Relationship between cavitation and water uptake in rose stems

Cavitation in rose stems ( Rosa hybrida L.) was assessed in both intact plants and excised flowers, by measurement of ultrasonic acoustic emissions at the stem surface and determination of the air-conductivity of 2.5-cm segments that were attached at one end to air at low pressure (0.01 MPa). On sunny days the stems of intact rose plants showed acoustic emissions and conductivity to air, starting early in the morning. In Cara Mia and Sonia rose plants the cavitations were repaired during the late afternoon; in Madelon plants this repair only occurred overnight. Water flow was seriously impaired in stems of Cara Mia roses cut around midday, on sunny days.
During dehydration of cut roses in air the onset of a high rate of acoustic emissions coincided with a low rate of water uptake when stems were subsequently placed in water. High emission frequency occurred after 2.4 ± 0.7 h, 6.8 ± 4.3 h and 19.8 ± 9.0 h of exposure to air in Cara Mia, Madelon and Sonia roses, respectively. A low rate of water uptake in excised stems placed in water was found after 3–4, 9–12 and 24–36 h of desiccation in air, respectively. The onset of the high emission frequency corresponded with a water potential of −1.7, −2.9 and −3.8 MPa in the three cultivars, respectively.
It is concluded that a high number of cavitations may occur in noncut stems of rose plants, leading to low water uptake immediately after excision, depending on the weather and the cultivar, and that the low rate of water uptake after a period of dry storage, among the three rose cultivars investigated, is correlated with the presence of a high number of cavitated xylem elements.     

Petal Abscission in Rose Flowers: Effects of Water Potential, Light Intensity and Light Quality

Petal abscission was studied in roses (Rosa hybrida L.), cvs.Korflapei (trade name Frisco), Sweet Promise (Sonia) and CaraMia (trade name as officially registered cultivar name). Unlikeflowers on plants in greenhouses, cut flowers placed in waterin the greenhouse produced visible symptoms of water stress,depending on the weather during the experiment and on the cultivar.Cut Frisco roses showed no visible signs of water stress andthe time to petal abscission was as in uncut flowers. In Soniaroses the symptoms of water stress varied from mild to severe,and the number of flowers in which the petals abscised variedfrom 100% (mild stress) to 0% (severe stress). An antimicrobialcompound in the vase water of Sonia roses, or removal of theleaves, alleviated the symptoms of water stress and increasedthe number of stems in which the petals abscised. Cut Cara Miaroses showed severe symptoms of water stress in all experimentsand petal abscission was found in only a few flowers, even whenthe stems were placed at 20 °C and low photon flux (15 µmolm^-2s^-1). Abscission in Sonia and Cara Mia roses was low or absentwhen the water potential of the leaves reached values below-2.0 MPa within the first 5 d of the experiment; such low valueswere not reached in Frisco roses. Addition of sucrose to the vase solution, together with an effectiveantimicrobial compound, had no effect on the time to petal abscission,at any light intensity. Placing flowers in far-red light alsohad no effect on abscission, compared with flowers placed inred light or white light of the same photon fluence. It is concluded that petal abscission in the rose cultivarsstudied is not affected by their water status unless the plantsreach a low water potential (about -2 MPa) early on during vaselife. Petal abscission is not inhibited by low light intensitynor affected by the Pr/Pfr ratio. Abscission; light intensity; petals; phytochrome; Rosa hybrida L.; rose; sugars; water potential     

Interaction between the effects of bacteria and dry storage on the opening and water relations of cut rose flowers

W.G. VAN DOORN AND K. D'HONT. 1994. Flowering stems of four rose cultivars (Sonia, Madelon, Jacaranda and Frisco) were placed in aqueous suspensions of bacteria at 10⁴ and 10⁸ colony-forming units (cfu) ml^-1 for 24 h at 5C, then stored dry or held in water for 24 h at 8C and subsequently placed in vase-water at 20C. The effects of these treatments on vase-water uptake were similar to the effects on flower opening. In Sonia and Madelon roses flower opening was negatively affected both by 10⁸ cfu ml^-1 of bacteria and by dry storage. No effect was found at 10⁴ cfu ml^-1, but this concentration had a detrimental effect on flower opening when combined with dry storage. Although flower development in Jacaranda roses was not affected by the bacteria treatments it was inhibited by dry storage. This inhibition was progressively greater when the stems had previously been pulse-treated with a larger number of bacteria. Flower opening in Frisco roses was not affected by even the highest concentration of bacteria, nor by the period of dry storage. It is concluded that placing flowers in water containing bacteria (up to 10⁸ cfu ml^-1) may not always have a negative effect on flower development in cut rose flowers but, together with the effects of dry storage, the presence of even a low number of exogenous bacteria (10⁴ cfu ml^-1) inhibits the development in several cultivars. Such bacterial counts are nearly always found in samples of water used for standing roses during distribution to the consumers.     

Comparison of endogenous polyamine content in hazel leaves and buds between the annual dormancy and flowering phases of growth

Upon exposure to air at 20°C and 60% relative humidity (RH), an occlusion to water uptake developed in the basal stem segment of cut, flowering Sonia roses. The development of the occlusion was delayed by removing the leaves before storage, indicating an effect of transpiration. Placing the stem ends at 100% RH during exposure to air had no effect, which shows that water loss through the cut end is not the cause. Scanning electron microscopy did not show plant gums or tyloses in the water conducting elements.
The hypothesis by Scholander et al. (1955) that water uptake into stems held in air and then placed in water occurs through conduit walls rather than the lumen was tested by using stems of which the cut surface was covered with laboratory grease and a ring of bark was removed (girdling). Girdled stems placed in water remained fully turgid when the girdled area was about 60 mm² or more. Water uptake was strongly inhibited when the girdled stems were exposed to air for 24–36 h, as in non-girdled controls exposed to air for the same period. Inclusion of a surfactant (nonylphenoxypolyethoxy ethanol) in the vase water, however, facilitated water uptake after dry storage of normal cut flowering stems but did not improve water uptake into the girdled stem system, which is inconsistent with the hypothesis.
It is concluded that the vascular occlusion developing upon exposure to air cannot be explained by decreased flow in the wall pathway for water.     

Cuticular transpiration and epicuticular lipids of primary leaves of barley (Hordeum vulgare)

Twenty cultivars of barley and 15 eceriferum mutants from one of the cultivars have been analysed for cuticular transpiration and epicuticular lipids of their primary leaves. The relative cuticular transpiration rates of the cultivars ranged from 0.61 to 1.98. In spite of this variation in transpiration most of the cultivars had almost the same amount of epicuticular lipids per leaf area, about 16 μg cm⁻². The eceriferum mutants showed a wider range in amount of epicuticular lipids, from 5.0 to 15.5 μg cm⁻². Nevertheless, most of the mutants transpired almost at the same rate. Only a weak correlation was found between cuticular transpiration and total amount of epicuticular lipids. None of the analysed lipid components (alkanes, aldehydes, primary alcohols, esters or fatty acids) was better correlated to the cuticular transpiration than the total amount of lipids. When the cultivars were exposed to a mild water stress their cuticular transpiration rates decreased by about 11%. This reduction was not accompanied by any corresponding increase in total amount of epicuticular lipids. The most pronounced effect of the water stress treatment was a stimulation in the ester formation and a reduced formation of primary alcohols. This shift in lipid composition could not be correlated to the decreased cuticular transpiration rates of the individual cultivars. From this investigation it is concluded that the cuticular transpiration is poorly correlated to the amount or composition of the epicuticular lipids in this barley material. As a consequence it was not possible to use any characteristic of the epicuticular lipids as a selection criterion in breeding for drought resistance.     

空气湿度与土壤水分胁迫对紫花苜蓿叶表皮蜡质特性的影响

选用2个抗旱性不同的紫花苜蓿品种,敖汉(强抗旱)和三得利(弱抗旱),设置空气湿度(45%-55%和75%-85%)和土壤水分胁迫(75%和35%田间持水量)处理,分析紫花苜蓿叶表皮蜡质含量、组分及晶体结构、气体交换参数、水势及脯氨酸含量的变化规律。结果表明,单独土壤水分胁迫时,紫花苜蓿叶表皮蜡质晶体结构及蜡质总量无显著变化;敖汉蜡质组分中烷类、酯类含量增加,醇类含量下降;三得利醇类含量下降,烷类、酯类含量变化不显著。低空气湿度胁迫时,两品种蜡质总量无显著变化,烷类和酯类含量显著增加,醇类含量显著下降,叶表皮片状蜡质晶体结构熔融呈弥漫性,扩大了对叶表面积的覆盖,其蒸腾速率显著低于正常湿度。复合胁迫处理时,叶表皮片状蜡质晶体结构继续呈弥漫性,烷类、酯类、未知蜡质组分含量均高于单独胁迫处理,醇类含量最低,而蜡质总量除三得利显著高于对照外,其余均无显著差异。紫花苜蓿叶表皮蜡质各组分含量(除醇类)及蜡质总量与光合速率呈显著负相关,与蒸腾速率无显著相关关系。蜡质总量与叶水势呈显著正相关。总体上,敖汉蜡质总量显著高于三得利,蜡质组分中烷类物质的增加有助于提高植株的抗旱性。在复合胁迫下,强抗旱品种主要通过气孔因素控制水分散失,而弱抗旱品种通过气孔和非气孔因素共同控制植物水分散失。     

Epi- and intracuticular lipids and cuticular transpiration rates of primary leaves of eight barley (Hordeum vulgare) cultivars

The major constituents of the epi- and intracuticular lipids of primary leaves of 8 cultivars of barley ( Hordeum vulgare L.) have been studied together with cuticular transpiration rates. The total amount of analysed cuticular lipids ranged from 9.6 to 13.4 μg cm⁻² and was dominated by the epicuticular fraction, which made up 73–84% of the total. There were variations in the percentages of the analysed lipid classes, alkanes, esters, aldehydes, β-diketones and alcohols, between epi- and intracuticular lipids among individual cultivars, but no clear tendency in these variations, except for the aldehydes, was found. The epicuticular lipids were richer in aldehydes than the intracuticular lipids. The cuticular transpiration rates were poorly correlated with the levels or composition of epi-, intra- or total cuticular lipids. The cuticular transpiration rates were considerably altered as a response to a water stress treatment, but these changes could not be correlated with any changes in amount or composition of the cuticular lipids. From these results it is concluded that some property other than amount or composition of cuticular lipids is the most important in regulation of water diffusion through the cuticle.     

Changes in cuticular transpiration rate and cuticular lipids of oat (Avena sativa) seedlings induced by water stress

Two cultivars of oat ( Avena sativa L. cvs Pendek and Stormogul II) were exposed to short periods of water-deficit stress on five consecutive days. The plants responded to the stress by decreasing their cuticular transpiration rate. After two stress periods the cuticular transpiration rate was reduced by 30% for Pendek and by 47% for Stormogul II, and after another three stress periods by 30% and 20%, respectively. These reductions were correlated neither to changes in the total amount of what is generally called epicuticular lipids, nor to changes in any of the major individual constituents of the epicuticular lipids (alkanes, free and esterified fatty acids or free primary alcohols). After removal of the epicuticular lipids the long chain free primary alcohols of the leaves were extracted and determined. The amount of these presumably intracuticular alcohols increased after stress and changed to shorter chain length. From these results it is concluded that the intra- as well as the epicuticular lipids must be taken into consideration when discussing leaf surface lipids as protecting agents against water loss.     

Inter-tracheid pitting and the hydraulic efficiency of conifer wood: the role of tracheid allometry and cavitation protection

Plant xylem must balance efficient delivery of water to the canopy against protection from air entry into the conduits via air-seeding. We investigated the relationship between tracheid allometry, end wall pitting, safety from air-seeding, and the hydraulic efficiency of conifer wood in order to better understand the trade-offs between effective transport and protection against air entry. Root and stem wood were sampled from conifers belonging to the Pinaceae, Cupressaceae, Podocarpaceae, and Araucariaceae. Hydraulic resistivity of tracheids decreased with increasing tracheid diameter and width, with 64 ± 4% residing in the end wall pitting regardless of tracheid size or phylogenetic affinity. This end-wall percentage was consistent with a near-optimal scaling between tracheid diameter and length that minimized flow resistance for a given tracheid length. There was no evidence that tracheid size and hydraulic efficiency were constrained by the role of the pits in protecting against cavitation by air-seeding. An increase in pit area resistance with safety from cavitation was observed only for species of the northern hemisphere (Pinaceae and Cupressaceae), but this variable was independent of tracheid size, and the increase in pit resistance did not significantly influence tracheid resistance. In contrast to recent work on angiosperm vessels, protection against air-seeding in conifer tracheids appears to be uncoupled from conduit size and conducting efficiency.     

Genotypic Variation in the Thickness of Silica Deposition on Flowering Rice Spikelets

Silicon may play an important role in regulating the transpirationrate of rice (Oryza sativa L.), particularly cuticular transpiration.The control of cuticular transpiration is important in ricespikelets because water stress at anthesis may severely disruptfertility and grain yield. Data on the quantitative variationamong rice cultivars in the thickness of the silica layer ofthe flowering spikelet were obtained in order to assess thepotential for genetic selection for silica layer thickness asa potential means for increasing spikelet resistance to waterloss. Flowering spikelets were collected from 17 genotypes and thicknessmeasurements were made of the major anatomical layers in crosssection. The silica layer of the lemma varied from 42 to 177µm among cultivars. Similar variation was observed inthe palea. Differences among cultivars were also found in thethickness of the epidermis and sclerenchyma-parenchyma layer. The total cross-sectional thickness of the lemma and palea averagedslightly over 100 µm. Total thickness was not significantlycorrelated with the length, width or product of length width.Thickness of silica deposition was significantly correlatedwith spikelet length width suggesting that these easily measuredparameters may be useful in screening for silica thickness. Oryza sativa L., rice, panicle, silicon, lemma, palea, husk     

Susceptibility of Cut Rose Flower Cultivars to Infections by Different Isolates of Botrytis cinerea

A method was developed for the inoculation of flowers of cut roses with conidia of Botrytis cinerea. Flower buds were inoculated by spraying of conidial supensions that were ultrasonicated for 10 s. The differences in susceptibility between 8 rose cvs to infections of isolate Bc-33 and the differences in pathogenicity between 14 5. cinerea isolates to cv. ‘Sonia’ were evaluated. Isolates obtained from rose flowers caused higher infection rates than those obtamed from various other hosts. The CVS ‘Madelon’, ‘Melody’ and ‘Sonia’ were found to be highly susceptible, whereas the cvs ‘Caramboie’, ‘Gabriella’, ‘Pasadena’ and ‘Rubinette’ were only slightly susceptible. The lower disease severity in the less susceptible cvs was based on a retardation of the growth of mfection hyphae in the petals, which may have been due to partial resistance. The formation of symptoms is effectuated already by the initial stages in the infection process. Thus, the apparent existing partial resistance to infections of B. cinerea cannot prevent the loss of ornamental value.     

油菜花丝枯萎机理的探讨

油菜（Brassica napus L.)花丝的输水系统由螺纹以及少量的环纹导管和管胞组成,它们随花丝的伸长而被拉长直至拉断。统计结果显示, 花开放后,大量的导管和管胞被拉断。输水组织的断裂终止了连续水柱的形成,使水分运输的重要动力－蒸腾拉力和内聚力不能发挥作用,引起了花丝组织的供水足,在蒸腾失水不断加剧的外部因素的相互作用下,最终导致了花丝的枯萎。     

Leaf cuticular waxes of potted rose cultivars as affected by plant development, drought and paclobutrazol treatments

The present work was carried out to evaluate how plant growth and cultural practices influence the amount and composition of cuticular waxes on leaves of rose cultivars. The total amount of cuticular wax per leaf area was higher for rose cultivar Apollo Parade than for Charming Parade. Both cultivars had waxes dominated by alkanes, with the major alkanes being the C₃₁ and C₃₃ homologues. Primary alcohols were the next most abundant constituent class, with C₂₆ as the dominant homologue. Compared with Charming Parade, Apollo Parade had higher proportions of its total wax load as primary alcohols but lower acids and aldehydes. The proportion of alkanes in the total load on these cultivars was similar. Commercially produced roses are routinely treated with paclobutrazol (PBZ) to retard growth. PBZ treatments caused a 10% increase in total wax load and changes in the proportions of certain wax constituents within 11 days of application. Notable was an increase in the total proportion of acids in the total load 25 days after PBZ application, primarily because of increased C₂₈ acids. An alternative method of retarding plant growth is production of roses under limited water availability. When Apollo Parade roses experienced periods of moderate drought stress during production, the wax load per leaf area increased 14 and 8% above control levels at 24 and 38 days after imposition of drought, respectively. Drought caused similar changes in the proportions of individual wax constituents as did PBZ application.     

A wilty mutant of rice has impaired hydraulic conductance

The rice CM2088 mutant is the wilty phenotype and wilts markedly under well-watered sunny conditions. The leaf water potential and epidermal (mainly stomatal) conductance of CM2088 plants decreased significantly under conditions that induced intense transpiration, as compared with those of wild-type plants, revealing that the wilty phenotype was not the result of abnormal stomatal behavior but was due to an increase in resistance to water transport. The resistance to water transport was dramatically elevated in the node and the sheath and blade of a leaf of the mutant, but not in the root or stem. The diameter of xylem vessels in the large vascular bundles of the leaf sheath and the internode tended to be small, and the numbers of vessel elements with narrowed or scalariform perforation plates in the leaf blade and sheath were greater in the mutant than in the wild type. Most xylem vessels were occluded, with air bubbles in the leaf sheath of the mutant during the midday hours under intense transpiration conditions, while no bubbles were observed in plants that were barely transpiring, revealing that the significant increase in resistance to water transport was a result of the cavitation. The additive effects of cavitation in xylem vessels and the decreased diameter and deformed plates of vessel elements might be responsible for the wilty phenotype of CM2088.     

Variations of cuticular wax in mulberry trees and their effects on gas exchange and post-harvest water loss

Though mulberry (Morus alba) tree shows great adaptations to various climate conditions, their leaf water status and photosynthesis are sensitive to climate changes. In the current study, seven widely planted mulberry cultivars in Chongqing, Southwest China, were selected to analyze leaf cuticular wax characteristics, gas exchange index, post-harvest leaf water status and their relationships, aiming to provide new theory in screening high resistant mulberry cultivars. Mulberry trees formed rounded cap-type idioblasts on the adaxial leaf surface. Film-like waxes and granule-type wax crystals covered leaf surfaces, varying in crystal density among cultivars. The stomatal aperture on the abaxial surface of cultivars with high wax amount was smaller than that of cultivars with low wax amount. The amount of total wax was negatively correlated with the net photosynthetic rate (P _N), transpiration rate (E) and stomatal conductance (g _s) and positively correlated with the moisture retention capacity. It suggested that both cuticular wax and stomatal factor might be involved in regulating water loss in mulberry leaves under field conditions. The variability in moisture retention capacity and cuticular wax characteristics might be important in evaluating and screening mulberry cultivars for increasing silk quality and silkworm productivity.     

XYLEM ANATOMY AND HYDRAULIC CONDUCTANCE OF COSTA RICAN BLECHNUM FERNS

Hydraulic conductivities of stems, stipes, and elongate leaf stipes were determined for greenhouse-grown Blechnum (B. fraxineum, B. fragile, B. buchtienii, B. sprucei) and Salpichlaena (S. volubilis) plants collected in tropical rain forests of Costa Rica. Organ conductivity was examined in relation to morphology and tracheid characteristics in order to gain an understanding of factors influencing water flow. Hydraulic conductivity of plant organs was determined by measurement of transpiration rates, leaf areas, and water potential gradients. Erect stemmed species develop larger whole plant water potential gradients than elongate stemmed species for a similar transpiration rate. Elongate leaves develop even smaller water potential gradients for a given transpiration rate. Stems have larger hydraulic conductivities but smaller leaf-specific conductivities (LSCs) than stipes. Small conductivities and small LSCs are associated with short, erect stems. Elongate structures have large conductivities and large LSCs. Of the tracheid characteristics examined, the most important characteristics determining the magnitude of organ hydraulic conductivity are diameter, pit aperture area between tracheids, taper length, and cell length. Large conductivities of S. volubilis climbing leaf stipes are associated with very large-diameter tracheids (some > 200 μm), large tracheid number, exceptionally long tracheids (some > 4 cm), large pit aperture area between tracheids, short tracheid taper, and smooth tracheid lumen walls. Hagen-Poiseuille estimates of hydraulic conductivity range from 1.1 to 3.3 times the measured values. Conductivity of stipes is highly correlated with leaf area supplied by stipes. Conductivities of stems and elongate leaf stipes also correlate with leaf area supplied by these structures. Estimated hydraulic conductivities of field-grown Blechnum and Salpichlaena demonstrate that larger conductivities are associated with larger plants. This study contributes toward our knowledge of fern water relations and extends previous growth form/hydraulic architecture characterizations by providing a more comprehensive comparison of closely related species. In addition, this study provides evidence for the relative importance of tracheid characteristics in determining the magnitude of organ hydraulic conductivity.     

Leaf gas exchange of upland and lowland rice cultivars

Leaf gas exchange of upland and lowland rice cultivars were measured during late vegetative and during grain filling stages in the field under upland and lowland growth conditions. The rate of photosynthesis and water use efficiency (the rate of photosynthesis/the rate of transpiration) under upland conditions decreased with ageing, but generally varied little among four cultivars. At mid-grain filling under lowland conditions, upland cultivars showed lower rates of photosynthesis and transpiration than the lowland cultivars with concomitant reduction in whole plant conductance. At this stage, water use efficiency was higher under upland conditions than under lowland conditions, particularly in the upland cultivars. Water stress reduced the rate of photosynthesis without altering water use efficiency.     

Effect of air and soil temperature on water balance of jojoba growing under controlled conditions

Jojoba [ Simmondsia chinensis (Link) Schneider] cuttings were grown in pots under constant light intensity and vapour pressure deficit at wir temperatures of 18 and 27°C in climate-controlled cabinets. Leaf conductance and transpiration rate decreased exponentially as the xylen water potential (Ψ_x) decreased concurrently with the drying out of the soil. At high Ψ_x'leaf conductance and transpiration rate were much higher at the higher air temperature, and as Ψ_x declined both parameters decreased more rapidly at 27°C than at 18°C. When soil temperatures were decreased from 27 to 13°C, leaf water potential was not affected at either air temperatures, but transpiration rate was reduced. A linear negative correlation was found between transpiration rates and soil temperatures. It is suggested that the low soil temperature may restrict reducion of water flux in turn reduces stomatal conductance and transpiration without affecting the water potential in the shoot. The releavance of the response to changes in soil or air temperature to the performance of the plant in its semi-arid habitat is discussed.     

A Method for Separating Cuticular and Stomatal Components of Gas Exchange by Amphistomatous Leaves: II. EXPERIMENTAL SOLUTION

The apparent cuticular component of transpiration of stomatabearing leaf epidermis was estimated by restricting stomataldiffusion by mass flow of air in the opposite direction. Thiswas achieved by applying an air pressure gradient across theamphistomatous leaf. Some assumptions of the previously suggestedmethod (antrcek and Slav?k, 1990) were experimentally verifiedusing maize leaves. The technique makes possible a quantitativeestimation of cuticular water loss including that of the externalperistomatal (i.e. vapour not passing through the pores) andthe respective conductance when the stomata are partially open. In addition to the fact that the cuticular portion of the totalleaf vapour loss (i.e. relative cuticular transpiration) dependson stomatal opening, even the absolute value of apparent cuticulartranspiration was (1) increased by lower vapour pressure deficitand (2) decreased with closing stomata. These changes, inducedby variations in a vapour pressure deficit of 2.45?0.35 kPa,ranged between 0.66?0.14µg cm ^–2 s^–1. Theabsolute value of apparent cuticular transpiration changed onaverage by a factor of 2.3 due to stomata opening or closingwhich was induced by turning the light on or by exogenous ABAapplication. Possible interference by residual vapour diffusingthrough the stomatal pore was evaluated by the model application.An attempt was also made to assess the cuticular component ofCO₂-uptake rate. Experimental results are discussed in contextwith the feedforward response of stomata to air humidity. Key words: Cuticular transpiration, cuticular CO₂-uptake, feedforward response, maize     

Identifying soybean lines differing in gas exchange sensitivity to humidity

The ratios of root length and root weight to leaf area differed within and between cultivars of soybean. Plants with low ratios of root length or weight to leaf area had leaf conductances and net photosynthetic rates more reduced by a given increase in the leaf to air water vapour pressure difference around a single leaf than plants with high ratios. Plant and root system conductances to water were estimated as transpiration rate per unit leaf area divided by the difference between substrate and leaf water potentials, and by the rate of water flow through pressurised root systems. These conductances were greater in plants with large, as compared with small, root systems per unit leaf area. Cultivar rankings in sensitivity of gas exchange to humidity were consistent in controlled environment chambers and in field tests.