Overexpression of AcWRKY31 Increases Sensitivity to Salt and Drought and Improves Tolerance to Mealybugs in Pineapple.

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.

Phenotypic clusters of type 2 diabetes mellitus among North Indians reveal higher levels of insulin deficiency along with insulin resistance.

BACKGROUND: Type 2 diabetes is now considered a heterogenous disease. Distinct clusters have been identified with patterns varying between Europeans and South Asians as well as between South Indians who have described a novel cluster; Combined Insulin-Resistant and Deficient Diabetes, and individuals from West and East India who have reported that insulin deficiency is the primary driver of heterogeneity. Therefore, North Indian patients may also have a distinct, novel clustering pattern due to unique genetic, epigenetic, and environmental factors. We aim to identify clusters of type 2 diabetes in North Indians and to describe the different characteristics of these clusters. METHODS: The K value for the optimal number of clusters was obtained from two-step clustering. K means clustering was done with this K value using SPSS 29.0 software. Variables used for clustering were age, BMI, HbA1c, HOMA-beta, HOMA-IR, and waist circumference. RESULTS: Four phenotypically different clusters were identified in 469 individuals with type 2 diabetes. Cluster 1 was severe insulin deficient diabetes (15%), Cluster 2 was severe insulin resistant diabetes (22%), Cluster 3 was moderate obesity-related diabetes (35%), and Cluster 4 was moderate age-related diabetes (27%). Clusters 1 and 2 were similar to earlier studies but in different proportions. Clusters 3 and 4 characteristics were different from earlier studies, with greater impairment in beta cell function and higher HbA1c levels. Significant insulin resistance was noted in all clusters. CONCLUSION: The phenotypic clusters of type 2 diabetes identified in the present study were characterized by high levels of insulin deficiency along with important contributions from insulin resistance.

Curriculum implementation challenges: Development and validation of an integrated curriculum implementation challenges tool.

Objective: To develop an instrument to identify the challenges faced by faculty while implementing an integrated curriculum in an undergraduate dentistry program. Methods: The study was conducted between September 2020 and October 2021 at the University College of Medicine and Dentistry (UCMD), University of Lahore (UOL). A preliminary questionnaire, developed through literature review and faculty interviews was sent to 10 medical education experts for content validation via the Delphi technique. Content Validity Index (CVI) was calculated for individual items (I-CVI) as well as for the composite scale (S-CVI). A panel agreement of more than 75% was considered as the criterion for the inclusion of items in the questionnaire. Cognitive pretesting of five faculty members was conducted and pilot testing was subsequently done with 27 faculty members. The reliability of the tool was determined by Cronbach's alpha. Results: After the Delphi process, the final Integrated Curriculum Implementation Challenges (ICIC) questionnaire had 42 items. S-CVI was 0.87 and the cut-off value for I-CVI was taken as 0.78 as the criterion for item deletion. Cognitive interviews and pretesting revealed good item interpretation. Cronbach's alpha for this tool was 0.87. Conclusion: ICIC is a useful instrument with good reliability and content validity. It can be used to identify the presence and extent of challenges faced by the faculty while implementing an integrated curriculum.

High level of psychological stress in COVID-19 recovered individuals: role of copeptin as a potential biomarker.

Background: Study aimed to assess stress in COVID-19 recovered individuals using a validated questionnaire PSS-10 score and stress biomarkers - salivary cortisol and serum copeptin. Methods: A total of 83 subjects of which 54 subjects (66.3%) who were hospitalized were recruited 8-20 weeks following recovery from COVID-19. Stress was assessed by PSS-10 stress-scale after a mean duration of 14.5 weeks after recovery. Sixty-eight subjects (81.9%) had new or persistent symptoms after recovery. Subjects were divided into two groups on the basis of PSS score; mild stress (PSS:0-13) and moderate to severe stress (PSS:>14) and levels of biomarkers (serum copeptin, DHEAS and salivary cortisol) were compared in the two groups. Results: Forty-four subjects (53%) had moderate to severe stress and 39 subjects (47%) had mild stress. Subjects with post COVID symptoms had significantly higher stress levels as compared to subjects who were asymptomatic [15 vs. 9; p = 0.003]. Serum copeptin levels were significantly higher among subjects with moderate to severe stress as compared to those with mild stress [0.41 vs. 0.67 ng/mL; p = 0.031]. Subjects with moderate to severe stress had higher median salivary cortisol compared to subjects with mild stress [1.03 vs. 1.44 nmol/L; p = 0.448]. Conclusion: Our study demonstrated moderate to severe stress in over half and some level of stress in nearly all COVID recovered individuals even after 3 months. Serum copeptin was found to be a useful biomarker to objectively measure stress in these subjects.

Pineapple SWEET10 is a glucose transporter.

SWEET transporters are a unique class of sugar transporters that play vital roles in various developmental and physiological processes in plants. While the functions of SWEETs have been well established in model plants such as Arabidopsis, their functions in economically important fruit crops like pineapple have not been well studied. Here we aimed to investigate the substrate specificity of pineapple SWEETs by comparing the protein sequences of known glucose and sucrose transporters in Arabidopsis with those in pineapple. Our genome-wide approach and 3D structure comparison showed that the Arabidopsis SWEET8 homolog in pineapple, AcSWEET10, shares similar sequences and protein properties responsible for glucose transport. To determine the functional conservation of AcSWEET10, we tested its ability to complement glucose transport mutants in yeast and analyzed its expression in stamens and impact on the microspore phenotype and seed set in transgenic Arabidopsis. The results showed that AcSWEET10 is functionally equivalent to AtSWEET8 and plays a critical role in regulating microspore formation through the regulation of the Callose synthase5 (CalS5), which highlights the importance of SWEET transporters in pineapple. This information could have important implications for improving fruit crop yield and quality by manipulating SWEET transporter activity.

Chromosome-scale genome sequence of Suaeda glauca sheds light on salt stress tolerance in halophytes.

Soil salinity is a growing concern for global crop production and the sustainable development of humanity. Therefore, it is crucial to comprehend salt tolerance mechanisms and identify salt-tolerance genes to enhance crop tolerance to salt stress. Suaeda glauca, a halophyte species well adapted to the seawater environment, possesses a unique ability to absorb and retain high salt concentrations within its cells, particularly in its leaves, suggesting the presence of a distinct mechanism for salt tolerance. In this study, we performed de novo sequencing of the S. glauca genome. The genome has a size of 1.02 Gb (consisting of two sets of haplotypes) and contains 54 761 annotated genes, including alleles and repeats. Comparative genomic analysis revealed a strong synteny between the genomes of S. glauca and Beta vulgaris. Of the S. glauca genome, 70.56% comprises repeat sequences, with retroelements being the most abundant. Leveraging the allele-aware assembly of the S. glauca genome, we investigated genome-wide allele-specific expression in the analyzed samples. The results indicated that the diversity in promoter sequences might contribute to consistent allele-specific expression. Moreover, a systematic analysis of the ABCE gene families shed light on the formation of S. glauca's flower morphology, suggesting that dysfunction of A-class genes is responsible for the absence of petals in S. glauca. Gene family expansion analysis demonstrated significant enrichment of Gene Ontology (GO) terms associated with DNA repair, chromosome stability, DNA demethylation, cation binding, and red/far-red light signaling pathways in the co-expanded gene families of S. glauca and S. aralocaspica, in comparison with glycophytic species within the chenopodium family. Time-course transcriptome analysis under salt treatments revealed detailed responses of S. glauca to salt tolerance, and the enrichment of the transition-upregulated genes in the leaves associated with DNA repair and chromosome stability, lipid biosynthetic process, and isoprenoid metabolic process. Additionally, genome-wide analysis of transcription factors indicated a significant expansion of FAR1 gene family. However, further investigation is needed to determine the exact role of the FAR1 gene family in salt tolerance in S. glauca.

Insights into the role of phytohormones in plant female germline cell specification.

Germline specification is a fundamental process in plant reproduction, and the Megaspore Mother Cell (MMC), is a critical cell that differentiates and develops into the female gametophyte. While numerous studies have investigated the molecular mechanisms underlying female germline specification, previous reviews have mainly focused on gene regulatory networks, epigenetic pathways, and small RNAs, neglecting the potential contribution of phytohormones to this process. This review aims to address this gap by highlighting recent advances in MMC formation and discussing the roles of specific phytohormones in female germline specialization. Here, we provide a comprehensive overview of the functions of phytohormones in the formation of MMC and their effects on female gametophyte development. Specifically, it examines the roles of gibberellins (GAs), brassinosteroids (BRs), auxins, and cytokinin, in MMC development. Understanding the function of phytohormones in MMC development is essential for comprehending the complex mechanisms underlying plant reproduction. This review adds valuable insights to the existing knowledge on MMC development, providing a new perspective for future research in the field of plant reproduction.

Can Bone-Specific Alkaline Phosphatase be a Marker of Vascular Calcification in Type 2 Diabetes Mellitus?

Background and Aims: Alkaline phosphatase (ALP) enzyme has been linked to vascular calcification. Unexplained elevations in serum ALP levels have been reported in patients with type 2 diabetes mellitus (T2DM). We assessed bone-specific alkaline phosphatase (BAP) levels in patients with T2DM who had unexplained ALP elevations and studied the association between BAP and other markers of vascular calcification. Methods: Patients with T2DM who had high serum ALP in the absence of known causes of ALP elevation were studied. The control group was T2DM patients with normal ALP. We measured the serum levels of BAP along with the leptin, fetuin-A, and vitamin K2 levels. Ankle-brachial index (ABI) was also measured in both groups. Results: Serum BAP levels were significantly higher in the group with high ALP when compared with the normal ALP group. A significant positive correlation was present between BAP and serum fetuin-A as well as between BAP and Vit K2 levels. There was no correlation between BAP and serum leptin. ABI was comparable between the two groups. Conclusions: Patients with T2DM may have unexplained elevation in ALP due to an increase in BAP. Elevation in BAP may be associated with other markers of vascular calcification suggesting an increased risk of vascular calcification.

Identification of passion fruit HSF gene family and the functional analysis of PeHSF-C1a in response to heat and osmotic stress.

Heat stress transcription factors (HSFs) are the major regulators of plant response to environmental stress, especially heat and drought stress. To gain a deeper understanding of the mechanisms underlying HSFs in the abiotic stress response of passion fruit, we conducted an in silico analysis of the HSF gene family. Through bioinformatics and phylogenetic analyses, we identified 18 PeHSF members and classified them into A, B, and C groups. Collinearity analysis results revealed that the expansion of the PeHSF gene family was due to the presence of segmental duplication. Furthermore, gene structure and protein domain analysis illustrated that PeHSFs in the same subgroup are relatively conserved. Conserved motif and function domain analysis suggested that PeHSF proteins possess typical conserved functional domains of the HSF family. A protein interaction network and 3D structure prediction were used to study the potential regulatory relationship of PeHSFs. Additionally, the subcellular localization results of PeHSF-A6a, PeHSF-B4b, and PeHSF-C1a were consistent with the predictions. RNA-seq and RT-qPCR analysis revealed the expression patterns of PeHSFs in different tissues of passion fruit floral organs. Promoter analysis and the expression patterns of the PeHSFs under different treatments demonstrated their involvement in various abiotic stress processes. Notably, overexpression of PeHSF-C1a consistently enhanced tolerance to drought and heat stress in Arabidopsis. Overall, our findings provide a scientific basis for further functional studies of PeHSFs that could contribute to improvement of passion fruit breeding.

Advances in micropropagation, somatic embryogenesis, somatic hybridizations, genetic transformation and cryopreservation for Passiflora improvement.

Passion fruit is an essential commercial plant in the tropics and subtropics, which has lately seen a rise in demand for high-quality fruits and large-scale production. Generally, different species of passion fruit (Passiflora sp.) are propagated by sexual reproduction. However, asexual reproduction, such as stem cuttings, grafting, or tissue culture, is also available and advantageous in many instances. Recent research on passion fruit has concentrated on improving and establishing methodologies for embryogenesis, clonal proliferation via (somatic embryos), homozygote regeneration (by anther culture), germplasm preservation (via cryopreservation), and genetic transformation. These developments have resulted in potentially new directions for asexual propagation. Even though effective embryo culture and cryogenics are now available, however the limited frequency of embryogenic callus transformation to ex-vitro seedlings still restricts the substantial clonal replication of passion fruit. Here, in this review the advancement related to biotechnological approaches and the current understanding of Passiflora tissue culture. In vitro culture, organogenesis, cryopreservation, breeding, and productivity of Passiflora will significantly improve with novel propagation approaches, which could be applied to a wider range of germplasm.

The genome size, chromosome number and the seed adaption to long-distance dispersal of Ipomoea pes-caprae (L.).

Ipomoeapes-caprae (L.) (IPC) is a common species in tropical and subtropical coastal areas and one of the world's most widely distributed plants. It has attracted researchers for its outstanding biological, ecological and medicinal values. It has been reported that the genetic diversity of IPCs located on different continents is very low because of their frequent gene flow. During the long journey of evolution, every aspect of the plant morphologies has evolved to the best adaptivity to the environment, seeking their survival and progeny expansion. However, the fundamental genetic characteristics of IPC and how their seed adapted to the success of population expansion remain unknown. In this study, the fundamental genetic characteristics, including the genome size and the chromosome number of IPC, were investigated. The results showed that IPC's genome size is approximately 0.98-1.08 GB, and the chromosome number is 2n=30, providing the basic information for further genome analysis. In order to decipher the long-distance dispersal secret of this species, the fruit and seed developments, seed morphology, and seed germination were extensively investigated and described. The results showed an exquisite adaptive mechanism of IPC seeds to fulfil the population expansion via ocean currents. The large cavity inside the seeds and the dense tomenta on the surface provide the buoyancy force for the seeds to float on the seawater. The hard seed coats significantly obstructed the water absorption, thus preventing the seed from germination during the dispersal. Meanwhile, the fully developed embryos of IPC also have physiological dormancy. The physical and physiological characteristics of IPC seeds provide insight into the mechanism of their long-distance dispersal across the oceans. Moreover, based on morphological observation and semi-section microscopy, the development pattern of IPC glander trichomes was described, and their physiological functions were also discussed.

Small Auxin Up RNA (SAUR) gene family identification and functional genes exploration during the floral organ and fruit developmental stages in pineapple (Ananas comosus L.) and its response to salinity and drought stresses.

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.

Signaling by the EPFL-ERECTA family coordinates female germline specification through the BZR1 family in Arabidopsis.

In most flowering plants, the female germline is initiated in the subepidermal L2 layer of ovule primordia forming a single megaspore mother cell (MMC). How signaling from the L1 (epidermal) layer could contribute to the gene regulatory network (GRN) restricting MMC formation to a single cell is unclear. We show that EPIDERMAL PATTERNING FACTOR-like (EPFL) peptide ligands are expressed in the L1 layer, together with their ERECTA family (ERf) receptor kinases, to control female germline specification in Arabidopsis thaliana. EPFL-ERf dependent signaling restricts multiple subepidermal cells from acquiring MMC-like cell identity by activating the expression of the major brassinosteroid (BR) receptor kinase BRASSINOSTEROID INSENSITIVE 1 and the BR-responsive transcription factor BRASSINOZOLE RESISTANT 1 (BZR1). Additionally, BZR1 coordinates female germline specification by directly activating the expression of a nucleolar GTP-binding protein, NUCLEOSTEMIN-LIKE 1 (NSN1), which is expressed in early-stage ovules excluding the MMC. Mutants defective in this GRN form multiple MMCs resulting in a strong reduction of seed set. In conclusion, we uncovered a ligand/receptor-like kinase-mediated signaling pathway acting upstream and coordinating BR signaling via NSN1 to restrict MMC differentiation to a single subepidermal cell.

Genome-wide identification and expression analysis of the bHLH gene family in passion fruit (Passiflora edulis) and its response to abiotic stress.

Passion fruit is a tropical fruit crop with significant agricultural, economic and ornamental values. The growth and development of passion fruit are greatly affected by climatic conditions. In plants, the basic helix-loop-helix (bHLH) gene family plays essential roles in the floral organ and fruit development, as well as stress response. However, the characteristics and functions of the bHLH genes of passion fruit remain unclear. Here, 138 passion fruit bHLH members were identified and classified into 20 subfamilies. The structural analysis illustrated that PebHLH proteins of the specific subfamily are relatively conserved. Collinearity analysis indicated that the expansion of the PebHLH gene family mainly took place by segmental duplication, and the structural diversity of duplicated genes might contribute to their functional diversity. PebHLHs, which potentially regulate different floral organ and fruit development, were further screened out, and many of these genes were differentially expressed under various stress treatments. The co-presence of different cis-regulatory elements involved in developmental regulation, hormone and stress responses in the promoter regions of PebHLHs might be closely related to their diverse regulatory roles. Overall, this study will be helpful for further functional investigation of PebHLHs and provides clues for improvement of the passion fruit breeding.

Identification and expression analysis of pineapple sugar transporters reveal their role in the development and environmental response.

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.

Strategic Electrochemical Determination of Nitrate over Polyaniline/Multi-Walled Carbon Nanotubes-Gum Arabic Architecture.

Significant agricultural and industrial activities necessitate the regular monitoring of nitrate (NO3-) ions levels in feed and groundwater. The current comparative study discloses an innovative user-friendly electrochemical approach for the determination of NO3- over polyaniline (PAni)-based modified electrodes. The electrochemical sensors concocted with PAni, multi-walled carbon nanotubes (CNT), and gum arabic (GA). The unique electrode material GA@PAni-CNT was synthesized by facile one-pot catalytic polymerization of aniline (Ani) with FeCl3/H2O2 in the presence of CNT and GA as integral components. As revealed by cyclic voltammetry (CV), the anchoring/retention of NO3- followed by reduction is proposed to occur when a GA@PAni-CNT electrode is immersed in phosphate buffer electrolyte containing NO3- that eventually results in a significantly higher redox activity of the GA@PAni-CNT electrode upon potential scan. The mechanism of NO3- anchoring may be associated with the non-redox transition of leucomeraldine salt (LS) into emeraldine salt (ES) and the generation of nitrite (NO2-) ions. As a result, the oxidation current produced by CV for redox transition of ES ↔ pernigraniline (PN) was ~9 times of that obtained with GA@PAni-CNT electrode and phosphate buffer electrolyte, thus achieving indirect NO3- voltammetric determination of the GA@PAni-CNT electrode. The prepared GA@PAni-CNT electrode displayed a higher charge transfer ability as compared to that of PAni-CNT and PAni electrodes. The optimum square wave voltammetric (SWV) response resulted in two linear concentration ranges of 1-10 (R2 = 0.9995) and 15-50 µM (R2 = 0.9988) with a detection limit of 0.42 µM, which is significantly lower. The GA@PAni-CNT electrode demonstrated the best detection, sensitivity, and performance among the investigated electrodes for indirect voltammetric determination of NO3- that portrayed the possibility of utilizing GA-stabilized PAni and CNT nanocomposite materials in additional electrochemical sensing applications.

Correction: Ye et al. Investigation of the JASMONATE ZIM-DOMAIN Gene Family Reveals the Canonical JA-Signaling Pathway in Pineapple. Biology 2022, 11, 445.

The authors would like to make the following correction to the published paper [...].

Genomic insights of the WRKY genes in kenaf (Hibiscus cannabinus L.) reveal that HcWRKY44 improves the plant's tolerance to the salinity stress.

The WRKY transcription factors (TFs) are among the most diverse TF families of plants. They are implicated in various processes related to plant growth and stress response. Kenaf (Hibiscus cannabinus L.), an important fiber crop, has many applications, including the phytoremediation of saline-alkaline soil. However, the roles of WRKY TFs in kenaf are rarely studied. In the present study, 46 kenaf WRKY genes were genome-widely identified and characterized by gene structure, phylogeny and expression pattern analysis. Furthermore, the HcWRKY44 gene was functionally characterized in Arabidopsis under salinity and drought stresses. HcWRKY44 is a nuclear-localized protein that is positively induced by salinity and drought, with roots showing maximum accumulation of its transcripts. Under NaCl and abscisic acid (ABA) stress conditions, plants overexpressing HcWRKY44 had higher germination rates, better root growth and increased survival than control plants; however, it did not improve the ability to withstand drought stress. Moreover, ABA signaling genes (ABI1, ABI2, and ABI5), ABA-responsive genes (ABF4, RD29B, COR15A, COR47, and RD22), stress-related genes (STZ, P5CS, and KIN1), and ionic homeostasis-related genes (SOS1, AHA1, AHA2, and HKT1) were positively induced in HcWRKY44 transgenic plants under NaCl treatment. These results suggest that HcWRKY44 improved plant's tolerance to salt stress but not osmotic stress through an ABA-mediated pathway. In summary, this study provides provided comprehensive information about HcWRKY genes and revealed that HcWRKY44 is involved in salinity tolerance and ABA signaling.