Temporal Changes in Biochemical Responses to Salt Stress in Three Salicornia Species.

Halophytes adapt to salinity using different biochemical response mechanisms. Temporal measurements of biochemical parameters over a period of exposure to salinity may clarify the patterns and kinetics of stress responses in halophytes. This study aimed to evaluate short-term temporal changes in shoot biomass and several biochemical variables, including the contents of photosynthetic pigments, ions (Na+, K+, Ca2+, and Mg2+), osmolytes (proline and glycine betaine), oxidative stress markers (H2O2 and malondialdehyde), and antioxidant enzymes (superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase) activities of three halophytic Salicornia species (S. persica, S. europaea, and S. bigelovii) in response to non-saline, moderate (300 mM NaCl), and high (500 mM NaCl) salinity treatments at three sampling times. Salicornia plants showed maximum shoot biomass under moderate salinity conditions. The results indicated that high Na+ accumulation in the shoots, coupled with the relative retention of K+ and Ca2+ under salt stress conditions, contributed significantly to ionic and osmotic balance and salinity tolerance in the tested Salicornia species. Glycine betaine accumulation, both constitutive and salt-induced, also seems to play a crucial role in osmotic adjustment in Salicornia plants subjected to salinity treatments. Salicornia species possess an efficient antioxidant enzyme system that largely relies on the ascorbate peroxidase and peroxidase activities to partly counteract salt-induced oxidative stress. The results also revealed that S. persica exhibited higher salinity tolerance than S. europaea and S. bigelovii, as shown by better plant growth under moderate and high salinity. This higher tolerance was associated with higher peroxidase activities and increased glycine betaine and proline accumulation in S. persica. Taking all the data together, this study allowed the identification of the biochemical mechanisms contributing significantly to salinity tolerance of Salicornia through the maintenance of ion and osmotic homeostasis and protection against oxidative stress.

Nano-Regulation of Gene Expression in Chlamydomonas reinhardtii: Harnessing AuNPs for Remotely Switchable Lipid Biosynthesis via Antisense Oligonucleotides.

Antisense oligonucleotide (ASO)-mediated gene silencing has broad applications, spanning from biomedicine to agriculture, involving molecular biology, synthetic biology, and genetic manipulation. This research harnessed nanotechnology to augment ASO-mediated gene silencing, introducing a remotely switchable gene expression system for precise temporal control. We targeted lipid biosynthesis and accumulation enhancement in the photosynthetic eukaryote Chlamydomonas reinhardtii. Gold nanoparticles (AuNPs) transported double-stranded DNA (dsDNA), forming dsDNA-AuNP complexes. These complexes comprised 3'-thiolated sense strands attached to AuNPs and fluorescent antisense oligonucleotides. To avoid harmful laser effects on cells, we adopted a light-emitting diode (LED). Confocal microscopy confirmed dsDNA-AuNP internalization in C. reinhardtii. LED-triggered antisense release led to an 83% decrease in Citrate Synthase 2 (CIS 2) expression. Thiolated sense strand attachment postillumination inhibited antisense reannealing, enhancing gene silencing. This led to significant lipid body accumulation in cells, verified through fluorometric and fluorescence microscopy. This union of nanotechnology and ASO-mediated silencing provides gene regulation opportunities across sectors like biomedicine and agriculture. The system's remote switching capability underscores its potential in synthetic biology and genetic engineering. Our findings substantiate the utility of this approach for enhancing lipid biosynthesis in C. reinhardtii but also underscores its broader applicability to other organisms, fostering the development of novel solutions for pressing global challenges in energy, agriculture, and healthcare.

Effects of drought stress and plant cultivar type on demographic characteristics of the rose-grain aphid, Metopolophium dirhodum (Hemiptera: Aphididae).

Drought is a substantial threat to cereal production under global climatic change scenarios, albeit its aftermath on arthropod pests is yet contentious. To address this issue, demographic characteristics of Metopolophium dirhodum (Walker, 1849) (Hemiptera: Aphididae) were studied on one drought-susceptible wheat cultivar and one drought-tolerant wheat cultivar under different water treatments. Some physiological and biochemical features of wheat cultivars including leaf soluble sugar and proline contents and antioxidant enzymes activities were also investigated. Significant differences occurred in the developmental period, survival, and fecundity of M. dirhodum between wheat cultivars under various water treatments. The impact of intermediate and severe water stress on M. dirhodum was neutral and negative for the tolerant cultivar and negative for the water-susceptible cultivar, respectively. Under severe water stress, on both wheat cultivars, the aphids had low net reproductive rates and finite and intrinsic rates of increase in comparison with those reared on unstressed plants. In total, drought resulted in lower growth of population and reduced survival of aphids. Hence, in the context of projected climatic changes, acute water deficiency could probably result in reducing the abundance and menace of outburst of M. dirhodum. However, it should be noted that the potential likelihood of M. dirhodum eruptions can be drastically affected by the degree of drought intensity and host plant cultivar.

Optimized gamma radiation produces physiological and morphological changes that improve seed yield in wheat.

To study the effect of gamma radiation on various morphological and agronomic characters of bread wheat (Triticum aestivum L.), seeds were subjected to different gamma radiation doses; and selected M5 and M6 generation lines were evaluated. The optimum doses to induce desirable changes in bread wheat were 100-200 Gy. Seed loss decreased while grain yield, yield components, fertile florets number, biological yield, plant height, harvest index and flag leaf area increased in all mutant lines. Shear strength increased in many lines. Selected mutant lines also showed reduced seed shattering that can greatly reduce seed loss at harvest. Some new phenotypic characters such as the appearance of bristles on the glume, important for drought tolerance, two spikelets at each rachis and more fertile florets at each spikelet. These can greatly increase yield, as seen in some mutant lines. Also, some physiological characteristics including photosynthetic rate, stomatal conductance, water use efficiency, and chlorophyll content improved in some mutant lines. About 95.8% of the total variation in grain yield was explained by three selected variables: flag leaf area, number of seeds per spike, and spike number per plant. Grain yield increased more than 45% in some mutant lines the highest ever reported using this approach to the genetic improvement of wheat. Wheat grain yield has increased 2.2 times in the last 50 years, which indicates that if mutagens are optimally used and the selection is carefully performed as described herein, it is possible to improve important economic traits, in a much shorter time. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01225-0.

Bioinformatics approaches for classification and investigation of the evolution of the Na/K-ATPase alpha-subunit.

BACKGROUND: Na,K-ATPase is a key protein in maintaining membrane potential that has numerous additional cellular functions. Its catalytic subunit (α), found in a wide range of organisms from prokaryotes to complex eukaryote. Several studies have been done to identify the functions as well as determining the evolutionary relationships of the α-subunit. However, a survey of a larger collection of protein sequences according to sequences similarity and their attributes is very important in revealing deeper evolutionary relationships and identifying specific amino acid differences among evolutionary groups that may have a functional role. RESULTS: In this study, 753 protein sequences using phylogenetic tree classification resulted in four groups: prokaryotes (I), fungi and various kinds of Protista and some invertebrates (II), the main group of invertebrates (III), and vertebrates (IV) that was consisted with species tree. The percent of sequences that acquired a specific motif for the α/ß subunit assembly increased from group I to group IV. The vertebrate sequences were divided into four groups according to isoforms with each group conforming to the evolutionary path of vertebrates from fish to tetrapods. Data mining was used to identify the most effective attributes in classification of sequences. Using 1252 attributes extracted from the sequences, the decision tree classified them in five groups: Protista, prokaryotes, fungi, invertebrates and vertebrates. Also, vertebrates were divided into four subgroups (isoforms). Generally, the count of different dipeptides and amino acid ratios were the most significant attributes for grouping. Using alignment of sequences identified the effective position of the respective dipeptides in the separation of the groups. So that 208GC is apparently involved in the separation of vertebrates from the four other organism groups, and 41DH, 431FK, and 451KC were involved in separation vertebrate isoform types. CONCLUSION: The application of phylogenetic and decision tree analysis for Na,K-ATPase, provides a better understanding of the evolutionary changes according to the amino acid sequence and its related properties that could lead to the identification of effective attributes in the separation of sequences in different groups of phylogenetic tree. In this study, key evolution-related dipeptides are identified which can guide future experimental studies.

Metabolic and genes expression analyses involved in proline metabolism of two rose species under drought stress.

An investigation was conducted to assess proline anabolism and catabolism pathway genes under drought stress. Treatments were irrigation in three levels (25, 50 and 100% field capacity) at 1, 3, 6 and 12 d and two rose species (Rosa canina L. and Rosa damascene Mill.). The results showed that the potential for proline accumulation in R. damascena was higher than R. canina under drought. Simultaneous with proline accumulation, expression of P5CS and P5CR genes increased from 1 to 12 d under 50% FC whereas their expression had an increasing trends from 1 to 6 d at 25% FC and expression of both genes decreased at 12 d in both species. The highest accumulation of proline was observed under 25% FC at 12 d, but expression of genes involved in proline synthesis main pathway decreased on this day. Furthermore, expression of genes (PDH and P5CDH) involved in proline catabolism pathway decreased in 50% FC from 1 to 12 d while their expression remarkably decreased from 1 to 6 d and increased at 12 d under 25% FC. These findings showed that under conditions of 50 and 25% FC, arginine accumulation resulted in the increased expression of the ARG gene, which led to ornithine production. Furthermore, ornithine accumulation increased OAT expression. Therefore, it seems that OAT-induced P5C is transported from the mitochondria to the cytosol and reduced to proline by the P5CR.

Regulation of stomatal aperture in response to drought stress mediating with polyamines, nitric oxide synthase and hydrogen peroxide in Rosa canina L.

To assess the role of genes involved in polyamines synthesis, nitric oxide synthase (NOS), copper amine oxidase activity (CuAO) and hydrogen peroxide (H2O2) in regulation of stomatal aperture to drought stress in Rosa canina L., a study was performed at three irrigating levels (25%, 50%, and 100% field capacity) with three replications at 1, 3, 6 and 12 days. The results showed that putrescine (Put) accumulation occurred under both 50% and 25% FC at 1 d. Furthermore, the role of the Put direct biosynthesis pathway ornithine decarboxylase (ODC) was more effective under 50% FC whereas in the 25% FC the Put indirect production pathway (agmatine iminohydrolase (AIH), N-carbamoyl putrescine amidohydrolase (CPA) and arginine decarboxylase (ADC)) was more effective. HPLC results showed that the accumulation of spermidine (Spd) and spermine (Spm) is consistent with the expression of S-adenosyl methionine decarboxylase (SAMDC), spermidine synthase (SPDS) and spermine synthase (SPMS) genes. Spd accumulation under both 50% and 25% FC occurred on the 3 d and then decreased in the other days. Spm content showed an increasing trend from 6 d under 50% FC and from 3 d under 25% FC. Our results suggest that among the measured polyamines, Put oxidation through CuAO activity increased resulted in an increase in H2O2 production. The H2O2 accumulation also as a secondary messenger led to enhance in NOS gene expression. Increase in NOS gene expression can act as a signal resulting in stomatal closure.

Genome-wide analysis of AP2/ERF transcription factors family in Brassica napus.

The AP2/ERF transcription factor family plays an important role in different biological processes such as growth, development and response to abiotic and biotic stresses in plants. The genome-wide analysis identified 531 AP2/ERF genes in Brassica napus (oilseed rape or canola) that ranged from 333 to 6440 bp in genomic and 273-2493 bp in coding DNA sequence length. We classified BnAP2/ERF proteins into five subfamilies including AP2 (58 genes), ERF (250 genes), DREB/CBF (194 genes), RAV (26 genes), and Soloist (3 genes). Furthermore, AP2/ERF proteins were subdivided into 15 groups according to the AP2/ERF classification in Arabidopsis. The number of exons in BnAP2/ERF genes was from one to eleven and most of these genes in the same subfamily had the same exon-intron pattern. The results also indicated that the composition of conserved motifs in most proteins in each group was similar. The intron-exon patterns and the composition of conserved motifs validated the BnAP2/ERF transcription factors phylogenetic classification. Based on the results of genome distribution, BnAP2/ERF genes were located unevenly on the 19 B. napus chromosomes. The results indicated that gene duplication may play an important role in genome expansion of B. napus. Furthermore, genome evolution of B. napus using orthologous and paralogous identification was studied. We found 278, 380 and 366 orthologous gene pairs between B. napus with A. thaliana, B. rapa and B. oleracea, respectively. The results of this study will be useful in investigation of functional role and molecular mechanisms of BnAP2/ERF transcription factors genes in response to different stresses.

Role of genes and metabolites involved in polyamines synthesis pathways and nitric oxide synthase in stomatal closure on Rosa damascena Mill. under drought stress.

In order to evaluate the genes involved in polyamines synthesis pathway and the role of nitric oxide synthase (NOS) and H2O2 in stomatal closure under drought stress, a research conducted with three irrigation levels (100, 50 and 25% field capacity) at 1, 3, 6 and 12 days on Rosa damascena Mill. HPLC and qPCR results showed that putrescine (Put) accumulation occurred at first day in both 50 and 25% of field capacity and then decreased the other days. Furthermore, Put accumulation in the indirect pathway (ADC, AIH and CPA) was more effective related to the direct pathway (ODC) under severe stress. Increased expression of genes involved in production of spermidine (Spd) and spermine (Spm) i.e., SAMDC, SPDS and SPMS correlated with the highest accumulation of Spd and Spm under 50% FC at 6 d and 25% FC at 12 d, respectively. Moreover, results showed that Put reduction simultaneously accumulated H2O2, which subsequently increased NOS expression suggesting a key signal for stomatal closure.

Microarray analysis of transcriptional responses to salt and drought stress in Arabidopsis thaliana.

Microarray expression profile analysis is a useful approach to increase our knowledge about genes involved in regulatory networks and signal transduction pathways related to abiotic stress tolerance. Salt and drought, as two important abiotic stresses, adversely affect plant productivity in the world every year. To understand stress response mechanisms and identify genes and proteins which play critical roles in these mechanisms, the study of individual genes and proteins cannot be considered as an effective approach. On the other hand, the availability of new global data provides us an effective way to shed some light on the central role of molecules involved in stress response mechanisms in the plant. A meta-analysis of salt and drought stress responses was carried out using 38 samples of different experiments from leaves and roots of Arabidopsis plants exposed to drought and salt stresses. We figured out the number of differentially expressed genes (DEGs) was higher in roots under both stresses. Also, we found that the number of common DEGs under both stresses was more in roots and also the number of common DEGs in both tissues under salt stress was more than drought stress. The highest percent of DEGs was related to cell and cell part (about 87%). Around 9% and 7% of DEGs in roots and leaves encoded transcription factors, respectively. Network analysis revealed that three transcription factor families HSF, AP2/ERF and C2H2, may have critical roles in salt and drought stress response mechanisms in Arabidopsis â€‹and some proteins like STZ may be introduced as a new candidate gene for enhancing salt and drought tolerance in crop plants.

Hydro-thermal priming enhance seed germination capacity and seedling growth in sugar beet.

Seed priming improves seed performance in many crop species. In this study, the influence of hydrothermal priming on seed parameters of sugar beet is investigated in both laboratory and field conditions. In the laboratory, the treatments consist of a combination of cultivars (Arya and Shokoofa), hydro-priming at two temperatures (10 and 15 °C) for 6, 10, 14, 18, 22 hours. Germination traits and seedling growth were measured for determination of optimum hydro-thermal priming. Also, the protein pattern in the optimum hydro-thermal priming treatments and unprimed seeds were compared by electrophoresis. In the field experiment, the percentage and rate of emergence of primed and unprimed seeds were measured. Results showed that hydro-thermal priming had a positive effect on final germination percentage, mean germination time and uniformity of germination. Optimum hydro-thermal priming time and the temperature were 6 and 10 hours at 15 °C for Shokoofa and Arya cultivars respectively. Hydro-thermal priming increased the seed emergence percentage in the field by 15%. There was no significant difference in protein pattern between primed and unprimed seeds. In general, hydro-thermal priming not only increases sugar beet seed germination in the laboratory but also has a more positive effect on the emergence in the field condition.

Meta-analysis of transcriptomic responses to biotic and abiotic stress in tomato.

A wide range of biotic stresses (BS) and abiotic stresses (AS) adversely affect plant growth and productivity worldwide. The study of individual genes cannot be considered as an effective approach for the understanding of tolerance mechanisms, since these stresses are frequent and often in combination with each other, and a large number of genes are involved in these mechanisms. The availability of high-throughput genomic data has enabled the discovery of the role of transcription factors (TFs) in regulatory networks. A meta-analysis of BS and AS responses was performed by analyzing a total of 391 microarray samples from 23 different experiments and 2,336 differentially expressed genes (DEGs) involved in multiple stresses were identified. We identified 1,862 genes differentially regulated in response to BS was much greater than that regulated by AS, 835 genes, and found 15.4% or 361 DEGs with the conserved expression between AS and BS. The greatest percent of genes related to the cellular process (>76% genes), metabolic process (>76% genes) and response to stimulus (>50%). About 4.2% of genes involved in BS and AS responses belonged to the TF families. We identified several genes, which encode TFs that play an important role in AS and BS responses. These proteins included Jasmonate Ethylene Response Factor 1 (JERF1), SlGRAS6, MYB48, SlERF4, EIL2, protein LATE ELONGATED HYPOCOTYL (LHY), SlERF1, WRKY 26, basic leucine zipper TF, inducer of CBF expression 1-like, pti6, EIL3 and WRKY 11. Six of these proteins, JERF1, MYB48, protein LHY, EIL3, EIL2 and SlGRAS6, play central roles in these mechanisms. This research promoted a new approach to clarify the expression profiles of various genes under different conditions in plants, detected common genes from differentially regulated in response to these conditions and introduced them as candidate genes for improving plant tolerance through genetic engineering approach.

Molecular characterization of Brassica napus stress related transcription factors, BnMYB44 and BnVIP1, selected based on comparative analysis of Arabidopsis thaliana and Eutrema salsugineum transcriptomes.

Many studies have been performed to identify regulatory circuit underlying plant stress tolerance. However, the reliability of some findings has been criticized because of exclusive use of stress sensitive plant species such as Arabidopsis thaliana. Sensitive plant species often harbor narrow defensive mechanisms and have relatively low capacity for adaptive responses. Therefore, it is useful to employ tolerant model plants, such as Eutrema salsugineum, to provide comprehensive insights into various mechanisms involved in response to abiotic stresses. In this study, comparative transcriptome and regulatory network analysis of stress-sensitive (A. thaliana) and -tolerant (E. salsugineum) model plants uncovered regulatory hierarchies underlying response to abiotic stresses and suggested the transcription factor genes, MYB44 and VIP1 as the candidate hub genes to perform molecular analyses on their Brassica napus homologs, BnMYB44 and BnVIP1. The full-length coding sequence of BnMYB44 and BnVIP1 with 891 and 969 bp long were cloned and sequenced. They shared high similarity with their counterparts in other plants at nucleotide and amino acid levels. The expression patterns of BnMYB44 and BnVIP1 genes of the two B. napus cultivars under drought and salt stress conditions coupled with the data obtained from the physiological measurements as well as analysis of the BnMYB44 and BnVIP1 promoters suggested that BnMYB44 and BnVIP1 genes may contribute to responses to drought and salt stresses in B. napus.

Microsatellite markers for the Triticum timopheevi-derived leaf rust resistance gene Lr18 on wheat 5BL chromosome.

Leaf rust, caused by Puccinia triticina, is a common wheat disease worldwide. Developing resistant cultivars through deploying new or pyramiding resistance genes in a suitable line, is the most effective approach to control this disease. However, to stack genes in a genotype, efficient and reliable markers are required. In the present study, F2 plants and their corresponding F3 families from a cross between the resistant line; Thatcher (Tc) Lr18, and the susceptible cultivar 'Boolani' were used to map rust resistance gene, Lr18 using SSR markers on chromosome 5BL of hexaploid wheat. The P. triticina pathotype no 15 was used to inoculate plants. Out of 20 primers tested, eight showed polymorphism between the two parents and were subsequently genotyped in the entire F2 population. The markers Xgpw7425 and Xwmc75 flanked the locus at a distance of 0.3 and 1.2 cM, respectively. Analysis of 81 genotypes from different backgrounds with these two markers confirmed their usefulness in screening absence or presence of Lr18. Therefore, these markers can be used for gene postulation and marker-assisted selection (MAS) of this gene in wheat breeding programs in future.

Mining expressed sequence tags of rapeseed (Brassica napus L.) to predict the drought responsive regulatory network.

It is of great significance to understand the regulatory mechanisms by which plants deal with drought stress. Two EST libraries derived from rapeseed (Brassica napus) leaves in non-stressed and drought stress conditions were analyzed in order to obtain the transcriptomic landscape of drought-exposed B. napus plants, and also to identify and characterize significant drought responsive regulatory genes and microRNAs. The functional ontology analysis revealed a substantial shift in the B. napus transcriptome to govern cellular drought responsiveness via different stress-activated mechanisms. The activity of transcription factor and protein kinase modules generally increased in response to drought stress. The 26 regulatory genes consisting of 17 transcription factor genes, eight protein kinase genes and one protein phosphatase gene were identified showing significant alterations in their expressions in response to drought stress. We also found the six microRNAs which were differentially expressed during drought stress supporting the involvement of a post-transcriptional level of regulation for B. napus drought response. The drought responsive regulatory network shed light on the significance of some regulatory components involved in biosynthesis and signaling of various plant hormones (abscisic acid, auxin and brassinosteroids), ubiquitin proteasome system, and signaling through Reactive Oxygen Species (ROS). Our findings suggested a complex and multi-level regulatory system modulating response to drought stress in B. napus.

A novel pairwise comparison method for in silico discovery of statistically significant cis-regulatory elements in eukaryotic promoter regions: application to Arabidopsis.

Cis regulatory elements (CREs), located within promoter regions, play a significant role in the blueprint for transcriptional regulation of genes. There is a growing interest to study the combinatorial nature of CREs including presence or absence of CREs, the number of occurrences of each CRE, as well as of their order and location relative to their target genes. Comparative promoter analysis has been shown to be a reliable strategy to test the significance of each component of promoter architecture. However, it remains unclear what level of difference in the number of occurrences of each CRE is of statistical significance in order to explain different expression patterns of two genes. In this study, we present a novel statistical approach for pairwise comparison of promoters of Arabidopsis genes in the context of number of occurrences of each CRE within the promoters. First, using the sample of 1000 Arabidopsis promoters, the results of the goodness of fit test and non-parametric analysis revealed that the number of occurrences of CREs in a promoter sequence is Poisson distributed. As a promoter sequence contained functional and non-functional CREs, we addressed the issue of the statistical distribution of functional CREs by analyzing the ChIP-seq datasets. The results showed that the number of occurrences of functional CREs over the genomic regions was determined as being Poisson distributed. In accordance with the obtained distribution of CREs occurrences, we suggested the Audic and Claverie (AC) test to compare two promoters based on the number of occurrences for the CREs. Superiority of the AC test over Chi-square (2×2) and Fisher's exact tests was also shown, as the AC test was able to detect a higher number of significant CREs. The two case studies on the Arabidopsis genes were performed in order to biologically verify the pairwise test for promoter comparison. Consequently, a number of CREs with significantly different occurrences was identified between the promoters. The results of the pairwise comparative analysis together with the expression data for the studied genes revealed the biological significance of the identified CREs.

Differential expression of BnSRK2D gene in two Brassica napus cultivars under water deficit stress.

The sucrose non-fermenting 1-related protein kinase 2 (SnRK2) family members are plant unique serine/threonine kinases which play a key role in cellular signaling in response to abiotic stresses. The three SnRK2 members including SRK2D, SRK2I and SRK2E are known to phosphorylate major abscisic acid (ABA) responsive transcription factors, ABF2 and ABF4, involved in an ABA-dependent stress signaling pathway in Arabidopsis. This study aimed to clone and sequence an ortholog of the Arabidopsis SRK2D gene from Brassica napus, designated as BnSRK2D. An 833bp cDNA fragment of BnSRK2D, which shared high amino acid sequence identity with its Arabidopsis counterpart, was obtained suggesting a possible conserved function for these genes. The expression pattern of BnSRK2D and its potential target gene B. napus ABF2 (BnABF2) were then analyzed in the two cultivars with contrasting reaction to water deficit stress. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) showed that BnSRK2D and BnABF2 were water-deficit stress responsive genes with similar expression profiles. The accumulation of the BnSRK2D and BnABF2 transcripts in the two cultivars was linked with their level of drought tolerance, as the drought tolerant cultivar had significantly higher expression levels of both genes under normal and water deficit stress conditions. These findings suggest that BnSRK2D and BnABF2 genes may be involved in conferring drought tolerance in B. napus.