Influence of Photoperiod and Leaf Age on Crassulacean Acid Metabolism in Portulacaria afra (L.) Jacq

The possibility that Crassulacean acid metabolism (CAM) is subject to long day photoperiodic control in Portulacaria afra (L.) Jacq., a facultative CAM plant, was studied. Periodic measurements of ¹⁴CO₂ uptake, stomatal resistance, and titratable acidity were made on plants exposed to long and short day photoperiods. Results indicates that waterstressed P. afra had primarily nocturnal CO₂ uptake, daytime stomatal closure, and a large diurnal acid fluctuation in either photoperiod. Mature leaf tissue from nonstressed plants under long days exhibited a moderate diurnal acid fluctuation and midday stomatal closure. Under short days, there was a reduced diurnal acid fluctuation in mature leaf tissue. Young leaf tissue taken from nonstressed plants did not utilize the CAM pathway under either photoperiod as indicated by daytime CO₂ uptake, lack of diurnal acid fluctuation, and incomplete daytime stomatal closure.

The induction of CAM in P. afra appears to be related to the water status of the plant and the age of the leaf tissue. The photosynthetic metabolism of mature leaves may be partly under the control of water stress and of photoperiod, where CAM is favored under long days.

     

Responses of succulents to plant water stress

Experiments were performed to test the hypothesis that succulents “shift” their method of photosynthetic metabolism in response to environmental change. Our data showed that there were at least three different responses of succulents to plant water status. When plant water status of Portulacaria afra (L.) Jacq. was lowered either by withholding water or by irrigating with 2% NaCl, a change from C₃-photosynthesis to Crassulacean acid metabolism (CAM) occurred. Fluctuation of titratable acidity and nocturnal CO₂ uptake was induced in the stressed plants. Stressed Peperomia obtusifolia A. Dietr. plants showed a change from C₃-photosynthesis to internal cycling of CO₂. Acid fluctuation commenced in response to stress but exogenous CO₂ uptake did not occur. Zygocactus truncatus Haworth plants showed a pattern of acid fluctuation and nocturnal CO₂ uptake typical of CAM even when well irrigated. The cacti converted from CAM to an internal CO₂ cycle similar to Peperomia when plants were water-stressed. Reverse phase gas exchange in succulents results in low water loss to carbon gain. Water is conserved and low levels of metabolic activity are maintained during drought periods by complete stomatal closure and continual fluctuation of organic acids.     

Characterization of carbon metabolism in Opuntia ficus-indica Mill. exhibiting the idling mode of Crassulacean acid metabolism

Upon transfer from well-watered conditions to total drought, long-day-grown cladodes of Opuntia ficus-indica Mill. shift from full Crassulacean acid metabolism (CAM) to CAM-idling. Experiments using ¹⁴C-tracers were conducted in order to characterize the carbon-flow pattern in cladodes under both physiological situations. Tracer was applied by ¹⁴CO₂ fumigations and NaH¹⁴CO₃ injections during the day-night cycle. The results showed that behind the closed stomata, mesophyll cells of CAM-idling plants retained their full capacity to metabolize CO₂ in light and in darkness. Upon the induction of CAM-idling the level of the capacity of phosphoenolpyruvate carboxylase (EC 4.1.1.31) was maintained. By contrast, malate pools decreased, displaying finally only a small or no day-night oscillation. The capacity of NADP-malic enzyme (EC 1.1.1.40) decreased in parallel with the reduction in malate pools. Differences in the labelling patterns, as influenced by the mode of tracer application, are discussed.Abbreviations CAM Crassulacean acid metabolism - PEP-Case phosphoenolpyruvate carboxylase     

Minor Physiological Response to Elevated CO(2) by the CAM Plant Agave vilmoriniana

One-year-old plants of the CAM leaf succulent Agave vilmoriniana Berger were grown outdoors at Riverside, California. Potted plants were acclimated to CO₂-enrichment (about 750 microliters per liter) by growth for 2 weeks in an open-top polyethylene chamber. Control plants were grown nearby where the ambient CO₂ concentration was about 370 microliters per liter. When the plants were well watered, CO₂-induced differences in stomatal conductances and CO₂ assimilation rates over the entire 24-hour period were not large. There was a large nocturnal acidification in both CO₂ treatments and insignificant differences in leaf chlorophyll content. Well watered plants maintained water potentials of −0.3 to −0.4 megapascals. When other plants were allowed to dry to water potentials of −1.2 to −1.7 megapascals, stomatal conductances and CO₂ uptake rates were reduced in magnitude, with the biggest difference in Phase IV photosynthesis. The minor nocturnal response to CO₂ by this species is interpreted to indicate saturated, or nearly saturated, phosphoenolpyruvate carboxylase activity at current atmospheric CO₂ concentrations. CO₂-enhanced diurnal activity of ribulose bisphosphate carboxylase activity remains a possibility.     

Shifts in the Carbon Metabolism of Xerosicyos danguyi H. Humb. (Cucurbitaceae) Brought About by Water Stress : II. Enzymology

Xerosicyos danguyi Humbert (Cucurbitaceae) is a leaf succulent endemic to Madagascar. Under well-watered conditions, the plant exhibited Crassulacean acid metabolism (CAM) but shifted to a dampened form of CAM, CAM-idling, when subjected to water stress. The purpose of this investigation was to examine the effects of a shift in carbon metabolism on phosphoenolpyruvate carboxylase and on NADP-malic enzyme in X. danguyi. Experiments were conducted to determine the diurnal patterns of enzyme activity and pH optima of both enzymes, as well as the approximate molecular mass, kinetic patterns, malate inhibition, and glucose-6-phosphate stimulation of phosphoenolpyruvate carboxylase. The two enzymes extracted from well-watered and water-stressed plants were similar in most parameters investigated; thus, CAM-idling appeared to be only a dampened form of CAM photosynthesis.     

Effects of abscisic acid on CAM in Portulacaria afra

Water stress induces Crassulacean acid metabolism (CAM) in Portulacaria afra as manifested by day stomatal closure, organic acid fluctuation, and night CO₂ uptake. We now have evidence that abscisic acid treatment of leaves causes partial stomatal closure that is accompanied by the induction of CAM in a manner similar to water stress. There appears to be an inverse relationship between exogenous CO₂ uptake and decarboxylation of organic acids in that organic acids remain high during the day providing stomata are open. When stomata close, there is consumption of organic acids by decarboxylation. The hypothesis is that stomatal opening controls CAM in this species.This material is based upon work supported by the Science and Education Administration of the USDA under Competitive Grant No. 5901-0410-8-0018-0.     

Characterization and Partial Purification of Aldose-6-phosphate Reductase (Alditol-6-Phosphate:NADP 1-Oxidoreductase) from Apple Leaves

The Pereskia are morphologically primitive, leafed members of the Cactaceae. Gas exchange characteristics using a dual isotope porometer to monitor ¹⁴CO₂ and tritiated water uptake, diurnal malic acid fluctuations, phosphoenolpyruvate carboxylase, and malate dehydrogenase activities were examined in two species of the genus Pereskia, Pereskia grandifolia and Pereskia aculeata. Investigations were done on well watered (control) and water-stressed plants. Nonstressed plants showed a CO₂ uptake pattern indicating C₃ carbon metabolism. However, diurnal fluctuations in titratable acidity were observed similar to Crassulacean acid metabolism. Plants exposed to 10 days of water stress exhibited stomatal opening only during an early morning period. Titratable acidity, phosphoenolpyruvate carboxylase activity, and malate dehydrogenase activity fluctuations were magnified in the stressed plants, but showed the same diurnal pattern as controls. Water stress causes these cacti to shift to an internal CO₂ recycling (“idling”) that has all attributes of Crassulacean acid metabolism except nocturnal stomata opening and CO₂ uptake. The consequences of this shift, which has been observed in other succulents, are unknown, and some possibilities are suggested.     

Correlation between CAM-Cycling and Photosynthetic Gas Exchange in Five Species of Talinum (Portulacaceae)

Photosynthetic gas exchange and malic acid fluctuations were monitored in 69 well-watered plants from five morphologically similar species of Talinum in an investigation of the ecophysiological significance of the Crassulacean acid metabolism (CAM)-cycling mode of photosynthesis. Unlike CAM, atmospheric CO₂ uptake in CAM-cycling occurs exclusively during the day; at night, the stomata are closed and respiratory CO₂ is recaptured to form malic acid. All species showed similar patterns of day-night gas exchange and overnight malic acid accumulation, confirming the presence of CAM-cycling. Species averages for gas exchange parameters and malic acid fluctuation were significantly different such that the species with the highest daytime gas exchange had the lowest malic acid accumulation and vice versa. Also, daytime CO₂ exchange and transpiration were negatively correlated with overnight malic acid fluctuation for all individuals examined together, as well as within one species. This suggests that malic acid may effect reductions in both atmospheric CO₂ uptake and transpiration during the day. No significant correlation between malic acid fluctuation and water-use efficiency was found, although a nonsignificant trend of increasing water-use efficiency with increasing malic acid fluctuation was observed among species averages. This study provides evidence that CO₂ recycling via malic acid is negatively correlated with daytime transpirational water losses in well-watered plants. Thus, CAM-cycling could be important for survival in the thin, frequently desiccated soils of rock outcrops on which these plants occur.     

Relationship between Respiration and CAM-Cycling in Peperomia camptotricha

Mature leaves of well-watered Peperomia camptotricha show Crassulacean acid metabolism (CAM). Young leaves show CAM-cycling in which CO₂ uptake occurs during the day concomitant with a marked diurnal fluctuation of organic acids as in CAM. Evidence is presented suggesting that respiration is the source of CO₂ for nocturnal acid synthesis in leaves exhibiting CAM-cycling. Respiratory quotients for these leaves were consistently much less than unity despite the fact that the leaves metabolize starch. The conservation of CO₂ by refixation into acids at night represents about 17% of the total photosynthetically fixed CO₂ and about 50% of the total respiratory CO₂.     

Effect of Severe Water Stress on Aspects of Crassulacean Acid Metabolism in Xerosicyos

Xerosicyos danguyi H.Humb. (Cucurbitaceae) is a Crassulacean acid metabolism (CAM) species native to Madagascar. Previously, it was shown that when grown under good water conditions, it is a typical CAM plant, but when water stressed, it shifts to a dampened form of CAM, termed CAM-idling, in which stomata are closed day and night but with a continued, low diurnal organic acid fluctuation. We have now studied the kinetics of some metabolic features of the shift from CAM to CAM-idling under severe water stress and the recovery upon rewatering. When water is withheld, there is a steady decrease in relative water content (RWC), reaching about 50%, at which point the water potential decreases precipitously from about -2 or -3 bars to -12 bars. Abscisic acid (ABA) increases sharply at about 75% RWC. Stomata close, which limits CO2 uptake, and there is a dampened diurnal organic acid fluctuation typical of CAM-idling. Throughout an extended stress period to 50% RWC, there is no change in chlorophyll, protein, and ribulose bisphosphate carboxylase activity compared with the well-watered plants. Despite the fact that the tissue was already in CAM, the stress is accompanied by an increase in phosphoenolpyruvate carboxylase (PEPc) mRNA, extractable PEPc activity, and PEPc protein (such that the specific activity remained approximately constant) and a decrease in the apparent Km(PEP). It is not known if the changes in Km(PEP) in response to drought are related to or are separate from the increases in PEPc protein and mRNA. The changes in Km(PEP) could be in response to the decreased endogenous levels of organic acids, but evidently are not an assay artifact. The increases in PEPc protein and mRNA appear to be related to the water-stress treatment and may result from the increased concentration of ABA or the decreased levels of endogenous organic acids. When rewatered, the metabolism quickly returns to the well-watered control typical of CAM.     

Comparison of photosynthesis and antioxidative protection in Sedum album and Sedum stoloniferum (Crassulaceae) under water stress

Photosynthetic gas exchange, dry mass production, water relations and inducibility of crassulacean acid metabolism (CAM) pathway as well as antioxidative protection during the C₃-CAM shift were investigated in Sedum album and Sedum stoloniferum from Crassulaceae under water stress for 20 days. Leaf relative water content (RWC), leaf osmotic and water potential decreased with increasing water stress in both studied species. Significant reduction in dry matter production and leaf thickness was detected only in S. stoloniferum after 20-d water stress. Δtitratable acidity and phosphoenolpyruvate carboxylase (PEPC) activity in S. album responded to drought at early stages of stress treatment, continued to increase throughout the entire stress period and reached levels 15 times higher than those in well-watered plants. In S. stoloniferum, however, both parameters responded later and after a transient increase declined again. In S. stoloniferum, in spite of increase by drought stress, net night-time CO₂ assimilation was negative resembling a C₃-like pattern of gas exchange. Catalase (CAT), ascorbate peroxidase (APX), and superoxide dismutase (SOD) activities increased in plants subjected to mild water stress while declined as the stress became severe. Although malondialdehyde (MDA) content was higher in drought-stressed S. stoloniferum, the increase in the concentration of hydrogen peroxide (H2O₂) that may act as a signal for C₃-CAM transition was higher in S. album compared with S. stoloniferum. In drought-stressed plants, SOD activity showed a clear diurnal fluctuation that was more steadily expressed in S. album. In addition, such pattern was observed for CAT only in S. album. We concluded that temporal and diurnal fluctuation patterns in the activity of antioxidant enzymes depended on duration of drought stress and was related to the mode of photosynthesis and degree of CAM induction. According to our results, S. stoloniferum developed a low degree of CAM activity, e.g. CAM-cycling metabolism, under drought conditions.     

Shifts in the Carbon Metabolism of Xerosicyos danguyi H. Humb. (Cucurbitaceae) Brought About by Water Stress : I. General Characteristics

Xerosicyos danguyi H. Humb. (Cucurbitaceae) is an unusual leaf succulent endemic to Madagascar. Under well-watered conditions the plant exhibited Crassulacean acid metabolism (CAM), as characterized by large diurnal changes in titratable acidity, predominantly nighttime stomatal opening and CO₂ uptake, and high δ¹³C values. When plants were exposed to water stress for a minimum of a month, they shifted to a mode of carbon metabolism previously labeled CAM-idling. Under this mode of metabolism, the plants exhibited reduced stomatal opening, reduced CO₂ uptake, dampened diurnal fluctuations in titratable acidity, and no apparent changes in the δ¹³C values. Additionally, investigations showed that the stress hormones 1-aminocyclopropane-1-carboxylic acid (an ethylene precursor) and abscisic acid increased as much as 6-fold in the water-stressed plants. The results are discussed in relation to physiological significance and evolution of the CAM-idling mode of metabolism.     

Fluorescence Quenching in the Varied Photosynthetic Modes of Portulacaria afra (L.) Jacq

The kinetics of chlorophyll fluorescence were measured in Portulacaria afra (L.) Jacq. when the plants were functioning in either Crassulacean acid metabolism (CAM) or C₃/CAM cycling (called cycling) modes, as determined by fluctuation in titratable acidity and gas exchange properties. Cycling plants showed primarily daytime CO₂ uptake typical of C₃ plants, but with a slight diurnal acid fluctuation, whereas CAM plants showed nocturnal CO₂ uptake, daytime stomatal closure, and a large diurnal acid fluctuation. Results from fluorescence measurements indicated no significant differences in photochemical quenching between cycling and CAM plants; however, sizable differences were detected in nonphoto-chemical quenching (q_n), with the largest differences being observed during the middle of the day. Cycling plants had lower q_n than CAM plants, indicating altered photosynthetic regulation processes. This q_n difference was believed to be related to reduced internal CO₂ concentration in the CAM plants because of daytime stomatal closure and reduced deacidification rates in the late afternoon when most of the malic acid has been utilized. Experimentally, higher external CO₂ given to plants in the CAM mode resulted in a decline in q_n in comparison to that measured in plants in the cycling mode. No changes were observed in photochemical quenching when CO₂ was added.     

Different CO<Subscript>2</Subscript> acclimation strategies in juvenile and mature leaves of <Emphasis Type="Italic">Ottelia alismoides</Emphasis>

The freshwater macrophyte, Ottelia alismoides, is a bicarbonate user performing C4 photosynthesis in the light, and crassulacean acid metabolism (CAM) when acclimated to low CO₂. The regulation of the three mechanisms by CO₂ concentration was studied in juvenile and mature leaves. For mature leaves, the ratios of phosphoenolpyruvate carboxylase (PEPC) to ribulose-bisphosphate carboxylase/oxygenase (Rubisco) are in the range of that of C4 plants regardless of CO₂ concentration (1.5–2.5 at low CO₂, 1.8–3.4 at high CO₂). In contrast, results for juvenile leaves suggest that C4 is facultative and only present under low CO₂. pH-drift experiments showed that both juvenile and mature leaves can use bicarbonate irrespective of CO₂ concentration, but mature leaves have a significantly greater carbon-extracting ability than juvenile leaves at low CO₂. At high CO₂, neither juvenile nor mature leaves perform CAM as indicated by lack of diurnal acid fluctuation. However, CAM was present at low CO₂, though the fluctuation of titratable acidity in juvenile leaves (15–17 µequiv g^?1 FW) was slightly but significantly lower than in mature leaves (19–25 µequiv g^?1 FW), implying that the capacity to perform CAM increases as leaves mature. The increased CAM activity is associated with elevated PEPC activity and large diel changes in starch content. These results show that in O. alismoides, carbon-dioxide concentrating mechanisms are more effective in mature compared to juvenile leaves, and C4 is facultative in juvenile leaves but constitutive in mature leaves.     

Induction of Crassulacean Acid Metabolism in the Facultative Halophyte Mesembryanthemum crystallinum by Abscisic Acid

The facultative halophyte, Mesembryanthemum crystallinum, shifts its mode of carbon assimilation from the C₃ pathway to Crassulacean acid metabolism (CAM) in response to water stress. In this study, exogenously applied abscisic acid (ABA), at micromolar concentrations, could partially substitute for water stress in induction of CAM in this species. ABA at concentrations of 5 to 10 micromolar, when applied to leaves or to the roots in hydroponic culture or in soil, induced the expression of CAM within days (as indicated by the nocturnal accumulation of total titratable acidity and malate). After applying ABA there was also an increase in phosphoenolpyruvate carboxylase and NADP-malic enzyme activities. The degree and time course of induction by ABA were comparable to those induced by salt and water stress. Electrophoretic analyses of leaf soluble protein indicate that the increases in phosphoenolpyruvate carboxylase activity during the induction by ABA, salt, and water stress are due to an increase in the quantity of the enzyme protein. ABA may be a factor in the stress-induced expression of CAM in M. crystallinum, serving as a functional link between stress and biochemical adaptation.     

Stimulation of CAM Photosynthesis in Kalanchoë blossfeldiana by Transferring to Nitrogen-Deficient Conditions

Kalanchoë blossfeldiana Poelln. cv Hikan plants were grown hydroponically with nutrient solution containing 5 millimolar NO₃⁻ (or NH₄⁺) for 1 to 2 months and then transferred to nutrient solution containing no nitrogen. CO₂ uptake at night, nocturnal increase in titratable acidity, and activity of phosphoenolpyruvate carboxylase increased after the transfer. Thus, transfer to nitrogen-deficient conditions stimulates Crassulacean acid metabolism (CAM photosynthesis) in K. blossfeldiana. The importance of the plant nitrogen status (nitrogen-withdrawal status) for induction and stimulation of CAM photosynthesis is discussed.     

Carbon Dioxide Fixation in Roots and Nodules of Alnus glutinosa: I. Role of Phosphoenolpyruvate Carboxylase and Carbamyl Phosphate Synthetase in Dark CO(2) Fixation, Citrulline Synthesis, and N(2) Fixation

Detached roots and nodules of the N₂-fixing species, Albus glutinosa (European black alder), actively assimilate CO₂. The maximum rates of dark CO₂ fixation observed for detached nodules and roots were 15 and 3 micromoles CO₂ fixed per gram dry weight per hour, respectively. The net incorporation of CO₂ in these tissues was catalyzed by phosphoenolpyruvate carboxylase which produces organic acids, some of which are used in the synthesis of the amino acids, aspartate, glutamate, and citrulline and by carbamyl phosphate synthetase. The latter accounts for approximately 30 to 40% of the CO₂ fixed and provides carbamyl phosphate for the synthesis of citrulline. Results of labeling studies suggest that there are multiple pools of malate present in nodules. The major pool is apparently metabolically inactive and of unknown function while the smaller pool is rapidly utilized in the synthesis of amino acids. Dark CO₂ fixation and N₂ fixation in nodules decreased after treatment of nodulated plants with nitrate while the percentage of the total ¹⁴C incorporated into organic acids increased. Phosphoenolpyruvate carboxylase and carbamyl phosphate synthetase play key roles in the synthesis of amino acids including citrulline and in the metabolism of N₂-fixing nodules and roots of alder.     

Light and dark CO2 fixation in Clusia uvitana and the effects of plant water status and CO2 availability

Summary In well-watered plants of Clusia uvitana, a species capable of carbon fixation by crassulacean acid metabolism (CAM), recently expanded leaves gained 5 to 13-fold more carbon during 12 h light than during 12 h dark periods. When water was withheld from the plants, daytime net CO₂ uptake strongly decreased over a period of several days, whereas there was a marked increase in nocturnal carbon gain. Photosynthetic rates in the chloroplasts were hardly affected by the water stress treatment, as demonstrated by measurements of chlorophyll a fluorescence of intact leaves, indicating efficient decarboxylation of organic acids and refixation of carbon in the light. Within a few days after rewatering, plants reverted to the original gas exchange pattern with net CO₂ uptake predominantly occurring during daytime. The reversible increase in dark CO₂ fixation was paralleled by a reversible increase in the content of phosphoenolpyruvate (PEP) carboxylase protein. In wellwatered plants, short-term changes in the degree of dark CO₂ fixation were induced by alterations in CO₂ partial pressure during light periods: a decrease from 350 to 170 bar CO₂ caused nocturnal carbon gain, measured in normal air (350 bar), to increase, whereas an increase to 700 bar CO₂, during the day, caused net dark CO₂ fixation to cease. The increased CAM activity in response to water shortage may, at least to some extent, be directly related to the reduced carbon gain during daytime.     

Ecophysiological Significance of CO(2)-Recycling via Crassulacean Acid Metabolism in Talinum calycinum Engelm. (Portulacaceae)

High levels of variability in gas exchange characteristics and degree of CAM-cycling were found in the same and different individuals of Talinum calycinum Engelm. collected from rock outcrops in Missouri. Differences in CO₂ assimilation were mostly correlated with differences in shoot conductance to CO₂ not shoot internal CO₂ concentration. As found previously, CAM acid fluctuations were evident in well-watered plants exhibiting C₃ gas exchange patterns (CAM-cycling) and also in drought-stressed plants with stomata closed, or nearly so, day and night (CAM-idling). Drought stress also resulted in rapid stomatal closure, conserving water during droughts. Maximal CO₂ uptake rates occurred below 35°C; higher temperatures induced decreases in CO₂ assimilation and conductance while shoot internal CO₂ concentrations remained similar. Plant water-use-efficiency was severely curtailed at temperatures above 30°C. Tissue acid fluctuations were the result of changes in malic acid concentrations. Calculations of the amount of water potentially conserved by CAM-cycling yielded values of approximately 5 to 44% of daytime water loss. Thus, CAM-cycling may be an important adaptation minimizing water loss by perennial succulents growing in shallow soil on rock outcrops.     

Effects of exogenous glycinebetaine on growth, CO2 assimilation, and photosystem II photochemistry of maize plants

Effects of exogenous glycinebetaine (GB, 2–50 mM) on growth, photosynthetic gas exchange, PSII photochemistry, and the activities of key enzymes involved in CO₂ fixation in maize plants were investigated. Growth, CO₂ assimilation rate, and stomatal conductance increased at low GB concentrations (2–20 mM) but decreased significantly at high GB concentrations (30–50 mM). Leaf relative water content and water potential remained unchanged at low GB concentrations but decreased at high GB concentrations. The maximal efficiency of PSII photochemistry was unchanged either at low or high GB concentrations. The actual PSII efficiency ( Φ _PSII) and photochemical quenching (q_P) increased at low GB concentrations but decreased at high GB concentrations. At low GB concentrations, there were no significant changes in the efficiency of excitation energy capture by open PSII reaction centres (F_v′/F_m′) and non‐photochemical quenching (q_N). At high GB concentrations, F_v′/F_m′ decreased while q_N increased significantly. There were no changes in the activities of phosphoenolpyruvate carboxylase, pyruvate phosphate dikinase, and ribulose‐1,5‐bisphosphate carboxylase in control and GB‐fed plants. However, there was a linear correlation between CO₂ assimilation rate and stomatal conductance in control and GB‐fed plants. Moreover, there were no significant differences in O₂ evolution rate between control and GB fed‐plants under saturated CO₂ conditions. The results suggest that exogenous GB application at certain concentrations can enhance CO₂ assimilation rate, which can be explained by an increased stomatal conductance.