fed with coconut juice vinegar, exhibited a higher population of CD4 + and CD8 + T-lymphocytes in the spleen, which was associated with greater levels of serum interleukin-2 and interferon-γ cytokines., Conclusions: Overall, the data suggested that not all vinegar samples cause acute and sub-chronic toxicity in vivo. Moreover, the in vivo immunity and organ antioxidant levels were enhanced, to varying extents, by the phenolic acids present in the vinegars. The results obtained in this study provide appropriate guidelines for further in vivo bioactivity studies and pre-clinical assessments of vinegar consumption. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

Background/aims: Ethylene is usually used to induce floral transition in pineapple. However, its successful induction in plants categorized as Cayenne is difficult or completely ineffective, and information concerned is limited. The present study was undertaken to investigate the molecular mechanisms underlying this obstacle., Methods: Transcriptome and proteome comparative analyses were performed to explore the important regulation and pathway variations after ethephon induction in the induction-easy 'Comte de Paris' (CP) and induction-hard 'Smooth Cayenne' (SC) cultivars via RNA-seq (RNA-sequencing) and iTRAQ (isobaric tags for relative and absolute quantification)., Results: CP and SC exhibited basic differences at the transcriptomic and proteomic levels before ethephon treatment, including the expression of genes and proteins related to ethylene signal transduction. After ethephon induction, the expression of genes and proteins involved in plant ethylene signal transduction and carbohydrate metabolism responded more strongly in CP than in SC. The expression of the floral meristem identity (FMI) genes AG, TFL and FT exhibited greater changes in CP, and more transcription factors responded in SC. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that many differentially expressed genes (DEGs) in CP were annotated to terms and pathways involved in photoperiodism and shared components involved in carbohydrate metabolism and plant hormone signal transduction., Conclusion: These findings contribute to the understanding of the molecular mechanism underlying the variation between CP and SC in response to ethephon-mediated floral induction., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Bromelain inhibitor, "bromein", is a proteinase-inhibitor specific to the cysteine proteinase bromelain from pineapple stem. In the stem, eight bromein isoforms are known to exist, and each isoform has a short peptide (light chain) and a long one (heavy chain) with five disulfide bonds. The three-dimensional structure of the sixth isoform (bromein-6) is composed of inhibitory and stabilizing domains, and each domain contains a three-stranded antiparallel β-sheet. The genomic sequence of a bromein precursor encodes three homologous bromein isoform domains, and each isoform domain has a signal peptide, three interchain peptides between the light chain and heavy chain, two interdomain peptides and a propeptide. Interestingly, at the protein level, bromein- 6 appears to share a similar folding and disulfide-bonding connectivity with Bowman-Birk serine proteinase inhibitors and shows weak inhibition toward chymotrypsin and trypsin. However, no significant similarity was found between them at the genomic level. This indicates that they have evolved convergently to possess such a structural similarity. To identify the essential reactive site(s) with bromelain, we investigated the inhibitory activity of 44 kinds of the single/double and insertion/ deletion mutants of bromein-6 towards stem bromelain. As a result, it was shown that both the appropriate positioning and the complete side-chain structure of Leu10 in the light chain are absolutely crucial for the inhibition, with an additional measure of importance for the preceding Pro9. Bromein and stem bromelain coexist in the acidic vacuoles of the stem tissue, and one of the key role of bromein appears to be the regulation of the bromelain activity., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Fusarium ananatum causes fruitlet core rot (FCR) in pineapple (Ananas comosus var. comosus) when the fruit reaches maturity. Hidden symptoms make it difficult to assess the disease, regardless of its stage, and basic questions concerning the involvement of the phenolic compounds in response to infection remain unknown. A direct inoculation method of F. ananatum in pineapple fruitlets was developed to monitor the growth of black spots and the changes in phenolic acids and ascorbic acid concentration under controlled conditions. After inoculation, infection began with a flesh discolouration at the inoculation point and then spread in a darker shade to form a black spot. Coumaroyl-isocitric and caffeoyl-isocitric acids levels respectively showed a 150- and 200-fold increase in infected fruitlet when compared to healthy fruitlet. These hydroxycinnamic acids increased minimally in the adjacent fruitlet and remained stable in the other parts of the fruit. By contrast, sinapic acid and hydroxybenzoic acid isomers (HBA) decreased after F. ananatum inoculation in the infected fruitlet, whereas they remained stable in the adjacent and healthy fruitlets. Ascorbic acid decreased to zero in the infected fruitlet. The antifungal activity of phenolic compounds and ascorbic acid was evaluated against the mycelial growth of F. ananatum. p-Coumaric acid exhibited a total inhibition of the mycelial growth at 1000 μg g -1 . Ferulic acid inhibited 64 % of mycelial growth at a concentration of 1000 μg g -1 . Caffeoylquinic acid, sinapic acid, and ascorbic acid also showed significant antifungal activity, but to a lesser extent. Finally, coinoculation of the hydroxycinnamic acids with the pathogen restrains its development in the fruit. This is the first study to highlight the involvement of phenolic compounds in the pineapple FCR disease., (Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Alterations of volatiles during accelerated light-induced ageing of pineapple juice were assessed by HS-SPME-GC-MS in a non-targeted profiling analysis over a 16-week period. Multivariate statistics permitted to reveal substantial chemical markers generally describing the effect of light storage. Volatiles generated comprised phenylpropenes, carbonyls, 2-methylthiophene, toluene, and furfural, while concentrations of methyl and ethyl esters, terpenes, and furanones decreased. In addition, the qualitative composition of phenolic compounds and glycoside-bound volatiles in selected samples was characterized by HPLC-DAD-ESI-MS n as well as HR-ESI-MS. The fresh juice contained unique pineapple metabolites such as S-p-coumaryl, S-coniferyl, S-sinapylglutathione, and structurally related derivatives. Among others, the presence of p-coumaroyl, feruloyl, and caffeoylisocitrate as well as three 4-hydroxy-2,5-dimethyl-3(2H)-furanone glycosides in pineapples could be substantiated by the HR-ESI-MS experiment. Mass spectrometric assignments of selected metabolites are presented, and putative linkages between volatiles and their precursors are established., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Vinegar is an inexpensive commodity, and economic considerations require that a relatively low-cost raw material be used for its production. An investigation into the use of a new, alternative substrate - pineapple waste - is described. This approach enables the utilization of the pineapple's (Ananas comosus) peels and core, which are usually discarded during the processing or consumption of the fruit. Using physical and enzymatic treatments, the waste was saccharified, and the resulting substrate was fermented with Saccharomyces cerevisiae for 7-10days under aerobic conditions at 25°C. This resulted in an alcohol yield of approximately 7%. The alcoholic medium was then used as a seed broth for acetic fermentation using Acetobacter aceti as the inoculum for approximately 30days at 32°C to obtain 5% acetic acid. Samples were analyzed at the beginning and end of the acetification cycle to assess the volatile and fixed compounds by GC-MS and UHPLC-QTOF-MS. The metabolomic analysis indicated that l-lysine, mellein, and gallic acid were significantly more concentrated in the pineapple vinegar than in the original wine. Higher alcohols, aldehydes, and ketones characterized the aroma of the final pineapple vinegar, whilst off-flavors were significantly reduced relative to the initial wine. This study is the first to highlight the metabolite profile of fruit vinegar with a slight floral aroma profile derived from pineapple waste. The potential to efficiently reduce the post-harvest losses of pineapple fruits by re-using them for products with added food values is also demonstrated., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Ultrasound treatment at different power output (0, 25 and 29W) and exposure time (10 and 15min) was used to investigate its effect on the phenolic metabolism enzymes, total phenolic content and antioxidant capacity of fresh-cut pineapple. Following ultrasound treatment at 25 and 29W, the activity of phenylalanine ammonia lyase (PAL) was increased significantly (P<0.05) by 2.0 and 1.9-fold, when compared to control. Meanwhile, both the activity of polyphenol oxidase (PPO) and polyphenol peroxidase (POD) in fresh-cut pineapple was significantly (P<0.05) lower than control upon subjected to ultrasound treatment. In the present study, induction of PAL was found to significantly (P<0.001) correlate with higher total phenolic content and thus higher antioxidant capacity in fresh-cut pineapple. Results suggest that hormetic dosage of ultrasound treatment can enhance the activity of PAL and total phenolic content and hence the total antioxidant capacity to encounter with oxidative stress., (Copyright © 2016. Published by Elsevier Ltd.)