Abstract:

Leuenbergeria bleo (Kunth) DC. (Cactaceae), previously classified as Pereskia bleo, represents a phylogenetically basal cactus species with a disjunct distribution across Central America, Southeast Asia, and southern China. Phytochemical investigations have traditionally emphasized small-molecule secondary metabolites, including phenolics, alkaloids, and terpenoids, which contribute to antioxidant and anti-inflammatory activities. However, recent peptidomic analyses have expanded this chemical space through the discovery of bleogens, a family of hyper-stable, cysteine-rich microproteins with specific antifungal and wound-healing properties. This review systematically integrates botanical characteristics, ethnomedicinal applications, and pharmacological profiles, providing a comparative analysis of the plant’s small-molecule constituents versus its peptidyl biologics. It identifies the co-existence of these distinct chemical classes as a defining feature of the plant’s efficacy while highlighting the need for future research into their potential interactions.

Abstract: Lavender has been cultivated in Bulgaria for over a century. The high essential oil content and quality of Bulgarian lavender varieties have established the country as a leading global producer. Studies into the crop's genetic diversity is essential for selecting varieties best suited to specific environmental conditions, maximising resilience and yield. Therefore, identifying appropriate genetic markers to monitor lavender diversity is a key prerequisite for developing effective crop selection strategies, particularly in response to the challenges posed by global climate change. In this study, we evaluate the versatility of markers for assessing genetic diversity of lavender genotypes. A total of 96, 97 and 96 bands were recorded using the 13 SCoT, 13 ISSR and 14 CEAP primers, respectively. All amplification programs used were successful in the studied genotypes. Additionally, were applied four informative primers of each marker systems for assessment of the within-field genetic variability in two lavender plantations from Bulgaria. This is the first report on the combined use and comparison of CEAP, SCoT and ISSR primers in lavender genotypes in Bulgaria.

Abstract:

Plants defend against pathogens such as fungi by detecting an attack and initiating both structural and chemical responses. Pathogen perception triggers rapid cytosolic calcium influx, calcium oscillations, and induces defense gene expression, yet the mechanisms by which these or other signals encode the external stressors or propagate signals plant-wide remain unclear. Here, we present a microfluidic system to examine intracellular calcium signals of the moss Physcomitrium patens upon precise and reversible exposure to fungal chitin oligosaccharides. Epifluorescent microscopy of juvenile moss cells expressing the calcium indicator GCaMP6f revealed a rapid, coordinated calcium response to chitin addition, followed by stereotyped oscillations that subsided quickly upon chitin removal. We developed an unbiased image segmentation algorithm to automatically locate regions with cell-specific oscillatory responses, using pixel-based k-means clustering, treating each time point as a separate dimension. Calcium dynamics were distinct across adjacent cells and distinguishable by cell type. Waves were dependent on time of day, adaptation time within the device, and stimulus timing. Cytosolic calcium waves, which rose and fell symmetrically within about 60 s, appeared spontaneously at night and with short adaptation time. Chitin increased wave frequency, amplitude, and duration, and repeated chitin pulses drove regular, plant-wide oscillations at a controlled frequency. This study complements prior investigations of whole plant and growth tip dynamics and provides new methods to comprehensively study calcium signaling in plants, including mechanisms of signal propagation and the role of oscillation frequency on gene expression.

Abstract:

Photo-oxidative stress results from an imbalance between light absorption and photosynthetic carbon utilization, posing a major threat to plant productivity and resilience under climate change. This review synthesizes recent advances in the molecular mechanisms of photo-oxidation, focusing on the dual role of reactive oxygen species (ROS) as both toxic byproducts and key signaling molecules. We outline the specific sites of ROS generation in chloroplasts, particularly singlet oxygen (¹O₂) at Photosystem II (PSII) and hydrogen peroxide (H₂O₂) at Photosystem I (PSI), and describe their distinct retrograde signaling pathways that regulate nuclear gene expression for acclimation. A systems perspective reveals how photo-oxidative damage propagates through interconnected cycles of impaired photosystem repair, lipid peroxidation, and protein oxidation, ultimately risking cellular collapse. To cope, plants employ a multi-layered photoprotective arsenal, including non-photochemical quenching (NPQ), alternative electron sinks, and integrated antioxidant networks. These mechanisms are further examined within an ecological and evolutionary context, highlighting natural variation and trade-offs between growth and defense. Finally, we discuss future directions for translating this knowledge into strategies for engineering climate-resilient crops, emphasizing the role of synthetic biology, multi-omics integration, and genomics-assisted breeding in supporting global food security.

Abstract:

Bacterial panicle blight (BPB) of rice, a disease caused by Burkholderia glumae and B. gladioli, threatens global rice yields and has recently emerged in Bangladesh. We analyzed 300 BPB-infected samples from 20 Bangladesh districts using S-PG medium and gyrB PCR amplification, identifying 46 B. gladioli and 5 B. glumae isolates. Twenty of these isolates were chosen for in-depth characterization. Pathogenicity tests identified B. glumae BD_21g as the most virulent strain, followed by B. gladioli BDBgla132A. Disease severity on rice strongly correlated with onion bulb assays, validating the assay as a rapid virulence-screening tool. Phenotypic characterization of the 20 isolates revealed substantial variation in toxoflavin production, lipase activity, polygalacturonase activity, motility, and type III secretion system. Comparative genomic analysis of virulence-associated genes between BDBgla132A and BD_21g showed high protein sequence identity, particularly in toxoflavin biosynthesis and transport genes, while genes encoding lipase (lipA/lipB), polygalacturonase (pehA/pehB), and those involved in motility, displayed moderate to high identity. Both strains retained virulence-related genes that are homologous to those of B. cepacia but displayed distinct pathogenic mechanisms. Real time RT-qPCR revealed significantly higher expression of toxoflavin and lipase-encoding genes in BD_21g compared with BDBgla132A, consistent with its elevated enzymatic activities. Conversely, BD_21g showed reduced expression of pectinolytic and flagellar genes over BDBgla132A, consistent with the enhanced pectinolytic activity and motility observed in BDBgla132A. These findings reveal that B. glumae BD_21g and B. gladioli BDBgla132A employ distinct virulence strategies to infect rice, providing critical insights for developing targeted BPB management approaches in Bangladesh.

Abstract: DNA methylation is a conserved regulatory mechanism of gene expression, genome stability, and development. DNA methylation modifications relate to effective induc-tion of defense responses for plant priming. In the Green Deal era, using plant defense inducers, compounds that activate defense and prime plants against imminent patho-gens attacks, is a safe and environmentally sustainable approach to support plants against pathogens. Here, salicylic acid loaded in chitosan nanoparticles, influenced hypomethylation on specific genomic regions that corresponded to defense-related genes, such as pectin lyases, defensins and leucine-rich repeat transmembrane protein kinases against the biotrophic fungal pathogen Podosphaera xanthii. A genomic region of the promoter of SKP1A, a core member of the SCF E3 ubiquitin ligase complex, was found to be a significantly hypomethylated DMR. Examination of this DMR revealed the presence of salicylic acid-, auxin-, and defense-related cis-elements. Investigation of proteins associated with the above cis-elements showed significant expression upreg-ulation after salicylic acid application. Moreover, association of the identified DMR with transcriptomics showed significant enrichment of the salicylic acid pathway. Overall, these findings shed light on the epigenetic mechanisms that underly salicylic acid- re-lated defense priming in plants.

Abstract: Phytopathogens threaten natural ecosystems and global food security. Horticultural trade is the main long-distance pathway causing the spread of these organisms and disease outbreaks worldwide. Most inspections for disease symptoms are conducted visually but this is insufficient given the large number of plants and the prevalence of asymptomatic infections. Therefore, there is increasing interest in the use of high-throughput sequencing (HTS) and environmental DNA (eDNA) for plant health surveillance. Many studies have used these technologies to detect phytopathogens, but fewer have done so in horticultural settings. Furthermore, much work has focused on the molecular and bioinformatic approaches for this work, with relatively little attention given to sample collection. This systematic review therefore provides an overview of the available sampling methods and their target plant pathogens, with a particular focus on the utility of these sampling methods in horticultural nurseries. It highlights some striking gaps in the literature and opportunities for further research, for example, the detection of bacterial phytopathogens using eDNA has received little attention despite having considerable potential as a surveillance and/or diagnostic tool.

Abstract: Diaporthe sojae is well known as a causal pathogen of pod and blight as well as seed decay in soybean (Glycine max), it is also known to cause root rot symptoms, though its role is poorly defined. During a 2024 survey in Manitoba, Canada, D. sojae was routinely isolated from roots exhibiting cortical decay and discoloration. The identity of 23 representative isolates was confirmed through morphological characterization and multi-locus phylogenetic analysis using internal transcribed spacer (ITS) and translation elongation factor 1-alpha (EF1-α) sequences. Phylogenetic analysis revealed that the ITS region provided superior resolution in distinguishing D. sojae from the closely related D. longicolla, whereas EF1-α lacked discriminatory power. Pathogenicity was evaluated for 19 isolates on soybean cv. Sperling in a greenhouse assay using colonized wheat-kernel inoculum. While all isolates were pathogenic, significant virulence diversity was observed among isolates, with Disease Severity Indices (DSI) ranging from 68.8% to 100%. Two distinct virulence phenotypes were identified: acute virulence isolates causing 100% seedling mortality (damping-off), and more chronic phenotype characterized by root necrosis and stunting without immediate plant death. This study constitutes the first confirmed report of D. sojae causing soybean root rot in Canada. These findings expand the known disease spectrum of D. sojae and highlight the necessity of including this pathogen in future root-rot diagnostic and management strategies.

Abstract: The remarkable plasticity of plants is best exemplified by the capacity of their somatic cells to regenerate entire organs or the organism itself. The molecular and cellular events underlying this ability are complex and multifaceted. The initial phase, that is triggered either by wounding or exogenous hormone application and leads to cell cycle reactivation, is often called dedifferentiation. This review proposes that the dedifferentiation of mature somatic cells is a two-step process. It involves a transition into a transient senescence-like state. This leads to a critical cellular condition; in the absence of proliferative signals, cells become committed to programmed cell death. If, however, it is succeeded by a second hormonal rescue step, when phytohormones, mainly auxin and cytokinin, override the death pathway and activate cell division, callus arises. Callus is not an undifferentiated homogenous mass of cells, but an unorganised tissue with at least some cells having ground-tissue-like molecular identity with high developmental potential. Callus, though, might also form from pre-existing, competent populations of cells, e.g. pericycle cells, with no senescence-like intermitting state. It is also discussed whether this “one-step” callus formation pathway can be referred to as dedifferentiation.

Abstract:

Sesame (Sesamum indicum L.) is a nutrient-rich oilseed valued for its high-quality oil and protein-rich seeds. Sesame breeding can be accelerated by unlocking the untapped genetic variation present in African landraces. This study integrated a global meta-quantitative trait loci (QTL) analysis with genome‑wide association study (GWAS) of Ethiopian germplasm to identify molecular markers for two key agronomic traits: plant height and seed coat color. To address inconsistencies among published studies, we explicitly documented the genetic maps, marker systems, mapping populations, linkage mapping and GWAS analysis methods used in each source study before conducting the meta-analysis. Only QTL whose markers could be reliably anchored to the sesame reference genome v3.0 were retained. Meta-analysis of eight published studies identified six conserved QTL hotspots on chromosomes 3, 4, 6, 8, 9, and 11. Field evaluation of 200 Ethiopian accessions over two seasons revealed wide phenotypic variation and high heritability (H² > 0.85). Using 3,633 genome-wide SNPs, GWAS detected 36 significant marker-trait associations, including multiple novel loci on chromosomes 12 and 13 not reported in Asian germplasm-focused studies. Key SNPs explained up to 14.2% (plant height) and 9.2% (seed coat color) of phenotypic variance. Candidate genes linked to significant SNPs included brassinosteroid-related CYP90B1 and ethylene-responsive AP2/ERF for plant height, and transcription factors WRKY23, DOF3.1, and SBP-like for seed coat color. Population structure showed two distinct groups (K = 2), and linkage disequilibrium decayed rapidly (~204 kb), enabling fine‑mapping. The study provides validated meta‑QTL intervals, trait-associated SNPs, and candidate genes that form a molecular foundation for marker-assisted selection in sesame improvement programs.

Abstract:

The Balkan Peninsula is a biodiversity hotspot where topographic and habitat heterogeneity have shaped genetic differentiation. Polyploidization significantly contributes to diversification within plant lineages, including the allopolyploid Veronica austriaca complex. We sampled 751 individuals from 50 Balkan and Central European populations belonging to the hexaploid V. austriaca and its putative diploid (V. dalmatica) and tetraploid progenitors. Diversity patterns were investigated through microsatellite markers (SSRs), plastid DNA sequences, ploidy estimations, morphological data and climatic niche differentiation analysis. Five lineages were detected within the complex according to nuclear DNA data. The plastid DNA haplotypes form two main groups that overall match those detected by SSRs data and could suggest that the hexaploid V. austriaca resulted from two different allopolyploid events. Our analyses evidence rapid and recent colonization of diverse mesic grassy habitats by an allopolyploid perennial herb across a large European scale. The enhanced dispersal abilities of the hexaploid V. austriaca (compared to its lower ploidy relatives) seem to result from higher genetic diversity and ecological niche differentiation, which may also be related to slight morphological differences of potential functional significance. Style length is a crucial character to distinguish diploids from polyploids, which may affect pollination biology within the complex.

Abstract: Tropical Andean forests are biodiversity hotspots that have been transformed by anthropogenic activities, making ecosystem regeneration and restoration essential for their recovery. This study evaluated floristic composition, forest structure, and diversity in three land cover types within tropical Andean ecosystems: riparian forest (RF), natural regeneration (RN), and ecological restoration areas (RE). Vegetation was inventoried using standardized plots, recording species composition, diameter, and height. Basal area, size class distribution, and vertical structure were estimated. The Shan-non-Wiener and Simpson indices were evaluated. RF exhibited greater structural com-plexity, a larger basal area, and defined vertical stratification, indicating advanced suc-cessional stages and functional stability. NR showed the highest diversity values and a predominance of individuals in lower diameter and height classes, reflecting active re-cruitment and intermediate successional stages. Segment E exhibited lower diversity and intermediate structural development, consistent with shorter recovery periods and limitations in restoration design. Overall, the integration of floristic, structural, and diversity attributes indicates distinct successional trajectories, conditioned by land-use history, disturbance intensity, and environmental heterogeneity. These findings high-light the great potential for natural regeneration under reduced anthropogenic pressure and emphasize the need to integrate passive and active restoration strategies to enhance biodiversity and resilience in Andean tropical forests.

Abstract:

Galeopsis tetrahit L. (Lamiaceae) is a traditional European medicinal species rich in phenolic compounds, among which verbascoside is a key bioactive marker with strong antioxidant potential. This study reports the standardization of a G. tetrahit leaf extract in verbascoside using a fully validated UHPLC–PDA method developed according to ICH Q2(R2) requirements. Leaves of wild-grown G. tetrahit collected from southwest Romania flora were extracted with 70% ethanol, yielding 17.28% dry extract. Chromatographic identification of verbascoside was confirmed by retention time, UV–PDA spectra, and QDa mass spectrometry (m/z 623.3 [M–H]^–). The method showed excellent performance, including high specificity, linearity over 1.875–60 μg/mL (r = 0.999955), low LOD and LOQ (0.2649 and 0.8028 μg/mL, respectively), and robust precision and accuracy. Dry extract contained 345.8 ± 28.3 mg verbascoside per g (34.6%, w/w), corresponding to approximately 59.8 mg/g in dried leaves. Antioxidant assays (DPPH, ABTS, FRAP), TPC and TFC confirmed notable radical scavenging and reducing activity, with pure verbascoside showing markedly stronger effects, supporting its major contribution to the extract’s antioxidant potential. These results demonstrate a reliable analytical approach and establish a verbascoside-based standardization framework for G. tetrahit extracts of documented Romanian origin.

Abstract: Despite over two centuries of debate, the morphological nature of the grass embryo’s parts remains unresolved. The bipartite interpretation of the grass cotyledon suggests that it is composed of two parts: the scutellum and the coleoptile. According to Wu et al. (2024, 2025), the cotyledon in maize is organized similarly to the vegetative leaf, comprising scutellum and coleoptile, homologous to the leaf blade and sheath, respectively. This view, which Wu et al. (2024, 2025) erroneously associate with the bipartite model of grass cotyledon, is morphologically impossible because within the embryo, the coleoptile is located above the scutellum and therefore cannot be homologous to the latter’s sheath that is suppressed. Within a bipartite context, the coleoptile may only be homologous to the leaf ligule or represent a de novo outgrowth of the scutellum’s reduced sheath, thus lacking homology with mature leaf structures. Molecular evidence from the mesocotyl and the leaf collar region is essential for evaluating the bipartite nature of the grass cotyledon. The genetic results of Wu et al. (2024) do, in fact, support the view that the coleoptile is the next leaf on the embryo’s axis after the scutellum, thus disproving the bipartite view of the cotyledon in Poaceae.

Abstract: Phosphate (P_i) deficiency causes broad transcriptional changes that enhance P_i uptake and utilization. Because sucrose transport from shoot to root mediates long-distance P_i-deficiency signaling, we asked whether soybean can still respond to –P when sucrose supply is restricted. To limit sucrose availability, we exposed hydroponically grown soybean plants to +P or –P conditions for 30 h under light or darkness, with darkness suppressing photosynthetic carbon production. Root transcriptomes were profiled using Oxford Nanopore cDNA sequencing. Across four biological replicates per treatment, ~90 million reads were generated, with >90% mapping to ~25,300 expressed genes (~54% of annotated coding genes). Principal component analysis showed that light accounted for most of the variance, while P_i status contributed a smaller component. Under light, P_i deficiency activated a strong transcriptional program, including canonical phos-phate-starvation markers such as PHT1, PHO1 and PTEN2α. In darkness, however, only a small number of genes were induced, including a MYB-domain transcription factor, and none overlapped with the light-responsive set. Together, these findings suggest that light is required for broad transcriptional responses to P_i deficiency, whereas a local Pi-responsive program becomes detectable only when systemic sucrose signaling is suppressed.

Abstract: Oxidative stress results from an imbalance of reactive oxygen species and antioxidant mechanisms and is associated with chronic and degenerative diseases. The use of natural and artificial antioxidants is commonly prescribed to manage it. In nature, some plants synthesize specialized metabolites with significant biological activity. This applies to Bixa orellana L., which contains carotenoids and terpenes. The present study purpose to per-form a phytochemical analysis of B. orellana seeds and determine their antioxidant activity using in vitro assays. The seed extract obtained by maceration in hexane, ethyl acetate, and methanol was analyzed by gas chromatography-mass spectrometry (GC-MS). In vitro antioxidant activity was also evaluated using the DPPH assay, with ascorbic acid as the reference control. A two-way ANOVA followed by Dunnett's multiple comparisons test was performed. A total of 141 volatile and semi-volatile compounds were identified, mainly terpenes, esters, and fatty acids, among which geranylgeraniol (27.50%), ish-warane (11.01%), and geraniol acetate (7.76%) were the most prominent. The extract showed significant dose-dependent antioxidant activity, with an IC50 of 0.5108 mg/mL-1, compared to ascorbic acid with an IC50 of 0.2266 mg/mL-1. The results indicate that the seeds of B. orellana L. possess metabolites capable of inhibiting free radicals such as DPPH.

Abstract: Eucommia ulmoides, a tree species native to China, holds considerable medicinal, ecological, and industrial importance. However, the absence of an efficient and stable genetic transformation system poses significant challenges to gene function studies and molecular breeding in E. ulmoides. Protoplasts, which lack cell walls, serve as effective receptors for transient transformation and are thus ideal for genetic engineering research. In this study, we identified the optimal conditions for callus induction using a full-factorial experimental design and accurately identified embryogenic callus through paraffin sectioning. Furthermore, we developed an efficient protoplast isolation and PEG-mediated transient transformation system using embryogenic callus as the starting material. Our findings revealed that the optimal medium for inducing embryogenic callus was B5, supplemented with 1.5 mg/L 6-BA, 0.5 mg/L NAA, 30 g/L sucrose, and 7 g/L agar. This medium achieved a callus induction rate of 97.50% and an embryogenic callus induction rate of 86.30%. For protoplast isolation, the best conditions involved enzymatic digestion with 1.5% cellulase R-10 and 1.0% macerozyme R-10 at an osmotic pressure of 0.6 M for 4 h, resulting in 1.82×10⁶ protoplasts/g FW with 91.13% viability. The highest transfection efficiency (53.23%) was attained when protoplasts were cultured with 10 µg of plasmid and 40% PEG4000 for 20 min. This study successfully established a stable and efficient system for protoplast isolation and transient transformation in E. ulmoides, offering technical support for exploring somatic hybridization and transient gene expression in this species.

Abstract:

This study aimed to characterize the variation and genetic architecture of traits with nutritional and health relevance in 156 pea (Pisum sativum L.) accessions representing diverse geographic origins. The traits included total phenolic compounds (TPC), two saponins (Ssβg, Ss1), sucrose, three raffinose-family oligosaccharides (RFOs) and in vitro antioxidant activity (AA). Analysis of variance revealed significant effects of regional germplasm pools for all traits. Accessions from West Asia showed the highest TPC and AA levels, while those from the East Balkans and the UK displayed the lowest values. High saponin and RFO concentrations characterized accessions from Germany and the UK. Correlation and PCA analyses highlighted strong associations within compound classes and an overall negative relationship between TPC/AA and saponins/RFOs. Hierarchical clustering separated accessions into seven metabolically distinct groups partially reflecting their geographic origin. Linkage disequilibrium decayed rapidly (average 4.7 kb). GWAS with FarmCPU and BLINK identified 37 significant SNPs, 35 within annotated genes, associated with the metabolites. The polygenic genetic architecture supported the development of genomic prediction models, which showed moderately high predictive ability (> 0.40) for all traits except raffinose content. Our findings can support line selection and the identification of genetic resources with a desired level of secondary metabolites.

Abstract: Cnidium monnieri (L.) Cusson is a species of Umbelliferae plants, and it is one of China's traditional medicinal herbs, widely distributing in China owing to its strong adaptability in fields. In this article, the research progress on the taxonomy, distribution, cultivation techniques, active components and the analysis methods, antibacterial and insecticidal properties, and ecological applications of C. monnieri were reviewed. The main active components in C. monnieri are coumarins (mainly osthole) and volatile compounds, exhibiting multiple pharmacological activities, e.g., anti-inflammatory, antibacterial, antioxidant, anti-tumor, and immune-regulating effects. Some modern analytical techniques (e.g., HPLC, GC-MS, and UPLC-QTOF-MS) have enabled more precise detection and quality control of these chemical components in C. monnieri. The specific active constituents in C. monnieri (e.g., coumarins and volatile components) exhibite significant inhibitory effects against various pathogenic fungi and insect pests. Simultaneously, the resources provided during its flowering stage (e.g., pollen and nectar) and the specific volatiles it releases can attract natural enemies, such as ladybugs, lacewings, and hoverflies, thereby enhancing ecological control of insect pests in farmland through a "push-pull" strategy. Additionally, C. monnieri has the ability to accumulate heavy metals, e.g., Zn and Cu, indicating its potentially valuable for ecological restoration in agroecosystem. Overall, C. monnieri has medicinal, ecological, and economic values. Future research should focus on regulating the active component synthesis, understanding ecological mechanisms, and developing standardized cultivation systems to enhance its application in modernized traditional Chinese medicine and green agriculture production.

Abstract:

Urban forests serve as representations of nature within city landscapes. Green Forest, spanning 5,198,412 square meters, has been incorporated into the Municipality of Timișoara’s public domain and designated as a forest park. This fact increased green space per capita and enriched biodiversity within Timișoara’s landscape architecture. This study explores the diversity of Green Forest trees and highlights their contribution to urban landscapes. Statistical methods, including comparative and linear relationships analyses, were employed to assess significant variations in the dendrometric parameters of the analyzed tree species: mean tree height, mean diameter at breast height (DBH), tree age, and stand density. Principal Component Analysis (PCA) and cluster analysis were applied to uncover underlying patterns in the data. Using ArchiCAD and Lumion, high-quality 3D visual representations were developed for an ecological education area, an active recreation region, and a passive recreation area within Green Forest. Due to their morphological characteristics and phenotypic traits, the predominant tree species include Quercus robur, Quercus cerris, Quercus rubra, Fraxinus excelsior, Acer platanoides, Acer pseudoplatanus, Ulmus campestris and Robinia pseudoacacia, contribute to Timișoara’s urban aesthetic. Moreover, the results of the dendrometric analysis provide a foundation for further research in urban ecology. A key practical application of this study is landscape design renderings, which provide detailed and realistic visualizations to effectively communicate the design and functionality of Green Forest’s spaces. If implemented, these developments will encourage public engagement with nature, promoting mental and physical well-being within the community.