Diurnal oscillatory movements of growing leaves of tobacco

The analysis of diurnal oscillatory movements of tobacco leaves was used in the diagnosis of viral infection of plants. The oscillatory helices circumscribed by a growing leaf of a healthy plant were regular, but some deviations, particularly in the transition points, were recorded. In order to make clear the cause of these irregularities of trajectory, the course of elongation of leaf petiole and blade in relation to localization and shift of zones of elongation during ontogenesis was analysed. The present analysis is similar to that described by the author's earlier experiments with pea roots. Oscillatory curves circumscribed by petiole, projected on a horizontal plane, were compared with curves circumscribed by the blade tip. The analysis of the leaves of different ages enabled us to study this process in dependence on growth rate. It was confirmed that oscillations are a result of elongation; the extent of oscillations is quantitatively dependent on the growth rate. An analysis of the zones of growth showed that in petiole the active meristems are localized near to its base while in the leaf lamina they move gradually during the ontogenesis from the apical to the basal part of the leaf blade. Active meristems of young rapidly growing leaves are localized approximately in the middle of the blade while those of old leaves were found in close proximity to the base of the lamina. The growth rate of petiole can be expressed in hundreds of mm per hour (4.8×10^?2 mm h^?1); half of this value was recorded for its apical part. The growth rate of leaf blade was found approximately ten times higher (3.2×10^?1 mm h^?1). The oscillatory movements of growing leaf consists of two integrate components: of oscillations originating in the base of the petiole and of oscillations of leaf blade the centrum of which is localized in the basal third of the blade. The arrangement of the experiments did not enable us to determine to what extent the phototropic response of leaf blade participates in leaf movements. The movements of leaves of an intact plant are evidently affected by rhythmic stem oscillations. Stem is an integral part of a system which participates in the transfer of information in plants.     

The different growth responses of the Arabidopsis thaliana leaf blade and the petiole during shade avoidance are regulated by photoreceptors and sugar

During the shade-avoidance response, leaf blade expansion is inhibited and petiole elongation is enhanced. In this study, we examined the roles of photoreceptors and sugar on the differential growth of the leaf blade and petiole in shade conditions. Under the conditions examined, cell expansion, not cell division, played a major role in the differential leaf growth. The enhanced cell expansion in the leaf blade is associated with an increase in the ploidy level, whereas cell elongation was stimulated in the petiole in dark conditions without an increase in the ploidy level. Analysis of phytochrome, cryptochrome and phototropin mutants revealed that phytochromes and cryptochromes specifically regulate the contrasting growth patterns of the leaf blade and petiole in shade. Examination of the effects of photo-assimilated sucrose on the growth of the leaf blade and petiole revealed growth-promotional effects of sucrose that are highly dependent on the light conditions. The leaf blades of abscisic acid-deficient and sugar-insensitive mutants did not expand in blue light, but expanded normally in red light. These results suggest that both the regulation of light signals and the modulation of responses to sugar are important in the control of the differential photomorphogenesis of the leaf blade and petiole.     

Spatial distribution of growth rates and of epidermal cell lengths in the elongation zone during leaf development in Lolium perenne L.

Relative elemental growth rates (REGR) and lengths of epidermal cells along the elongation zone of Lolium perenne L. leaves were determined at four developmental stages ranging from shortly after emergence of the leaf tip to shortly before cessation of leaf growth. Plants were grown at constant light and temperature. At all developmental stages the length of epidermal cells in the elongation zone of both the blade and sheath increased from 12 m at the leaf base to about 550 m at the distal end of the elongation zone, whereas the length of epidermal cells within the joint region only increased from 12 to 40 m. Throughout the developmental stages elongation was confined to the basal 20 to 30 mm of the leaf with maximum REGR occurring near the center of the elongation zone. Leaf elongation rate (LER) and the spatial distributions of REGR and epidermal cell lengths were steady to a first approximation between emergence of the leaf tip and transition from blade to sheath growth. Elongation of epidermal cells in the sheath started immediately after the onset of elongation of the most proximal blade epidermal cells. During transition from blade to sheath growth the length of the blade and sheath portion of the elongation zone decreased and increased, respectively, with the total length of the elongation zone and the spatial distribution of REGR staying near constant, with exception of the joint region which elongated little during displacement through the elongation zone. Leaf elongation rate decreased rapidly during the phase when only the sheath was growing. This was associated with decreasing REGR and only a small decrease in the length of the elongation zone. Data on the spatial distributions of growth rates and of epidermal cell lengths during blade elongation were used to derive the temporal pattern of epidermal cell elongation. These data demonstrate that the elongation rate of an epidermal cell increased for days and that cessation of epidermal cell elongation was an abrupt event with cell elongation rate declining from maximum to zero within less than 10 h.Abbreviations LER leaf elongation rate - REGR relative elemental growth rates     

Leaf development and phloem transport in Cucurbita pepo: Transition from import to export

Summary The capacity of a growing leaf blade of Cucurbita pepo L. to import ¹⁴C-labelled photoassimilate is lost in a basipetal direction. Import into the lamina tip stops when the blade is 10% expanded. Development of the leaf progresses linearly with time and the lamina base stops importing when the blade is 45% expanded. Export capacity also develops basipetally and follows immediately the loss of import capacity, at least in the lamina base. The small amount of material initially exported from the leaf tip is redistributed to the still-importing leaf base, delaying export from the lamina until the blade is 35% expanded. Loss of import capacity by the petiole is both basipetal and dorsoventral. The proximal, adaxial portion of the petiole is the last region to cease importing ¹⁴C. Leaves of Beta vulgaris L. and Nicotiana tabacum L. also lose import capacity in a basipetal direction.     

FOLIAR ONTOGENY AND HISTOGENESIS IN MAGNOLIA GRANDIFLORA L. I. APICAL ORGANIZATION AND EARLY DEVELOPMENT

Foliar ontogeny of Magnolia grandiflora was studied to elucidate possible unique features of evergreen leaves and their development. The apex of Magnolia grandiflora is composed of a biseriate or triseriate tunica overlying a central initial zone, a peripheral zone and a pith rib meristem. Leaf primordia are initiated by periclinal divisions on the apical flank of the tunica in its second layer. This initiation and expansion is seasonal just as in related deciduous magnolias. Following leaf initiation, a foliar buttress is formed and the leaf base gradually extends around the apex. As growth continues, separation of the leaf blade primordium from the stipule proceeds by intensified anticlinal divisions in the surface and subsurface layers near the base. Marginal growth begins in the blade primordium when it reaches approximately 200 μm in height and results in the formation of two wing-like extensions, the lamina. This young blade remains in a conduplicately folded position next to the stipule until bud break.     

Relationships between the blade and sheath growth in the same leaf and in successive leaves of winter barley

Winter barley (Hordeum vulgare L. cv. Efra) plants were grown till the stage of the fourth leaf under controlled conditions at constant temperatures 26.0 °C, 21.8 °C, 19.6 °C and 15.3 °C. The relationships between the sheath and blade growth was studied. The leaf sheath began to be discernible when it was 0.1 mm long and the blade length was 20 mm. In this stage a correlation (r = 0.812) was found between the length of blade and that of shearth. The sheath length in 20 mm long leaf increased in dependence on leaf insertion. At the time of the beginning of sheath discernibility the elongation growth of the subsequent leaf was initiated. In this stage the sheath length and the length of the subsequent leaf were correlated (r = 0.911). At the beginning of the growth of the subsequent leaf the length of the preceding sheath increased in dependence on insertion. Other relations were derived graphically and a hypothetical model of relationships between the cereal leaf growth and development was formulated.     

The stimulating effects of ethylene and auxin on petiole elongation and on hyponastic curvature are independent processes in submerged Rumex palustris

The flooding-tolerant plant species Rumex palustris (Sm.) responds to complete submergence with stimulation of petiole elongation mediated by the gaseous hormone ethylene. We examined the involvement of auxin in petiole elongation. The manipulation of petiolar auxin levels by removing the leaf blade, or by addition of synthetic auxins or auxin transport inhibitors, led to the finding that auxin plays an important role in submergence-induced petiole elongation in R. palustris. A detailed kinetic analysis revealed a transient effect of removing the auxin source (leaf blade), explaining why earlier studies in which less frequent measurements were taken failed to identify any role for auxin in petiole elongation. We previously showed that the onset of stimulated petiole elongation depends on a more upright petiole angle being reached by means of hyponastic (upward) curvature, a differential growth process that is also regulated by ethylene and auxin. This raised the possibility that both ethylene and auxin stimulate elongation only indirectly by influencing hyponastic growth. We show here that the action of ethylene and auxin in promoting petiole elongation in submerged R. palustris is independent of the promoting effect that these hormones also exert on the hyponastic curvature of the same petiole.     

Peroxidase Activity in the Leaf Elongation Zone of Tall Fescue : II. Spatial Distribution of Apoplastic Peroxidase Activity in Genotypes Differing in Length of the Elongation Zone

Previous work suggested that cell wall peroxidase activity increased as cells were displaced through the elongation zone in leaf blades of tall fescue (Festuca arundinacea Schreb.). In this study, two genotypes that differ in length of the elongation zone were used to examine the relationship between peroxidase activity in apoplastic fluid of intact leaf blade segments and the spatial distribution of leaf growth. Apoplastic fluid was extracted by vacuum infiltration and centrifugation, and peroxidase activity was assayed spectrophotometrically. Isoelectric focusing was used to characterize the isoforms of apoplastic peroxidase within the region of elongation and in the region of secondary cell wall deposition, which is distal to the elongation zone. A striking correlation was found in each genotype between both the location and timing of increase in apoplastic peroxidase activity and the onset of growth deceleration. Only cationic isoforms of apoplastic peroxidase could be identified in the elongation zone, whereas additional anionic isoforms appeared in the region of secondary cell wall deposition. We conclude that cessation of elongation growth in tall fescue leaf blades is likely to be related to the secretion of cationic isoforms of peroxidase into the cell wall.     

PHANTASTICA regulates development of the adaxial mesophyll in Nicotiana leaves

Initiation and growth of leaf blades is oriented by an adaxial/abaxial axis aligned with the original axis of polarity in the leaf primordium. To investigate mechanisms regulating this process, we cloned the Nicotiana tabacum ortholog of PHANTASTICA (NTPHAN) and generated a series of antisense transgenics in N. sylvestris. We show that NSPHAN is expressed throughout emerging blade primordia in the wild type and becomes localized to the middle mesophyll in the expanding lamina. Antisense NSPHAN leaves show ectopic expression of NTH20, a class I KNOX gene. Juvenile transgenic leaves have normal adaxial/abaxial polarity and generate leaf blades in the normal position, but the adaxial mesophyll shows disorganized patterns of cell division, delayed maturation of palisade, and ectopic reinitiation of blade primordia along the midrib. Reversal of the phenotype with exogenous gibberellic acid suggests that NSPHAN, acting via KNOX repression, maintains determinacy in the expanding lamina and sustains the patterns of cell proliferation critical to palisade development.     

A model co-ordinating the elongation of all leaves of a sorghum cultivar was applied to both Mediterranean and Sahelian conditions

Sorghum leaf development was analysed at plant level by analysing the time-course of elongation and identifying the beginning and end of the elongation phases of each leaf blade. This was done with destructive and non-destructive measurements in 14 experiments carried out during several growing periods in Southern France and Sahelian Africa. Elongation of each blade was characterized by the succession of a nearly exponential phase and a linear phase. For a given blade and provided that time was expressed in thermal units, initiation, beginning and end of the linear phase, and time-courses of elongation rate were strikingly similar in all experiments, except in environments with a maximum air temperature close to 40 degrees C and a maximum vapour pressure deficit close to 6 kPa. The relative elongation rate during the exponential phase declined with leaf number from 0.08 to 0.02 degrees Cd(-1), while the duration of this phase increased from 140 to 320 degrees Cd. By contrast, the absolute elongation rate during the linear phase was nearly constant from leaf 8 onwards. This phase was shorter than the exponential phase regardless of leaf position, but accounted for the largest part of blade length. A strict pattern of leaf development was observed at the whole plant level, whereby dates of elongation events and leaf and ligule appearance, represented on a thermal time scale, were linearly related to phytomer number. This pattern exhibited a simultaneous elongation cessation of the last-formed leaves and a mismatch between real and apparent (from leaf to ligule appearance) elongation duration.     

phyB-1 sorghum maintains responsiveness to simulated shade, irradiance and red light: far-red light

The sorghum [Sorghum bicolor (L.) Moench] phyB-1 mutant exhibits a constitutive shade-avoidance phenotype including excessive shoot elongation. It was previously shown that this mutant also overproduces ethylene. Although phytochrome B (phyB) is assumed to be the pigment most important in sensing and transducing shade signals, the sorghum phyB-1 mutant still responds to light signals characteristic of shade. Specifically, it was determined that the leaf blade : leaf sheath elongation of phyB-1 is responsive to red : far red (R : FR), but this response is opposite that of wild type (WT). Reducing the photosynthetic photon flux density (PPFD) strongly reduced the leaf blade : leaf sheath of WT but did not affect phyB-1, demonstrating a role for phyB in sensing PPFD. Using light-emitting diode (LED) lighting, it was found that WT ethylene production was increased with low R : FR while PPFD had no effect. Conversely, phyB-1 ethylene production increased only with high PPFD, high R : FR which was the treatment resulting in the least ethylene production by WT. Elevated ethylene production inhibits shoot elongation, but may contribute to shade avoidance by reducing leaf blade : leaf sheath elongation. Ethylene responses to light treatments designed to promote or reduce phytochrome A (phyA) activity, and the analysis of PHYA levels in the two cultivars suggests that phyA could be involved in transducing shade signals in light-grown sorghum. Responses potentially tranduced by phyA are elevated in phyB-1 which also over-expresses PHYA.     

Regulation of leaf growth of grass by blue light

Changes in light quality occur naturally within a canopy when a plant grows from unshaded to shaded conditions, and the reverse occurs after a cut that reduces shading. These changes in light quality could be responsible for the variation in leaf elongation and appearance rates of grasses. The role of blue light in leaf growth was investigated in tall fescue (Festuca arundinacea Schreb.) and perennial ryegrass (Lolium perenne L.). Leaf length was measured daily following a decrease or an increase in blue light to evaluate effects on duration of leaf growth, leaf elongation and the rate of leaf appearance rate. A reduction in blue light increased sheath length by 8 to 14% and lamina length by 6 to 12% for both species. These increases could be reversed by enrichment of blue light. With low blue light treatment, final leaf length was increased due to a greater leaf elongation rate. In tall fescue, but not in perennial ryegrass, this effect was coupled with a greater phyllochron and a longer duration of leaf elongation. Development of successive leaves on a tall fescue tiller were co-ordinated. A decrease in blue light increased the duration of elongation in the oldest growing leaf and also delayed the appearance of a new leaf, maintaining this co-ordination. We conclude that final leaf size and phyllochron for tall fescue can be significantly modified by blue light. Perennial ryegrass appeared less responsive, except for displaying longer sheaths and laminae in low blue light, as also occurred for tall fescue. We hypothesize that leaf length could be regulated by the quality of the light reaching the growing region itself.     

Assessment of spatial distribution of growth in the elongation zone of grass leaf blades

Knowledge about the spatial distribution of growth is essential for understanding the leaf growth process. In grasses the elongation zone is located at the base of the leaf blade and is enclosed by sheaths of older leaves. Assessment of spatial growth distribution, therefore, necessitates use of a destructive method. We used a fine needle to make holes through bases of tillers at the location of the leaf elongation zone of tall fescue (Festuca arundinacea Schreb.), then measured the displacement of the holes after a 6 or 24 h interval. Needle holes caused a 22 to 41% decrease in daily leaf elongation so experiments were conducted to investigate if the spatial distribution of growth in the elongation zone was altered. Leaf elongation rate was reduced similarly when needle holes were made within or above the zone where cell elongation occurs. Distribution of elongation within the zone was the same when estimated by displacement of needle holes or ink marks placed on the epidermis of the elongation zone after surrounding tissue had been removed. Making holes at different locations within the elongation zone did not differentially affect the relative contribution of the damaged or undamaged parts to leaf elongation. These findings demonstrate that needle holes or ink marks in paired leaves can be used to estimate the relative distribution of growth in the elongation zone of undamaged tall fescue leaf blades.     

Peroxidase activity in the leaf elongation zone of tall fescue : I. Spatial distribution of ionically bound peroxidase activity in genotypes differing in length of the elongation zone

Cessation of cell expansion has been associated with cell wall cross-linking reactions catalyzed by peroxidase. This study utilized two genotypes of tall fescue (Festuca arundinacea Schreb.) that differ in length of the leaf elongation zone to investigate the relationship between ionically bound peroxidase activity and the spatial distribution of leaf elongation. Peroxidase activity was also localized histochemically in transverse sections of the leaf blade using 3,3′ -diaminobenzidine. Soluble or soluble plus ionically bound peroxidase activities were extracted from homogenized segments of the elongating leaf blade and assayed spectrophotometrically. Activity of the ionically bound fraction, expressed per milligram fresh weight or per microgram protein, increased as cells were displaced through the distal half of the elongation zone, corresponding to the region in which the elongation rate declined. In both genotypes, the initial increase in activity preceded the onset of growth deceleration by about 10 hours. In the basal region where elongation began, histochemical localization showed that peroxidase activity was found only in vascular tissues. As cells were displaced farther through the elongation zone, peroxidase activity appeared in walls of other longitudinally continuous tissues such as the epidermis and bundle sheaths. Increase in ionically bound peroxidase activity and changes in localization of peroxidase activity occurred at comparable developmental stages in the two genotypes. The results indicate that cessation of elongation followed an increase in cell wall peroxidase activity.     

HETEROPHYLLIC DEVELOPMENT IN MUEHLENBECKIA (POLYGONACEAE)

Flowering shoots of Muehlenbeckia platyclados Meisn. bear only reduced scale leaves which resemble the membranous sheath portion (ochrea) of leaves of other members of the Polygonaceae. Shoots propagated from cuttings bear enlarged foliage leaves with distinct lamina, petiole, and ochrea zones. The developmental basis for this heterophylly is explored in order to determine whether scale leaves resemble foliage leaves in their pattern of ontogeny or are developmentally unique. SEM and histological analyses have shown that scale leaves and foliage leaves are distinctive from inception. The scale leaf arises as a collarlike growth and extends over the shoot apex as a hooded sheath without evidence of blade initiation. By contrast, the first stage of foliage-leaf ontogeny is the differentiation of the distal lamina from the future leaf base. As the foliage-leaf ochrea encircles the stem axis, the lamina grows erect and projects from the abaxial surface of the sheath. Lamina reduction coupled with ochrea elaboration in intermediate leaf types indicate a homology between the entire scale leaf and foliage-leaf ochrea. Despite this homology, the production of the bladeless scale leaf does not involve a mere suppression of the foliage-leaf lamina. Erect growth of the saccate ochrea of the foliage leaf contrasts with the hooded expansion of the scale. Early histological differences, including contrasting rates of cell differentiation, also distinguish the two organs. This disparity in modes of growth and differentiation from inception results from separate, predetermined courses of ontogeny. Unlike other plants studied, leaf size and degree of leaf elaboration decrease with shoot meristem enlargement in Muehlenbeckia. Leaf packing does increase with shoot development and may contribute to variations in leaf morphology. It is concluded that the peculiarities of the heterophyllic leaf sequence in Muehlenbeckia are a property of the shoot system as a whole.     

Expansion growth of isolated leaf blades of Todea barbara

Summary Etiolation of Todea barbara sporophytes and the subsequent deetiolation of excised leaf blades have been studied. In etiolated plants leaf blade growth is arrested, petiole and stem growth is enhanced, and root growth is decreased. De-etiolation permits the resumption of leaf blade growth and differentiation, and the resulting de-etiolated blade appears comparable to its light-grown counterpart in every respect but cell number. Only two factors are required to attain maximum surface area growth in cultured leaf blades; these are light and sucrose. The addition of plant growth substances does not increase the final area attained. However, the inhibition of leaf blade growth with growth retardants and its partial reversal by exogenously supplied gibberellic acid demonstrates a role for gibberellins in leaf expansion in Todea.     

The cellular parameters of leaf development in tobacco: a clonal analysis

The cellular parameters of leaf development in tobacco (Nicotiana tabacum L.) have been characterized using clonal analysis, an approach that provides unequivocal evidence of cell lineage. Our results indicate that the tobacco leaf arises from a group of around 100 cells in the shoot apical meristem. Each of these cells contributes to a unique longitudinal section of the axis and transverse section of the lamina. This pattern of cell lincage indicates that primordial cells contribute more or less equally to the growth of the axis, in contrast to the more traditional view of leaf development in which the leaf is pictured as arising from a group of apical initials. Clones induced prior to the initiation of the lamina demonstrate that the subepidermal layer of the lamina arises from at least six files of cells. Submarginal cells usually divide with their spindles parallel to the margin, and therefore contribute relatively little to the transverse expansion of the lamina. During the expansion of the lamina the orientation and frequency of cell division are highly regulated, as is the duration of meristematic growth. Initially, cell division is polarized so as to produce lineages that are at an oblique angle to the midrib; later cell division is in alternating perpendicular planes. The distribution of clones generated by irradiation at various stages of development indicates that cell division ceases at the tip of the leaf when the leaf is about one tenth its final size, and then ceases in progressively more basal regions of the lamina. Variation in the mutation frequency within the lamina reflects variation in the frequency of mitosis. Prior to the mergence of the leaf the frequency of mutation is maximal near the tip of the leaf and extremely low at its base; after emergence, the frequency of mutation increases at the base of the leaf. In any given region of the lamina the frequency of mutation is highest in interveinal regions, and is relatively low near the margin. Thus, both the orientation and frequency of cell division at the leaf margin indicate that this region plays a minor role in the growth of the lamina.Abbreviation MF mutation frequency     

Rapid and tissue-specific accumulation of solutes in the growth zone of barley leaves in response to salinity

The aim of the present study was to test whether rapid accumulation of solutes in response to salinity in leaf tissues of barley (Hordeum vulgare L.) contributes to recovery and maintenance of residual elongation growth. Addition of 100 mM NaCl to the root medium caused an immediate reduction close to zero in elongation velocity of the growing leaf 3. After 20–30 min, elongation velocity recovered suddenly, to 40–50% of the pre-stress level. Bulk osmolality increased first, after 60 min, significantly in the proximal half of the elongation zone. Over the following 3 days, osmolality increases became significant in the distal half of the elongation zone, the adjacent, enclosed non-elongation zone and finally in the emerged portion of the blade. The developmental gradient and time course in osmolality increase along the growing leaf was reflected in the pattern of solute (Cl, Na and K) accumulation in bulk tissue and epidermal cells. The partitioning of newly accumulated solutes between epidermis and bulk tissue changed with time. Even though solute accumulation does not contribute to the sudden and partial growth recovery 20–30 min after exposure to salt, it does facilitate residual growth from 1 h onwards. This is due to a high sink strength for solutes of the proximal part of the growth zone and its ability to accumulate solutes rapidly and at high rates.Abbreviations EDX analysis Energy-dispersive X-ray analysis - LEV Leaf elongation velocity - LVDT Linear variable differential transformer - REGR Relative elemental growth rate     

Long-term inhibition by auxin of leaf blade expansion in bean and Arabidopsis

The role of auxin in controlling leaf expansion remains unclear. Experimental increases to normal auxin levels in expanding leaves have shown conflicting results, with both increases and decreases in leaf growth having been measured. Therefore, the effects of both auxin application and adjustment of endogenous leaf auxin levels on midrib elongation and final leaf size (fresh weight and area) were examined in attached primary monofoliate leaves of the common bean (Phaseolus vulgaris) and in early Arabidopsis rosette leaves. Aqueous auxin application inhibited long-term leaf blade elongation. Bean leaves, initially 40 to 50 mm in length, treated once with alpha-naphthalene acetic acid (1.0 mm), were, after 6 d, approximately 80% the length and weight of controls. When applied at 1.0 and 0.1 mm, alpha-naphthalene acetic acid significantly inhibited long-term leaf growth. The weak auxin, beta-naphthalene acetic acid, was effective at 1.0 mm; and a weak acid control, benzoic acid, was ineffective. Indole-3-acetic acid (1 microm, 10 microm, 0.1 mm, and 1 mm) required daily application to be effective at any concentration. Application of the auxin transport inhibitor, 1-N-naphthylphthalamic acid (1% [w/w] in lanolin), to petioles also inhibited long-term leaf growth. This treatment also was found to lead to a sustained elevation of leaf free indole-3-acetic acid content relative to untreated control leaves. Auxin-induced inhibition of leaf growth appeared not to be mediated by auxin-induced ethylene synthesis because growth inhibition was not rescued by inhibition of ethylene synthesis. Also, petiole treatment of Arabidopsis with 1-N-naphthylphthalamic acid similarly inhibited leaf growth of both wild-type plants and ethylene-insensitive ein4 mutants.