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Enhanced Organic Phosphorus Assimilation Promoting Biomass and Shoot P Hyperaccumulations in Lolium multiflorum Grown under Sterile Conditions Nilesh C. Sharma*,† and Shivendra V. Sahi† †
Department of Biology, Western Kentucky University, Bowling Green, 1906 College Heights Boulevard, Kentucky 42101-1080, United States ABSTRACT: Search for plant species prodigious in P use is important for both P-sufficient and -deficient conditions. Gulf and Marshall ryegrass (Lolium multiflorum), grown in sterile media containing different organic P substrates (AMP, ATP, GMP, and IHP), exhibited high rates of growth and shoot P concentrations. Growth increase in Gulf was significantly greater on IHP relative to other sources of organic P substrates. Growth was also dependent on an increasing concentration of IHP (020 mM) in this cultivar. P accumulations in Gulf exceeded 1% shoot dry weight from IHP, AMP, and ATP— equivalent to the P accrual from equimolar Pi source. Plants supplied with IHP had phytase activity in root extracts comparable to that in Pi-fed plants or control (no P). The extracellular phytase, however, increased by about 100% relative to that observed in root extracts- for both ryegrass cultivars, but there were no significant differences (P < 0.05) between plant groups grown on different substrates (IHP, Pi or control). No significant differences in phosphomonoesterase activities were evident between plant groups supplied with organic P (IHP, G1P) and inorganic source or control. This study establishes the high P-use efficiency in ryegrass, irrespective of P source.
’ INTRODUCTION Soil chemistry of phosphorus (P) has attracted more attention in recent times than in previous decades, especially in relation to temperate climate agriculture with over application of organic manures rich in P. Soils typically contain a significant quantity of P, but only a small proportion (less than 1%) of the total P is available to plants.1 Organic and inorganic fractions constitute the sum total of soil P. Organic P varies in a wide range (3080%) depending on the type of soils,2 phosphomonoesters constituting the bulk (up to 90%) of the organic P fraction.3 Phytate, the derivative of inositol hexakis-phosphate (IHP), is the most abundant form of phosphomonoesters present in most of the soils, particularly in temperate climates.4 Besides phosphomonoesters, sugar phosphates and phosphate diesters also occur in soils, however, in small quantity, about 5% of total organic P.2 Plants are known to uptake only phosphate anions to meet their P requirement. Thus to be available to plants, organic P must be hydrolyzed by the activity of phosphatase enzymes to release phosphate. Phosphatase activity has also been used as an indicator of organic phosphorus mineralization.5 Phosphatase activity in soil is mainly controlled by soil microorganisms6 although the contribution of plants to the pool of soil phosphatase cannot be undermined. Plants secrete extracellular phosphatase in rhizospheres.7,8 In natural ecosystems, mineralization of soil organic P is considered to provide a major source of plant-available P.9,10 Controlled studies have also demonstrated that mineralization of organic P occurs significantly in soils to affect phosphate uptake by plants.11,12 However, the direct r 2011 American Chemical Society
contribution of hydrolyzed organic P to plant P nutrition remains unclear. Phosphatases with differing substrate specificities for phosphomonoesters and phosphodiesters have been characterized in plant roots.8 6-Phytase (EC 3.1.3.26) is a class of acid phosphatase with high affinity for phytate that may constitute up to 80% of a total organic P2 and may thus be especially important for the hydrolysis of organic P in soils. The presence of extracellular phosphatase in plant roots is supported further by other studies that have directly measured many-fold increases in phosphomonoesterase activity in root zones of plants.6,13 Nevertheless, it is difficult to separate the direct contribution of plants to an observed increase in the activity of phosphatases within the rhizosphere because of the fact that simultaneous increase in microbial population and activity also occurs.14,15 Therefore, the following studies focused on assessing organic P utilization by plants grown in sterile media or under controlled conditions. Wheat seedlings, when grown aseptically in media containing different forms of organic P, had differential ability of P acquisition from the different organic P substrates.16,17 These studies also demonstrated a correlation between organic P use and the activities of phytase and acid phosphomonoesterase. Transgenic Arabidopsis plants modified with phytase gene (phyA) derived from Aspergillus niger demonstrated improved growth and Received: March 21, 2011 Accepted: October 28, 2011 Revised: September 30, 2011 Published: October 28, 2011 10531
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Environmental Science & Technology P nutrition when supplied with phytate as a source of P.18 Improved utilization of phytate was also reported in transgenic clover and barrel medic that expressed phytase genes, phyA and MtPHY1, respectively, and released extracellular phytase.19,20 Annual ryegrass (Lolium multiflorum), closely related to perennial ryegrass (Lolium perenne), is grown all over the world as a key forage grass.21 Marshall and Gulf ryegrass different cultivars of Lolium multiflorum were recently shown to grow and accumulate P in their shoots when seedlings were grown in hydroponic media and soils containing high concentrations of inorganic P.21,22 Using scanning electron microscopy and electron-dispersive X-ray spectroscopy, it was also shown that the accumulation of P occurs in the cortical and stellar regions of ryegrass shoot.21 These studies formed the basis of evolving a P-phytoremediation strategy using above cultivars for the soils loaded with organic manure and P. Mining of soil P, which includes harvesting P taken up from the soil by a crop grown without external P application, has been proposed as a possible management strategy for P-enriched soils.23 In the above backdrop, particularly keeping in view the unique feature of annual ryegrass, it was interesting to reveal in this species the pattern of P nutrition using organic P sources. In the present study, experiments were thus designed to determine i) plant growth and ii) the quantity of total P stored in shoots and elucidate iii) the relationship between organic P use and root activities of phytase and acid phosphomonoesterase when Marshall- and Gulf ryegrass were grown aseptically in media fortified with a range of organic P substrates (AMP, ATP, GMP, and phytate).
’ EXPERIMENTAL SECTION Seed Germination. Seeds of Marshall- and Gulf ryegrass (Lolium multiflorum cultivars), provided by USDA Lab, Starkville, MS, were sterilized with sodium hypochlorite (1% v/v) and rinsed several times with sterile deionized water. They were then transferred to water-agar (0.8%) medium in Magenta boxes and maintained at 25 ( 2 °C under 12/12 light/dark regime in a growth chamber. Ten day-old seedlings isolated from agar medium were rinsed with deionized water before the aseptic transfer in different experiments. Growth of Seedlings. Half strength Modified Hoagland’s salts mixture (115 mg/L ammonium nitrate, 2.86 mg/L boric acid, 656.4 mg/L calcium nitrate, 3.0 mg/L ferric chloride, 240.7 mg/L magnesium sulfate, 1.81 mg/L manganese chloride, 0.016 mg/L molybdenum trioxide, 400.6 mg/L potassium nitrate, and 0.22 mg/L zinc sulfate) containing 0.8% (w/v) agar was used as a basal growth medium. To examine the effect of different P substrates, medium was supplemented either with no P (control) or 5 mM each of α-D-glucose 1-phosphate disodium salt (G1P), adenosine 30 :50 cyclic monophosphate sodium salt (AMP), adenosine-50 -triphosphate disodium salt (ATP), myoinositol hexakis-phosphoric acid dodecasodium salt (IHP), or KH2PO4 (Pi) [Sigma Chemical Co., St. Louis, MO]. Another experiment was set up to measure the effect of increasing concentrations (020 mM) of IHP. Three plants were transferred aseptically to each culture tube (15 cm 2.5 cm) containing 15 mL of the medium. Plants were grown in a growth chamber at 25 ( 2 °C in 16/8 light/dark (180200 μmol m2 s1 of cool fluorescent light) for different periods (2 and 5 weeks). Each treatment was replicated three times and experiments were repeated (n = 6).
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Determination of Fresh and Dry Weight. Plants harvested either from 2 weeks of growth (on G1P, AMP, ATP, IHP or Pi) or from 5 weeks of growth (on 020 mM IHP) were measured for the growth of biomass. Harvested shoots pooled from different treatment replicates were blotted with the filter paper before the fresh weight determination and dried in an oven at 70 °C for 48 h for dry weight determination. The average weight of three plants per replicate was determined. Growth was measured by the difference in the final and initial weights of each replicate. Analysis of P in Plant Tissue. Plants from different treatments were harvested, washed thoroughly with deionized water, divided into root and shoot biomass, and dried in an oven at 70 °C for two days. The ground samples were then weighed and placed in 15 mL Teflon beakers. Three mL of concentrated HNO3 was added to the sample, and the beaker was placed on a hot plate set at 100 °C overnight, until evaporated to dryness. The samples were allowed to cool and made up gravimetrically to a volume of 20 mL with 2% HNO3. A VG Elemental Plasma Quad (model PQZ) ICP-AES was used for all data acquisition. Analyses were performed using an external calibration procedure, and internal standards were included to correct for matrix effects and instrumental drift corrections.21,22 Preparation of Root Extracts and Phosphomonoesterase Assay. After 2 weeks of growth on different P treatments, plants were harvested and washed thoroughly with DI water followed by rinse in 2-morpholinoethanesulfonic acid, monohydrate (MES) buffer solution (pH 5.5). Roots were separated, chilled on ice, and homogenized with a mortar and pestle in 15 mM MES buffer (pH 5.5, 0.5 mM CaCl2 3 H2O, 1 mM EDTA). The buffer was added at a ratio of 1:5 (root fresh weight: extraction buffer volume). The extract was centrifuged (13,000 g; 15 min at 4 °C), and the supernatant was used for enzyme assay.16 For the assay of phosphomonoesterase activity, enzyme extract (50 μL) was incubated in a total volume of 500 μL of 15 mM MES buffer (pH 5.5, 0.5 mM CaCl2) in the presence of 10 mM p- nitrophenylphosphate, disodium salt (Sigma-Aldrich, St. Louis, MO).16,18 The assay was conducted over 30 min, and reactions were terminated by equal volumes of 0.25 M NaOH. The enzyme activity was calculated from the release of p-nitrophenol (pNP), determined at 412 nm (relative to standard solutions) by a UVvis spectrophotometer (Model Ultrospec 3000, Pharmacia Biotech, USA). Phytase Assay. Harvested plant roots were treated and homogenized as described above. To assay for phytase activity, a 500 μL enzyme extract was incubated in a total volume of 1 mL of 15 mM MES buffer (pH 5.5, 0.5 mM CaCl2) in the presence of 2 mM myo-inositol hexaphosphoric acid (Sigma-Aldrich, St. Louis, MO).16,18 The assay was conducted over 60 min, and reactions were terminated by addition of equal volumes of ice-cold 10% trichloroacetic acid (TCA). Solutions were subsequently centrifuged to remove precipitated material, and their phosphate concentrations were determined by measuring absorbance at 882 nm using the molybdenum-blue reaction.16 Phosphate concentrations were recorded at a fixed time within 1 h following addition of the color reagent to samples, to minimize possible interference. The enzyme assays were conducted at 26 °C using three replicates. Phosphomonoesterase and phytase activities were expressed in mU g1 root fresh weight (FW), where 1 U is defined as the release of 1 μmol of Pi min1 under the assay conditions. Extracellular phytase activity was determined for plants harvested from media containing 5 mM each of IHP, Pi, or control 10532
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Figure 1. (A-B). A. Fresh weight of Gulf (a-c) and Marshall ryegrass (d-f) B. Dry weight of Gulf (a-d) and Marshall ryegrass (e-f) grown in sterile media containing 5 mM each of G1P, AMP, ATP, IHP, Pi, or no P (control) for a period of 2 weeks. Treatment means (n = 6) labeled with not the same notations are significantly different (P < 0.05) in each cultivar.
(no P). After the growth period (2 weeks), plants were carefully removed from the agar and incubated in 10 mL of modified halfstrength Hoagland’s solution (containing no P) for another 16 h under light to collect root exudate. Aliquots of root exudate were analyzed for the enzyme activity as described above. Phytase activity was expressed in mU g1 root fresh weight/day, where 1 U is defined as the release of 1 μmol of Pi min1 under the assay conditions. Statistical Analysis. The data were analyzed by one-way analyses of variance using SYSTAT (Version 9 for Windows, 1999, Systat Software Inc., Richmond, CA). Where variance ratios were significant (p < 0.05), treatment means were compared using LSD (P = 0.05) for each cultivar separately.
’ RESULTS Plant Growth on Media Containing Different P Substrates or No P for 2 Weeks. Gulf ryegrass registered differential fresh
weight growth on different organic substrates (Figure 1A). Growth on G1P (236 mg) and ATP (250 mg) was not significantly different than in control in both cultivars (Figure 1A). However, Gulf displayed growth significantly greater on AMP (312 mg) and IHP (490 mg) than control. IHP-supplemented medium supported even greater increase in fresh weight than Pi containing medium (381 mg). The pattern of growth in Marshall ryegrass was different; when growth on G1P (213 mg) was comparable to control (180 mg), it declined on AMP (156 mg) (Figure 1A). Further, Marshall registered significantly greater growth on ATP (251 mg) and IHP (282 mg) with respect to control but not with respect to Pi (266 mg). A similar trend was noticed in respect of dry weight increase in both cultivars (Figure 1B). Dry weight increase in Gulf was significantly greater on organic substrates (25.357.8 mg) than control (18.2). Gulf growth in IHP (57.8 mg) was again significantly greater than in other organic substrates (Figure 1B). Marshall registered dry weight growth in a relatively narrow range of 19.731.3 mg; IHP growth not being significantly greater than Pi growth (Figure 1B) similar to the fresh weight pattern. Plant Growth on Media Containing 020 mM of IHP or 20 mM of Pi for 5 Weeks. Both cultivars displayed a significant increase (P < 0.05) in fresh weight depending on increasing concentrations of IHP (Figure 2A). Fresh weight growth ranged from 396 to 584 mg on increasing concentrations of IHP (120 mM). Again, growth in Gulf was greater than in Marshall on all concentrations of IHP (Figure 2A). A similar trend was noticeable with respect to dry weight increase in Gulf (Figure 2B). In
Figure 2. (A-B). A. Fresh weight of Gulf (a-d) and Marshall ryegrass (ef) B. Dry weight of Gulf (a-d) and Marshall ryegrass (e-f) grown in sterile media containing (020 mM) of IHP or 20 mM of Pi for a period of 5 weeks. Treatment means (n = 6) labeled with not the same notations are significantly different (P < 0.05) in each cultivar.
Marshall, dry weight increased with an increase in IHP concentration up to 10 mM and declined thereafter. However, Marshall dry weight gain at 10 mM IHP was comparable to that of 20 mM Pi. Accumulation of P by Plants Grown on Media Containing Different P Substrates or No P for 2 Weeks. Shoot accumulation of P was significantly higher (P < 0.05) on all media than control (no P). Accumulations of P from organic substrates (AMP, ATP, and IHP) ranged from 11,47011,950 mg/kg greater than 1.1% shoot dry weight in Gulf. These accumulations were comparable to the accumulation from inorganic P source, KH2PO4 (11,890 mg/kg) (Figure 3A). Marshall ryegrass exhibited a differential pattern with a maximum P accumulation of 11,930 mg/kg over 1.1% shoot dry weight from ATP-containing medium, followed by accumulation from AMP- medium (Figure 3A). The accumulation from IHP medium was not significantly different (P > 0.05) than from Pi in this cultivar as well. Accumulation of P by Plants Grown on Media Containing 020 mM of IHP or 20 mM of Pi for 5 Weeks. In both cultivars, P accumulation was dependent on increasing concentrations up to 10 mM IHP, after which accumulation leveled off (Figure 3B). However, accumulations in Gulf were significantly greater (P < 0.05) than in Marshall up to 10 mM IHP. There were no significant differences in accumulations from 20 mM IHP and 20 mM Pi in both cultivars (Figure 3B). Phytase and Phosphomonoesterase Activities in Plant Roots Grown on Different P Substrates or No P for 2 Weeks. Figure 4A shows a differential pattern of phytase activities. Both cultivars grown on IHP exhibited a similar activity of phytase 10533
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Figure 3. (A-B). Shoot P concentration in Gulf (a-c) and Marshall ryegrass (d-f) grown in sterile media containing A. 5 mM each of G1P, AMP, ATP, IHP, Pi, or no P (control). B. 020 mM of IHP or 20 mM of Pi for a period of 5 weeks. Treatment means (n = 6) labeled with not the same notations are significantly different (P < 0.05) in each cultivar.
Figure 4. (A-B). A. Phytase activity. B. Phosphomonoesterase activity in Gulf (a-b) and Marshall ryegrass (c-e) grown in sterile media containing 5 mM each of G1P, AMP, ATP, IHP, Pi, or no P (control) for a period of 2 weeks. Treatment means (n = 6) labeled with not the same notations are significantly different (P < 0.05) in each cultivar.
comparable to control (no P) and Pi-plants, ranging from 67.3 mU/g (FW). However, phytase activities were reduced, approximately, 3-fold in media enriched with G1P, AMP, or ATP. Phosphomonoesterase activities of plants grown in G1P, IHP, or Pi were not significantly different than control (without P), varying in a range of 600740 mU/g FW (Figure 4B). Similar to phytase activities, plants grown in AMP or ATP had lower values of phosphomonoesterase activity (400450 mU/g FW). Extracellular Phytase Activity in Plants Harvested from IHP, Pi, or Control Media. Secreted phytase activity in Gulf and Marshall ryegrass grown on media containing 5 mM each of IHP, Pi, or control (no P) was measured. The secreted activity ranged from 13.018.2 mU/g FW/day in both cultivars (Table 1). Phytase activities among different groups of Gulf were not significantly different, while activity was significantly different in Marshall grown in IHP and Pi.
’ DISCUSSION Both cultivars of annual ryegrass registered significant fresh and dry weight growth when grown in the presence of different P substrates with respect to control (without P) (Figure 1A-B). However, biomass increase in Gulf ryegrass was generally higher than in Marshall ryegrass on organic and inorganic P (KH2PO4) sources. Growth in these grasses also correlated with increasing concentrations (120 mM) of IHP (Figure 2A-B). An interesting pattern noticed in case of Gulf ryegrass was that its growth on IHP (20 mM) was comparable to that in equimolar Pi medium.
However, it cannot be undermined that equimolar IHP can release three times more Pi on enzyme-catalyzed hydrolysis. Growth profile of these cultivars certainly indicates that these plants can draw P from organic substrates, particularly IHP, as efficiently as from an inorganic source to reach an optimum level of growth. Several studies show that growth had significantly reduced when Arabidopsis,18 Trifolium,19 and Solanum24 wild-type plants were grown with organic substrates, particularly IHP. Wheat plants grown in IHP, in similar conditions, exhibited more than 40% reduction in mean shoot dry weight compared to Pi-fed controls.16 However, shoot growth in wheat was not significantly different (P < 0.05) for other sources of P treatments (AMP, G1P, ATP, and PGA) relative to Pi-fed controls. Figure 3(A-B) clearly indicates that both cultivars had significantly high shoot P accumulations from organic and inorganic P sources. Gulf plant acquisitions from IHP, AMP, and ATP exceeded 1% (shoot DW) similar to its uptake from Pi. Marshall exhibited a maximum shoot P concentration (>1% DW) when grown on ATP; P acquisition from IHP not being significantly (P < 0.05) different than from Pi. When ryegrass P use from IHP surpassed many wild-type species (Arabidopsis, Solanum, Trifolium) tested in similar conditions, it compared well with transgenic lines of those species expressing Aspergillus phytase gene, phyA.18,19,24 Transgenic Arabidopsis lines expressing ex: phyA demonstrated several-fold increase in P use and plant growth when grown in IHP-supplemented media.18 Shoot P concentrations in these lines exceeded 1% (shoot DW) as observed in our study. Similarly, P acquisition from phytate in sterile media 10534
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Table 1. Secreted Phytase Activity (mU/g Root Fresh Weight/Day) in Gulf and Marshall Ryegrass Grown on Media Containing 5 mM Each of IHP, Pi, or no P (Control)a media containing IHP KH2PO4 control (0P)
Gulf ryegrass
Marshall ryegrass
a
14.3a
a
18.2b
a
16.9a
16.0 13.7
13.0
a
Values represent the mean of 6 replicates and, within each column, those not followed by the same letter are significantly different (P < 0.05).
was increased substantially in a transgenic potato24 and Arabidopsis25 expressing root hair-specific phytase—similar to our observations in this study. High rates of P accumulation in the present study are also consistent with earlier reports when Gulf and Marshall ryegrass was characterized as a potential P accumulator using different regimes of inorganic P.21,22 It is evident from Figure 4A that plants supplied with IHP had phytase activity in soluble extracts comparable to activities in Pifed plants and control (no P). The activity in Gulf and Marshall ryegrass was unlike wheat where plants grown with no P had significantly greater activity (P < 0.05) than those grown in IHP and Pi.16 Nevertheless, wild-type Arabidopsis had phytate activity trend comparable to our observations in this study.18 It was also observed that phytase activity in ryegrass was significantly reduced on G1P, AMP, and ATP relative to IHP. Similar reduction, however, was not uncommon to wheat and Arabidopsis grown on G1P.16,18 The phytase activity in the external solution (secreted phytase), however, increased by about 100% relative to that observed in soluble extracts in both of the cultivars, but there were no significant differences (P < 0.05) between plant groups (IHP, Pi, or control). Figure 4B shows a different pattern of phosphomonoesterase activities in ryegrass. No significant differences in phosphomonoesterase activities were evident between plant groups that were supplied with no P or inorganic or organic P sources (IHP, G1P)—similar to the trend reported in wheat and Arabidopsis.16,18 From the above account it is evident that ryegrass, compared to many plant species, exhibits high efficiency in IHP utilization as reflected in appreciable shoot biomass and P accumulations despite having phytase activity equivalent to 12% of total phosphomonoesterase. Interestingly, the level of P and biomass accumulations in IHP-plants matched to the plants grown in inorganic P. However, some intriguing questions remain with regard to AMP- and ATP-plants where a reduced phosphomonoesterase activity negatively correlated (P < 0.05) to the increased P accumulation. It can be understood that AMP, belonging to the class of phosphodiesters, requires phosphodiesterases to be catalyzed, and the same was not estimated in the present study. Several transgenic lines of Trifolium,19 Solanum,24 and Arabidopsis25 overexpressing (several-fold) extracellular phytase have been reported to acquire P from IHP equal or comparable to the level of contributions that accrue from inorganic source of P. From this standpoint, ryegrass is unique with efficient P nutrition and biomass production utilizing IHP at the expense of a small amount of phytase (12% of total phosphomonoesterases). The observation that enzyme activities are independent of P supply (deficiency in case of control- no P- and P sufficiency in the case of IHP or Pi) makes Gulf ryegrass more interesting with respect to P nutrition. It also points to the probability of
constitutive expression of phosphatase genes, influencing P metabolism in this species. Only further studies can answer this hypothesis. Many researchers recently attempted to answer the question as why several plant species demonstrate poor ability to utilize phytate from soil and have scanty phytase production despite possessing the phytate gene (phyA). The consensus view that emerges is that the evolutionary pressure for utilization of P from phytate is constrained by factors such as i) low availability of phytate in soil solution or rhizosphere that can be accessed by plants and ii) propensity of phytate to undergo precipitation and sorption reactions in soil environments.18 In this context, what makes plant phytase a critical player in the use of P from phytate is its copious secretion into soil to reach out its substrate. This fact is further substantiated by the demonstrated role of transgenic lines (overexpressing extracellular phytase) belonging to a number of species discussed earlier.19,24,25 Thus the search for wild-type crop species or development of transgenics that are more efficient in P utilization from soil would be particularly beneficial to agriculture in reducing the dependence on P-based fertilizers with diminishing natural resource. Another dimension of P use-efficient species is in their application in P mining from P-loaded soils P phytoremediation, as argued by Novak and Chan.23 Though there are few species reported so far as an effective P remover from soils, annual ryegrass is an interesting candidate for P phytoremediation.22 Studies have shown annual ryegrass efficacy in P removal from P-enriched conditions: hydroponic medium21 to soils acidic, alkaline,22 to soils enriched with poultry litter.26 The instant study while confirming earlier reports of high P use and accumulation in annual ryegrass provides evidence for a biochemical basis of P nutrition from organic P sources, particularly phytate. Hayes et al.,9 when compared P nutrition between some pasture legume and grass species, observed that the legume species (Trifolium spp. and Medicago polymorpha) were more efficient in P use and had higher levels of phytase activity compared to grass species. Higher P acquisition efficiency in legumes was also implicated with plant attributes such as follows: I. high storage capacity for inorganic P; II. a favorable ratio of P uptake per unit root length; and III. a high activity of acid phosphatase in the root and capacity to use P from organic P sources when they were grown in soils enriched with organic P.27 This study, in conjunction with previous observations on ryegrass, amply shows that annual ryegrass possesses all these attributes to be efficient in P use. In an interesting comparative study, when Duo grass (Duo festulolium) a hybrid between fescue and ryegrass was grown in similar conditions, sterile solutions containing IHP and other organic substrates, it exhibited comparable phytase and significantly greater (
