Salt tolerance of Casuarina equisetifolia and Frankia Ceq1 strain isolated from the root nodules of C. equisetifolia

Effects of NaCl on the seed germination and growth of Casuarina equisetifolia seedlings and multiplication of the Frankia Ceq1 strain isolated from the root nodules of C. equisetifolia were examined. The germination rate of the seeds markedly decreased as the NaCl concentration increased and germination did not occur at 300 mM NaCl. The fresh weight of both shoots and roots of the seedlings treated with NaCl for 6 weeks apparently decreased as the NaCl concentration increased. However, root nodules were formed by inoculation with the Frankia Ceq1 strain in some seedlings treated with 300 mM NaCl and the viability of the seedlings at 500 mM NaCl was almost the same as that of the seedlings not subjected to the NaCl treatment. The Na⁺ concentration in the shoots sharply increased with the elevation of the NaCl concentration in the ambient solution, but the level was approximately 300 mM even in the seedlings treated with 500 mM NaCl for 6 weeks. On the other hand, the increase of the Na⁺ concentration in the roots by the NaCl treatment was much smaller than that in the shoots and the level was less than 150 mM. The growth of the free-living Frankia Ceq1 strain was approximately linearly suppressed as the NaCl concentration in the medium increased and the hyphae became somewhat thicker and shorter or disintegrated in the medium containing NaCl at a concentration above 150 mM. The Na⁺ concentration in the cells increased as the NaCl concentration in the medium increased, but the level was maintained at less than 30 mM even in the medium containing 500 mM NaCl. The cells whose growth was suppressed by the NaCI treatment grew actively again at almost the same rate as the control cells (not subjected to the NaCl treatment) when they were transferred to NaCl-free medium. These results strongly suggested that both C. equisetifolia seedlings and Frankia Ceq1 strain are highly tolerant to salt and this symbiotic system is useful for the recovery of the vegetation in areas with severe salt accumulation.     

Formulation of humic substances coated fertilizer and its use to enhance K fertilizer use efficiency for tomato under greenhouse conditions

A greenhouse experiment was conducted at Land Resources Research Institute, NARC, Islamabad to examine the impact of humic substances (HSs) coating on potassium fertilizers use efficiency. Tomato variety “Rio Grande” was used. The treatments applied were T1= Control (N, P at 250 and100?mg kg^?1 respectively), T2?=?N, P?+?K at 200?mg kg^?1 as SOP, T3?=?N, P?+?K at 200?mg kg^?1 as NPK blend, T4?=?N, P?+?K at 200?mg kg^?1 HSs coated SOP) and T5?=?N, P?+?K at 200?mg kg^?1 HSs coated NPK blend. Results indicated a positive impact of sole and HSs coated products on agronomic traits, nutrient concentration, fruit quality traits, flower number, fruit umber, fruit weight, chlorophyll contents, fresh and dry biomass, tissue water contents, diameter and fruit mineral composition. The response of afore said traits to applied treatment varied.     

Formation of methyl mercaptan in paddy soils I

It is well known that methyl mercaptan is porduced by the microbiological decomposition of methionine1),2),3). According to Kondo ⁴⁾ and Onitake ¹⁾not only hydrogen sulfide, but also methyl mercaptan were produced from cystine by E. coli and Proteus vulgaris in the medium containing one of glucose, lactose, sucrose, glycerin or histidine. Moreover, Onitake ¹⁾ found that methyl mercaptan was produced by the action of E. coli in the medium containing hydrogen sulfide and a trace of ethyl alcohol, and that evolution of methyl mercaptan began only 5 minutes after the start of experiment in the medium containing methionine, but it began after 12hrs in the medium containing 1-cystine and glucose. According to Birkinshaw, Findlay and Webb⁵⁾ methyl mercaptan was found in the medium containing glucose, sulfate and other mineral salts, inoculated by Schizophyllum commune. In the same cultural condition as given above, methyl mercaptan, dimethyl sulfide, and dimethyl disulfide were detected by Challenger and Chartons ⁵⁾ from the data presented above, in addition to microbiological formation of methyl mercaptan from methionine, the possibility cannot be excluded of methyl mercaptan formation by microbes from cystine, sulfate or hydrogen sulfide in the medium containing one of organic compounds such as sugars, glycerin, histidine and ethyl alcohol, etc.     

Negative correlation between the ratio of K+ to Na+ and proline accumulation in tobacco suspension cells

Abstract

The concentrations of K⁺, Na⁺, and proline and the ratio of K⁺ to Na⁺ (K⁺ / Na⁺) were analyzed in NaCl-unadapted and NaCl-adapted tobacco (Nicotiana tabacum) cells in suspension culture. At 3 to 5 d after inoculation, the NaCl-unadapted cells cultured in 100 mmol L^?1 NaCl saline culture medium (Na100 medium) accumulated 28.7 mmol L^?1 proline with a low ratio of K⁺ to Na⁺ (= 2.8) and the NaCl-adapted cells cultured in the Na100 medium contained 6.28 mmol L^?1 proline with a high K⁺ / Na⁺ ratio (≧ 7.5). The contents of amino acids for the NaCl-adapted cells in the Na100 medium were similar to those for the NaCl-unadapted cells in a modified LS medium (standard medium). At 14 d after inoculation, the NaCl-unadapted cells in the Na100 medium contained 4.77 mmol L^?1 proline and restored the K⁺ / Na⁺ ratio from 2.8 to 6.2. These results indicate the presence of a negative correlation between the K⁺ / Na⁺ ratio and proline accumulation and suggest that a balance between the K⁺ / Na⁺ ratio and proline accumulation may be the factor involved in determining the salt tolerance of plant cells.     

Alleviation of manganese toxicity and manganese-induced iron deficiency in barley by additional potassium supply in nutrient solution

Barley plants were grown hydroponically at two levels of K (3.0 and 30 mm) and Fe (1.0 and 10 μm) in the presence of excess Mn (25 μm) for 14 d in a phytotron. Plants grown under adequate K level (3.0 mm) were characterized by brown spots on old leaves, desiccation of old leaves, interveinal chlorosis on young leaves, browning of roots, and release of phytosiderophores (PS) from roots. These symptoms were more pronounced in the plants grown under suboptimal Fe level (1.0 p,M) than in the plants grown under adequate Fe level (10 μm). Plants grown in 10 μm Fe with additional K (30 mm) produced a larger amount of dry matter and released less PS than the plants grown under adequate K level (3.0 mm), and did not show leaf injury symptoms and root browning. On the other hand, the additional K supply in the presence of 1.0 μM Fe decreased the severity of brown spots, prevented leaf desiccation, and increased the leaf chlorophyll content, which was not sufficient for the regreening of chlorotic leaves. These results suggested that the additional K alleviated the symptoms of Mn toxicity depending on the Fe concentration in the nutrient solution. The concentration (per g dry matter) and accumulation (per plant) of Mn in shoots and roots of plants grown in 10 μm Fe and 30 mm K were much lower than those of the plants grown in 10 μm Fe and 3.0 mm K, indicating that additional K repressed the absorption of Mn. The concentration and accumulation of Fe in the shoots and roots of the plants grown in 10 μm Fe and 30 mm K were higher than those of the plants grown in 10 μm Fe and 3.0 mm K, indicating that the additional K increased the absorption of Fe under excess Mn level in the nutrient solution. The release of PS, chlorophyll content, and shoot Fe concentration were closely correlated.     

Phosphate solubilising bacteria

Most reports of the studies on solubilisatioh of phosphate in liquid culture and soil suggested that organic acids produced by microorganisms are responsible for promoting the dissolution of phosphate. Sperber (11, 12), Louw and Webley (7) and Duff, Webley and Scott (2) studied the production of acids by cultures of some phosphate dissolving fungi. actinomycetes and bacteria and observed that among the acids produced in liquid medium, hydroxy carboxylic acids such as lactic and 2-ketogluconic acids were responsible for solubilisation of phosphates. In the present studies, apart from the culture of Bacillus megaterium var. phosphaticum isolated from phosphobacterin which had been obtained from USSR. Indian strains of Bacillus megaterium, Bacillus circulans and Escherichia freundii isolated and identified by Sundara Rao and Sinha (13) were used. These studies were undertaken to find out different types of nonvolatile acids produced in Pikovskaya's (9) liquid medium.     

Photoassimilate partitioning in hypernodulation mutant of soybean (Glycine max (L.) Merr.) NOD1-3 and its parent williams in relation to nitrate inhibition of nodule growth

Nodule growth of a hypernodulating soybean (Glycine max (L.) Merr.) mutant line NOD1-3 was compared to that of its wild-type parent cv. Williams from 14 to 18 days after planting (DAP) in the absence of nitrate treatment (hereafter referred to as “0 mM treatment”) or with 5 mM nitrate treatment. The growth rate determined by increase in the diameter of the nodules was relatively lower in the mutant NOD1-3 than that of the parent Williams under nitrogen-free conditions (0 mM nitrate). The inhibition of nodule growth by 5 mM nitrate started at 1 d after the onset of the nitrate treatment in Williams, while the inhibition did not occur before the application of the nitrate treatment for 2 d in NOD1-3. The nodule growth was completely inhibited after 2 d in Williams and after 3 d in NOD1-3 during the 5 mM nitrate treatment period. After 4 d of 5 mM nitrate treatment, the nodule dry weight decreased by 22% in NOD1-3 and by 58% in Williams, respectively. The treatment with 5 mM nitrate decreased the acetylene reduction activity (ARA) in NOD1-3 by 60% per plant and by 50% per nodule g DW and these parameters were less sensitive to the treatment than those in Williams in which the inhibition rate was 90% per plant and 80% per nodule g DW. These results indicate that NOD1-3 is partially nitrate-tolerant in terms of individual nodule growth as well as total nodule dry weight and Nz fixation activity. A whole shoot of Williams and NOD1-3 plants was exposed to ¹⁴CO₂ for 120 min followed by 0 or 5 mM nitrate treatment for 2 d, and the partitioning of the photoassimilates among the organs was analyzed. Under 0 mM nitrate treatment, the percentages of the distribution of ¹⁴C radioactivity between the nodules and roots were 63 and 37% in Williams and 89 and 11% in NOD1-3. Under the 5 mM nitrate conditions, the percentages of the distribution of ¹⁴C between the nodules and roots changed to 14 and 86% in Williams and 39 and 61% in NOD1-3, respectively. These results indicated that the hypernodulating mutant NOD1-3 supplied a larger amount of photoassimilates to the nodules than to the roots under nitrogen-free conditions, and that the nitrate depression of photoassimilate transport to the nodules was less sensitive than that of the parent line.     

Effect of physiological activities on aluminum uptake in carrot (Daucus carota L.) cells in suspension culture

Responses to aluminum (AI) of carrot (Daucus carota L.) cells in suspension culture (hereafter referred as “carrot suspension cells”) were investigated under different physiological activities. The respiration rate of the cells at the logarithmic growth phase decreased when the external Al concentration increased. A similar tendency was observed for the ATP content of the cells when the external Al concentration of the medium increased. Decrease of about 50% of the respiration rate and of about 35% of the ATP content of the cells was. observed at 300 µm AlCl₃ at 24 h, respectively. The Al content (total and citrate-insoluble AI) in cells increased when the external Al concentration increased. Both the respiration rate and the ATP content in cells were reduced when the culture temperature decreased or ATP synthesis inhibitors, namely CCCP (carbonylcyanide-m-chlorophenylhydrazone) and DNP (2,4-dinitrophenol) were added. The addition of SHAM (salicylhydroxamic acid) did not affect the ATP content in cells. The Al content of the cells treated with 50 µm Al together with SHAM was similar to that of the cells treated with only AI. However, the Al content of the cells increased when the cells were treated with 50 µm Al together with CCCP, DNP, or a low temperature (15°C). The content of citrate Al content in cells increased rapidly when ATP synthesis inhibitors were present or the culture temperature was low. There was a negative linear correlation between the Al content and the ATP content in cells. These results suggest that the cells containing a large amount of ATP and showing normal functions of the plasma membranes may display an apparent tolerance to Al by suppressing Al absorption into their cytoplasm at a cell suspension level.     

Amino acid metabolism in plant leaf

Recent studies have shown that the incorporation of ammonium nitrogen into amino acids in the leaves is strictly dependent on light (1-4). It is speculated that the effect of light on ammonium assimilation may be through the synthesis of the precursors of amino acids, or by the supply of the energy required for amination and amidation with organic acids. In the Vicia faba chloroplasts Givan et al. (1) exhibited that the synthesis of glutamic acid from a-ketoglutarate was linked with the generation of reduced pyridin nucleotide by photosynthetic electron transport. Mitchell and Stocking (2) suggested the direct coupling of glutamine formation with photophosphorylation in the pea chloroplasts. On the other hand. the processes of nitrate assimilation are more indebted to light than those of ammonium assimilation, because the former ones involve the reduction of nitrate to ammonium which is believed to be light-dependent (5). Canvin and Atkins (6). and Atkins and Canvin (7) reported that the incorporation of ¹⁵N-labeled ammonium and nitrate into amino acid fractiom was depressed by the dark treatment and by photosystem inhibitors; 3-(3′,4′-dichlrophenyl)-1-1-dimethylurea (DCMU) and carbonyl-cyanide-m-chlorophenyl-hydrazone(CCCP).     

Occurrence of 4-methyleneglutamine and 2-oxo-4-methyl-3-pentene-1,5-dioic acid in leaves and stem of tulip plants

Abstract

The presence of 4-MeGln in the tulip plant was discovered by ZACHARIUS et al. (1954), and it was found that this amide generally occurred in the leaves of almost all the species of genus Tulipa (FOWDEN and STEWARD 1957a). The 4-MeGln compound has been detected in every part of the tulip plant, i.e., bulb scales, roots, basal plate, young shoots, leaves, stern, and flower (FOWDEN and STEWARD 1957a, b; OHYAMA 1986; OHYAMA et al. 1985, 1988a, b; ZACHARIUS et al. 1954, 1957). Especially 4-MeGln was found to be a major soluble N constituent in the leaves and stem of tulip of the flowering stage (OHYAMA et al. 1985; OHYAMA 1986).     

Suitability of extractant for soil available silicon and silicon response to upland paddy grown on alkaline soils of central India

Abstract

Several silicon (Si) extractants are being employed in different countries mostly for lowland acidic soils. Present investigation was conducted to evaluate suitable extractants for upland paddy grown on alkaline soils. Available Si was extracted by using ten different extractants. Tris buffer pH 7.0 (1:10) in Inceptisols showed positively highest and significant correlation with grain yield (r?=?0.870), grain Si uptake (r?=?0.887), straw yield (r?=?0.852), and straw Si uptake (r?=?0.919). However, 0.5?M acetic acid (1:2.5) in Vertisols showed positively highest and significant correlation with grain yield (r?=?0.810), grain Si uptake (r?=?0.852), straw yield (r?=?0.850), and straw Si uptake (r?=?0.929). The application of Si @ 200?kg ha^?1 along with chemical fertilizers significantly increased yield and nutrient uptake of upland paddy on Vertisols. Tris buffer pH 7.0 (1:10) and 0.5?M acetic acid (1:2.5) were suitable extractant for Inceptisols and Vertisols, respectively based on its correlation with yield and nutrient uptake.     

Effect of nutrient deficiencies on the activities of glutamic acid decarboxylase in tobacco plant

It has been well known that the glutamic acid decarboxylase occured¹ and γ-aminobutylic acid was contained₂ in the higher plant. In the tobacco plant, the property of this enzyme was investigated in detail by Tomita ³, and Noguchi ⁴ showed. the comparatively higher concentration of γ-aminobutylic acid in tobacco leaves.     

Responses of Polygonatum odoratum seedlings in aeroponic culture to treatments of different ammonium: nitrate ratios

The objective was to determine the most favorable nitrogen (N) source ratio of ammonium (NH₄⁺) to nitrate (NO₃^?) for aeroponic culture of Chinese fragrant solomonseal Polygonatum odoratum (Mill.) Druce seedlings. Seedlings were cultured with solutions based on 50% Hoagland formula containing 0:100, 10:90, 20:80, and 30:70 NH₄⁺:NO₃^? ratios for 21 days. Activities of anti-oxidant enzymes and glutathione contents of leaves with treatments of 10:90 and 20:80 NH₄⁺:NO₃^? ratios were higher than that of all-nitrate treatment, and malondialdehyde (MDA) concentrations were lower than that of all-nitrate treatment. The maximum quantum yield ?P_o value was normal among all of the treatments, whereas the absorption energy ABS/CSm value with 20:80 NH₄⁺:NO₃^? treatment was highest and the heat dissipation DIo/CSm value with 20:80 NH₄⁺:NO₃^? treatment was lowest among all of the treatments. These results supported that moderate proportion of 20% NH₄⁺ in the solution provided optimal growth condition for (P) P. odoratum aeroponic culture.     

Rapid and reversible nitrate inhibition of nodule growth and N2 fixation activity in soybean (Glycine max (L.) Merr.)

Nodulated soybean (Glycine max. (L) Merr. cv. Williams) plants were hydroponically cultured, and various combinations of 1-week culture with 5 or 0 mm nitrate were applied using 13-d-old soybean seedlings during three successive weeks. The treatments were designated as 0-0-0, 5-5-5, 5-5-0, 5-0-0, 5-0-5, 0-5-5, and 0-0-5, where the three sequential numbers denote the nitrate concentration (mm) applied in the first-second-third weeks. The size of the individual nodule was measured periodically using a slide caliper. All the plants were harvested after measurement of the acetylene reduction activity (ARA) at the end of the treatments. In the 0-0-0 treatment, the nodules grew continuously during the treatment period. Individual nodule growth was immediately suppressed after 5 mm nitrate supply. However, the nodule growth rapidly recovered by changing the 5 mm nitrate solution to a 0 mm nitrate solution in the 5-0-0 and 5-5-0 treatments. In the 5-0-5 treatment, nodule growth was completely inhibited in the first and the third weeks with 5 mm nitrate, but the nodule growth was enhanced in the second week with 0 mm nitrate. The nodule growth response to 5 mm nitrate was similar between small and large size nodules. After the 5-5-5, 5-0-5, 0-0-5, and 0-5-5 treatments, where the plants were cultured with 5 mm nitrate in the last third week, the ARA per plant was significantly lower compared with the 0-0-0 treatment. On the other hand, the ARA after the 5-0-0 and 5-5-0 treatments was relatively higher than that after the 0-0-0 treatment, possibly due to the higher photosynthate supply associated with the vigorous vegetative growth of the plants supplemented with nitrate nitrogen. It is concluded that both soybean nodule growth and N₂ fixation activity sensitively responded to the external nitrate level, and that these parameters were reversibly regulated by the current status of nitrate in the culture solution, possibly through sensing of the nitrate concentration in roots and / or nodules.     

H2 15O translocation in rice was enhanced by 10 μm 5-aminolevulinic acid as monitored by positron emitting tracer imaging system (PETIS)

5-Aminolevulinic acid (ALA) acts to increase chlorophyll biosynthesis, photosynthesis, cold stress tolerance, and salt tolerance at low concentrations. We studied the effects of ALA on H₂ ¹⁵O translocation from the roots to the shoots of rice plants (Oryza sativa L. cv. Nipponbare) in real time by a positron-emitting tracer imaging system (PETIS). When the plant was treated with 10 μm ALA, the velocity of the H₂ ¹⁵O translocation from 2 to 12 min after absorption increased to 126, 137, 140% that of the control at 1.5, 2.5, and 3.5 h after ALA treatment, respectively. However, ALA did not affect the H₂ ¹⁵O translocation within 0.5 h of treatment. When the plant was treated with 0.1 mM ABA at 4 h after 10 μm ALA treatment, the velocity of the H₂ ¹⁵O translocation decreased at 0.5 h after ABA treatment. Those observations suggested ALA might be absorbed and transported to the guard cells within 1.5 h and functioned to expand the stomatal aperture.     

Fractionation of foliar absorbed phosphorus by paper electro-phoresis

Fractional analysis of phosphorus compounds in plant tissue has been carried out by many workers, and reported on tobacco plant by Holden (1) and Komatsu (2). The fractional distribution of foliar absorbed phosphorus was investigated by Yatazawa (3) who indicated that the phosphorus was first converted into the phosphate-ester as an intermediate substance on the phosphorus metabolism. The methods of fractional analysis used by these authors were based on the method proposed by Page and Umbereit (4) in which they used trichloroacetic acid as the first extracting reagent. Recently, in order to separate the protein bound phosphorus, Wildmann, Campell and Bonner (5) adopted the salting-out method without using trichloroacetic acid. They reported that the protein-bound phosphorus plays an important role and is in the dynamic state. Fujiwara and Kadowaki (6) paid attention to the phosphorus bound to cytoplasmic protein as energy-rich phosphate compounds, and adopted the method so as to separate such compounds. In order to reveal the utilization of foliar absorbed phosphorus by the tobacco plant, the fractional distribution of phosphorus was examined. In this paper, the degree of change to organic phosphorus was taken as an index of phosphorus utilization. Paper electrophoresis was used for this purpose because the inorganic and the protein-bound phosphorus could be separated easily.     

Amino acid composition of rice callus tissue grown with different kinds of nitrogen sources

There have been several papers dealing with the difference in chemical composition between callus tissue and normal parent tissue. WEINSTEIN, TULECKE, NICKELL, and LAURENCOT (1–3) revealed, in a series of papers, that the contents of amino acids, sugars, and nucleic acids often differed strikingly between callus and normal tissue of Agave toumeyana Trel. (1), Ginkgo biloba, L. (2), and PAUL's scarlet rose (3). STEWARD, THOMPSON, and POLLARD (4) also reported that the content of some amino acids of rapidly growing and randomly proliferating tissue is outstandingly different from that of normal tissue.     

Interaction effect of nitrogen,phosphorus, and zinc fertilization on growth,yield, and nutrient contents of aromatic rice varieties

Abstract

Studies on nutrient interactions in aromatic rice are needed for proper understanding of impact of imbalanced use of nutrients in the era of multi and micro-nutrient deficiencies. A pot experiment was conducted during the rainy/wet season (June–October) of 2013 at New Delhi, to study the interaction effects of two levels each of nitrogen (N) (0 and 120?kg?ha^?1), phosphorus (P; 0 and 25.8?kg?ha^?1), and zinc (Zn; 0 and 5?kg?ha^?1) in two aromatic rice (Oryza sativa L.) varieties, viz. Pusa Rice Hybrid 10 and Pusa Basmati 1121. Application of N, P, and Zn resulted in increase of dry matter (0.91, 0.32, and 0.24?g plant^?1, respectively) 60?days after sowing (DAS) and grain yield of rice (3.68, 1.67, and 1.17?g plant^?1). The increase in yield of rice owing to N application was relatively higher by 0.98, 0.22, and 1.05?g plant^?1, respectively, when either P or Zn or both were applied with N than alone application of N, indicating synergetic effect of P and Zn application with N. The higher concentration and uptake of K in grain (0.25% vs 0.10%) and straw (1.32% vs 0.94%) were observed in the treatment received N than no N, though K was applied uniformly in all the treatments. It indicates positive interaction of N and K. The higher uptake of P in grain and straw was observed when P was applied with N and Zn (3.34 and 2.15?mg plant^?1), or with N (3.26 and 2.11?mg plant^?1) signifying positive effect of N on P uptake in rice.     

Adsorption strength of zinc for soil humus

Soil humus plays a significant role in the cation exchange of a soil. YOSHIDA (1) showed that, as a general rule, divalent ions such as calcium and magnesium were adsorbed more strongly onto humus than monovalent ions such as ammonium and potassium in an ion-exchange reaction. He did not, however, describe the behavior of heavy metal ions. BREMNER et al. (2) first suggested that soil organic matter forms complexes with polyvalent cations. HIMES and BARBER (3) found that soil organic matter reacts with divalent metal ions in a manner similar to the chelation reaction. Reviews of the soil organic matter-metal complex have been written by BREMNER et al. (2) and KAWAGUCHI, MATSUO and KYUMA (4).     

Isolation and characterization of a novel Arabidopsis thatiana mutant that requires a high concentration of boron

We isolated a novel Arabidopsis thaliana mutant line that requires high levels of boron (B) for normal growth. Line 8–21 was identified from ethyl methanesulfonate-mutagenized M2 population. When grown in medium containing 3 μm or less B in the form of boric acid, the fresh weights of aerial portions of the mutant were about a half of those of the wild type, but in that containing 300 μm B, the growth appeared normal. The mutant plants did not shown any difference in root growth from the wild-type plants in the range of B concentrations tested. When grown with 30 μm B, the B concentration in shoots of the line 8–21 was similar to that in the wild-type, suggesting that the mutant could not utilize B efficiently. Line 8–21 was not allelic to bor1-1 (Noguchi et al., Plant Physiol., 115, 901–906, 1997). A significant portion of F2 plants from the crosses between the wild-type and the mutant grew poorly on a low B media, suggesting segregation of the mutation.