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Pineapple is a globally significant tropical fruit, but its cultivation faces numerous challenges due to abiotic and biotic stresses, affecting its quality and quantity. WRKY transcription factors are known regulators of stress responses, however, their specific functions in pineapple are not fully understood. This study investigates the role of AcWRKY31 by overexpressing it in pineapple and Arabidopsis. Transgenic pineapple lines were obtained using Agrobacterium-mediated transformation methods and abiotic and biotic stress treatments. Transgenic AcWRKY31-OE pineapple plants showed an increased sensitivity to salt and drought stress and an increased resistance to biotic stress from pineapple mealybugs compared to that of WT plants. Similar experiments in AcWRKY31-OE, AtWRKY53-OE, and the Arabidopsis Atwrky53 mutant were performed and consistently confirmed these findings. A comparative transcriptomic analysis revealed 5357 upregulated genes in AcWRKY31-OE pineapple, with 30 genes related to disease and pathogen response. Notably, 18 of these genes contained a W-box sequence in their promoter region. A KEGG analysis of RNA-Seq data showed that upregulated DEG genes are mostly involved in translation, protein kinases, peptidases and inhibitors, membrane trafficking, folding, sorting, and degradation, while the downregulated genes are involved in metabolism, protein families, signaling, and cellular processes. RT-qPCR assays of selected genes confirmed the transcriptomic results. In summary, the AcWRKY31 gene is promising for the improvement of stress responses in pineapple, and it could be a valuable tool for plant breeders to develop stress-tolerant crops in the future. [ABSTRACT FROM AUTHOR]

SWR1 chromatin remodeling complex (SWR1-C) plays a crucial role in gene regulation during growth and development of eukaryotes. ACTIN-RELATED PROTEIN 6 (ARP6), an essential subunit of SWR1-C, is implicated in flowering and stress response. However, the functions of pineapple ARP6 have not been investigated, which limits its potential use in molecular breeding. Here, pineapple ARP6 is functionally characterized and its role in flowering, salt stress, drought, nutrient deficiency and immunity is verified. Phylogenetic analysis showed that AcARP6 is homologous to ARP6 of rice and Arabidopsis. Expression analysis revealed that AcARP6 gets induced by salt, osmotic and nutrient deficiency. Additionally, ectopic expression of AcARP6 in Arabidopsis delayed flowering and increased tolerance to salinity, drought and nutrient deficiency, but displayed sensitivity against Sclerotinia sclerotiorum infection. Furthermore, AcARP6 complemented the phenotypic defects of the arp6 mutation in Arabidopsis. These results provide evidence for the involvement of AcARP6 in multiple stress responses and advocate that AcARP6 could be used as a potential candidate gene for improving tolerance against abiotic stresses. [ABSTRACT FROM AUTHOR]

In plants, sugars are required for several essential functions, including growth, storage, signaling, defense and reproduction. Sugar transporters carry out the controlled movement of sugars from source (leaves) to sink (fruits and roots) tissues and determine the overall development of the plant. Various types of sugar transporter families have been described in plants, including sucrose transporters (SUC/SUT), monosaccharide transporter (MST) and SWEET (from "Sugar Will Eventually be Exported Transporters"). However, the information about pineapple sugar transporters is minimal. This study systematically identified and classified 45 MST and 4 SUC/SUT genes in the pineapple genome. We found that the expression patterns of sugar transporter genes have a spatiotemporal expression in reproductive and vegetative tissues indicating their pivotal role in reproductive growth and development. Besides, different families of sugar transporters have a diel expression pattern in photosynthetic and non-photosynthetic tissues displaying circadian rhythm associated participation of sugar transporters in the CAM pathway. Moreover, regulation of the stress-related sugar transporters during cold stress indicates their contribution to cold tolerance in pineapple. Heterologous expression (yeast complementation assays) of sugar transporters in a mutant yeast strain suggested that SUT1/2 have the ability to transport sucrose, and STP13, STP26, pGlcT-L2 and TMT4 are able to transport glucose, whereas SWEET11/13 transport both sucrose and fructose. The information provided here would help researchers further explore the underlying molecular mechanism involved in the sugar metabolism of pineapple. [ABSTRACT FROM AUTHOR]

Simple Summary: JASMONATE ZIM-DOMAIN (JAZ) proteins are key components of the jasmonate (JA) signaling pathway in response to biotic and abiotic stresses in plants. Information about the JAZ gene family in pineapple (AcJAZ) is limited. In this study, 14 AcJAZ genes were identified in the pineapple genome. A complete overview of AcJAZ genes is presented, including the chromosome locations, phylogenetic relationships, gene structures, conserved motifs and cis-regulatory elements, and expression patterns at different developmental stages and under various stress conditions, and their possible involvement in diverse functions is suggested. Furthermore, the BiFC analysis revealed direct binary interactions between AcJAZs and crucial JA-signaling regulators in vivo. These results suggest that AcJAZs and other vital players function in the JA-signaling pathway in response to abiotic stresses in pineapple. JASMONATE ZIM-DOMAIN (JAZ) proteins are negative regulators of the jasmonate (JA)-signaling pathway and play pivotal roles in plant resistance to biotic and abiotic stresses. Genome-wide identification of JAZ genes has been performed in many plant species. However, systematic information about pineapple (Ananas comosus L. Merr.) JAZ genes (AcJAZs) is still not available. In this study, we identified 14 AcJAZ genes and classified them into five groups along with the Arabidopsis and rice orthologs. The AcJAZ genes have 3–10 exons, and the putative AcJAZ proteins have between two and eight conserved regions, including the TIFY motif and Jas domain. The cis-acting element analysis revealed that the putative promoter regions of AcJAZs contain between three and eight abiotic stress-responsive cis-acting elements. The gene-expression analysis suggested that AcJAZs were expressed differentially during plant development and subjected to regulation by the cold, heat, salt, and osmotic stresses as well as by phytohormones. Moreover, the BiFC analysis of protein interactions among the central JA-signaling regulators showed that AcJAZ4, AcMYC2, AcNINJA, and AcJAM1 could interact with AcJAZ5 and AcJAZ13 in vivo, indicating a canonical JA-signaling pathway in pineapple. These results increase our understanding of the functions of AcJAZs and the responses of the core players in the JA-signaling pathway to abiotic stresses. [ABSTRACT FROM AUTHOR]

Understanding germline specification in plants could be advantageous for agricultural applications. In recent decades, substantial efforts have been made to understand germline specification in several plant species, including Arabidopsis, rice, and maize. However, our knowledge of germline specification in many agronomically important plant species remains obscure. Here, we characterized the female germline specification and subsequent female gametophyte development in pineapple using callose staining, cytological, and whole-mount immunolocalization analyses. We also determined the male germline specification and gametophyte developmental timeline and observed male meiotic behavior using chromosome spreading assays. Furthermore, we identified 229 genes that are preferentially expressed at the megaspore mother cell (MMC) stage during ovule development and 478 genes that are preferentially expressed at the pollen mother cell (PMC) stage of anther development using comparative transcriptomic analysis. The biological functions, associated regulatory pathways and expression patterns of these genes were also analyzed. Our study provides a convenient cytological reference for exploring pineapple germline development and a molecular basis for the future functional analysis of germline specification in related plant species. [ABSTRACT FROM AUTHOR]

Transcription factors (TFs), such as heat shock transcription factors (HSFs), usually play critical regulatory functions in plant development, growth, and response to environmental cues. However, no HSFs have been characterized in pineapple thus far. Here, we identified 22 AcHSF genes from the pineapple genome. Gene structure, motifs, and phylogenetic analysis showed that AcHSF families were distinctly grouped into three subfamilies (12 in Group A, seven in Group B, and four in Group C). The AcHSF promoters contained various cis-elements associated with stress, hormones, and plant development processes, for instance, STRE, WRKY, and ABRE binding sites. The majority of HSFs were expressed in diverse pineapple tissues and developmental stages. The expression of AcHSF-B4b/AcHSF-B4c and AcHSF-A7b/AcHSF-A1c were enriched in the ovules and fruits, respectively. Six genes (AcHSF-A1a, AcHSF-A2, AcHSF-A9a, AcHSF-B1a, AcHSF-B2a, and AcHSF-C1a) were transcriptionally modified by cold, heat, and ABA. Our results provide an overview and lay the foundation for future functional characterization of the pineapple HSF gene family. [ABSTRACT FROM AUTHOR]

Investigation of the JASMONATE ZIM-DOMAIN Gene Family Reveals the Canonical JA-Signaling Pathway in Pineapple. Text Correction In the original publication, there was a mistake in the subtitle of 2.8. The scale on the left is for chromosomes in megabases (B) Phylogenetic relationship of JAZ proteins from Arabidopsis thaliana, Oryza sativa, and pineapple. [Extracted from the article]

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. • AcCIPK5 expression is highly regulated against various environmental conditions. • In AcCIPK5 expressing lines salt, drought and cold stress are positively regulated. • Heat and S. sclerotiorum infection is negatively regulated in AcCIPK5 lines. • AcCIPK5 regulates ABA signaling and ROS homeostasis to mediate stress response. [ABSTRACT FROM AUTHOR]

In plants, calcineurin B-like proteins (CBLs) and their interacting kinase, CBL-interacting protein kinases (CIPKs), perform essential roles in mediating responses to various extracellular cues, including biotic and abiotic stresses. Several CIPK genes have been functionally characterized in different plant species. However, to date, the functions of pineapple CIPKs have not been investigated, which limits their potential use in molecular breeding. Here we functionally characterized an AcCIPK18 of pineapple and verified its role in salt, drought, heat stresses and immunity. Bioinformatics studies and phylogenetic analysis showed that AcCIPK18 is homologous to CIPK6 of Arabidopsis. Expression analysis shows that AcCIPK18 gets induced by salt, osmotic, heat and ABA. Besides, the ectopic expression of AcCIPK18 increased tolerance to salt, drought and heat stress but displayed sensitivity against fungal infection in Arabidopsis. We found that overexpression of AcCIPK18 changes the ROS homeostasis by altering the expression of several ABA signaling genes. These results provide evidence for the involvement of AcCIPK18 in multiple stress responses and advocates that AcCIPK18 could be used as a potential candidate gene for improving tolerance against multiple abiotic stress. • AcCIPK18 is a CBL interacting protein kinase whose expression is highly regulated in pineapple during stress conditions. • Ectopic expression of AcCIPK18 positively regulates salt, drought and heat tolerance and negatively regulates S. sclerotiorum infection. • AcCIPK18 mediates tolerance to salt, drought and heat by regulating ABA signaling and susceptibility to fungal infection by ROS accumulation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 Arabidopsisswc6 by pineapple SWC6 suggested the conserved function of SWC6 in plants. [ABSTRACT FROM AUTHOR]

The cellulose synthase (Ces) and cellulose synthase-like (Csl) gene families belonging to the cellulose synthase gene superfamily, are responsible for the biosynthesis of cellulose and hemicellulose of the plant cell wall, and play critical roles in plant development, growth and evolution. However, the Ces/Csl gene family remains to be characterized in pineapple, a highly valued and delicious tropical fruit. Here, we carried out genome-wide study and identified a total of seven Ces genes and 25 Csl genes in pineapple. Genomic features and phylogeny analysis of Ces/Csl genes were carried out, including phylogenetic tree, chromosomal locations, gene structures, and conserved motifs identification. In addition, we identified 32 pineapple AcoCes/Csl genes with 31 Arabidopsis AtCes/Csl genes as orthologs by the syntenic and phylogenetic approaches. Furthermore, a RNA-seq investigation exhibited the expression profile of several AcoCes/Csl genes in various tissues and multiple developmental stages. Collectively, we provided comprehensive information of the evolution and function of pineapple Ces/Csl gene superfamily, which would be useful for screening out and characterization of the putative genes responsible for tissue development in pineapple. The present study laid the foundation for future functional characterization of Ces/Csl genes in pineapple. [ABSTRACT FROM AUTHOR]

Ca2+ 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 Ca2+ 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 AcCBL and 21 AcCIPK 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 AcCBL and AcCIPK genes were expressed differentially in different tissues. Further, the expression analysis of AcCBL1 in different tissues showed significant changes under various abiotic stimuli. Additionally, the ectopic expression of AcCBL1 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. [ABSTRACT FROM AUTHOR]

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 AcoSAUR s. 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. • 52 AcoSAUR genes were identified and characterized in the Ananas comosus genome. • Both tandem and segmental duplications contributed to the AcoSAUR gene expansion and evolution in pineapple. • AcoSAUR genes differentially expressed during the pineapple floral organ and fruit development. • AcoSAUR6 , AcoSAUR11 , AcoSAUR36 , and AcoSAUR50 played progressive roles during the pineapple fruit development. • AcoSAUR12 , AcoSAUR24 , and AcoSAUR50 positively responded to the salinity and drought stresses treatment. [ABSTRACT FROM AUTHOR]