Trees (2004) 18:175–183 DOI 10.1007/s00468-003-0267-x

ORIGINAL ARTICLE

Shaoliang Chen · Moitaba Zommorodi · Eberhard Fritz · Shasheng Wang · Aloys H
ttermann

Hydrogel modified uptake of salt ions and calcium in Populus euphratica under saline conditions Received: 27 April 2002 / Accepted: 1 April 2003 / Published online: 8 November 2003
Springer-Verlag 2003

Abstract The effects of hydrogel on growth and ion relationships of a salt resistant woody species, Populus euphratica , were investigated under saline conditions. The hydrogel used was Stockosorb K410, a highly crosslinked polyacrylamide with about 40% of the amide group hydrolysed to carboxylic groups. Amendment of saline soil (potassium mine refuse) with 0.6% hydrogel improved seedling growth (2.7-fold higher biomass) over a period of 2 years, even though plant growth was reduced by salinity. Hydrogel-treated plants had approximately 3.5-fold higher root length and root surface area than those grown in unamended saline soil. In addition, over 6% of total roots were aggregated in gel fragments. Tissue and cellular ion analysis showed that growth improvement appeared to be the result of increased capacity for salt exclusion and enhancement of Ca2+ uptake. X-ray microanalysis of root compartments indicated that the presence of polymer restricted apoplastic Na+ in both young and old roots, and limited apoplastic and cytoplastic Cl in old roots while increasing Cl compartmentation in cortical vacuoles of both young and old roots. Collectively, radical transport of salt ions (Na+ and Cl) through the cortex into the xylem was lowered and subsequent axial transport was limited. Hydrogel treatment enhanced uptake of Ca2+ and microanalysis showed that enrichment of Ca2+ in root tissue mainly occurred in the apoplast. In conclusion, enhanced Ca2+ uptake and the increased capacity of P. euphratica to exclude salt were the result of improved Ca2+/Na+ concentration of soil solution available to the plant. Hydrogel amendment improves the quality of soil solutions by lowering salt level as a result of its salt-buffering capacity and S. Chen ()) · S. Wang College of Biological Sciences and Technology, Beijing Forestry University, Box 162, 100083 Beijing, PR China e-mail: [email protected] M. Zommorodi · E. Fritz · A. Httermann Forstbotanisches Institut, Universitt Gttingen, Bsgenweg 2, 37077 Gttingen, Germany

enriching Ca2+ uptake, because of the polymer’s cationexchange character. Accordingly, root aggregation allows good contact of roots with a Ca2+ source and reduces contact with Na+ and Cl, which presumably plays a major role in enhancing salt tolerance of P. euphratica. Keywords Polymer · Growth · Root · Ion compartmentation · Populus euphratica

Introduction Hydrogels, which were developed to increase water holding capacity of amended media, have been used to aid plant establishment and growth in dry soils. Plants grown in substrates amended with hydrophilic polymers were slower to wilt than those in unamended medium (Still 1976; Conover and Poole 1979; Gehring and Lewis 1980; Baasiri et al. 1986; Woodhouse and Johnson 1991). There is other evidence showing that the presence of polymers prolongs the survival of plants (Callaghan et al. 1988; Woodhouse and Johnson 1991), and increases water use efficiency and dry matter production during periods of drought (Azzam 1983, 1985; Wooodhouse and Johnson 1991). Recently, the effects of application of hydrogel on crops grown in substrates have been investigated under saline conditions. Hydrogel amendment improved emergence of wheat (Saleh and Hussein 1987), germination of maize pollen (El-Sayed and Kirkwood 1992), and salt tolerance and growth of certain horticultural crops, including barley, tomato, cucumber and lettuce (Awad et al. 1986; Szmidt and Graham 1990; El-Sayed et al. 1991). In most of these studies, cross-linked waterinsoluble but swellable hydrogels, e.g. poly(ethylene oxide) hydrogel (Szmidt and Graham 1990), polyacrylamide hydrogel (Awad et al. 1986) and cross-linked poly(ethylene oxide)-co-polyurethane hydrogel (El-Sayed et al. 1991; El-Sayed and Kirkwood 1992) were usually used under saline conditions. However, little is known

176

about the effect of hydrogel on trees already under salt stress. In order to explore the possibility of establishing forest in a potassium mine in Bischofferode (Germany), a new generation of polymer Stockosorb K 410, a highly crosslinked polyacrylamide with about 40% of the amide group hydrolysed to carboxylic groups, was used as a soil conditioner (Httermann et al. 1997). Stockosorb K 410 does not interact directly with the soil matrices, but form aqueous gels which have diameters in the range of centimetres and act as a water reservoir for the plant-soil system (Buoranis et al. 1995). The result showed that amendment of mine refuse with Stockosorb K 410 significantly improved survival of woody species at a high level of salinity (Httermann et al. 1997). The presence of hydrogel might change the ion composition of soil water since polyacrylamide gels are a cation absorbing polymer (Martin et al. 1993). The contribution of changes in ion relations to the differences in growth exhibited by trees in the presence or absence of polymer needs to be elucidated. Seedlings of Populus euphratica were used in our experiment since it has been well demonstrated to be a salt tolerant tree species (Ma et al. 1996, 1997; Chen et al. 2001a, 2001b, 2002a, 2002b). Salt tolerance of P. euphratica is attributed to its greater ability for salt exclusion, nutrient balance maintenance and accumulation of compatible solutes under salt stress (Chen et al. 2001a, 2001b, 2002b). Ion compartmentation within the roots is likely to play a key role in salt exclusion. P. euphratica effectively sequestered Cl in root cortical vacuoles at high salinity (Chen et al. 2002a). This limited ion loading into the xylem during radial transport, contributed to the restriction of subsequent axial transport (Chen et al. 2002a). Similarly, Jeschke (1984) found that Na+ compartmentation in root vacuoles of barley lowered its flow to the shoot. The objective of this study was to elucidate the effects of hydrogel on plant ion relations and the relevance to salinity tolerance of P. euphratica. In addition to tissue ion analysis, we used the X-ray microanalysis technique to furnish direct evidence of element uptake and transport at the cellular level.

planted in individual pots (1 l) containing nursery soil (Einheitserde) and placed in a greenhouse at the Institute of Forest Botany, Gttingen University (Germany). These plants were kept wellwatered for 1 month until leaves developed. Then they were randomly transferred to 15 l containers filled with nursery soil (no salt control) or saline soil (mine refuse) amended with 0.0% (control) or 0.6% Stockosorb K410 (by weight). The mine refuse was collected from a potassium mine in Bischofferode (Germany) and aqueous extraction (1:2, w/v) contains the following elements in mg/l: Na+: 3,301, K+: 16.32, Ca2+: 902, Mg2+: 2.58, Fe3+: 0, Mn2+: 0.01, Cl–: 5,535, NO3–: 0.03, SO42–: 742, PO43–: 0.39 (Httermann et al. 1997). All plants were placed in a greenhouse in March 1997 and kept well irrigated by maintaining the soil water content at 70–100% of field capacity (FC) irrespective of substrate types. To keep soil water content close to the target value (70–100% FC), all containers were weighed twice a week and watered as required [the amount of water for each pot was estimated according to the full field capacity of substrates (three types of substrates: nursery soil, mine refuse amended with 0.0% or 0.6% Stockosorb K410) and the mass basis]. During the 1997 growing season, enhancement of survival was observed in plants grown in saline soil amended with 0.6% hydrogel polymer (Httermann et al. 1997). The surviving plants were placed outdoors in November 1997 and irrigated by natural precipitation until harvest on 24 April 1998. Plant harvest Three replicate trees were harvested from each of three treatments: which were (1) those growing in nursery soil (no salt control) (2) saline soil treated with 0.0% Stockosorb (salt) or (3) 0.6% Stockosorb (salt + hydrogel). Leaves and woody tissues, including new twigs (new year’s growth from 1998) and old branches (2- and 3-year-old) were harvested separately, and roots were separated into fine roots (diameter <1 mm) and main roots after being quickly washed free of soil with deionized water. No visible damage was seen in roots of salinized plants regardless of polymer treatments. Hydrogel-covered roots were firstly separated from uncovered roots, and then gel fragments were carefully removed by hand under a light microscope. The root system (ranging from 0–5 mm in diameter) of each plant, including main roots, long pioneer roots with laterals that largely suberised except for the distal region of root were scanned with a ScanJet 4C/T scanner (Hewlett Packard). Root length and surface area were estimated using software WinRHIZO (Version 3.6). Finally, hydrogel and plant tissues (leaf, root and branch) were oven-dried (70C for 4 days) and dry weight was obtained. Dried samples were ground into powder and stored for mineral analysis. Ion analysis of plant tissue Cation

Materials and methods Hydrogel The polymer used was Stockosorb K 410 (Stockhausen, Krefeld, Germany), a highly cross-linked polyacrylamide with about 40% of the amide group hydrolysed to carboxylic groups. Plant material and treatment Two-year-old seedlings of P. euphratica were obtained in winter from Xinjiang Uygur Autonomous Region of China in 1996. In order to break dormancy, the seedlings were kept in a cold room (4–7C) for 2 weeks and then immersed with their roots in 50 ppm IAA (indole-3-acetic acid) solution for 24 h. Afterwards, they were

Samples (0.1–0.2 g) were digested with HNO3 in Teflon pressure vessels. Na+, K+, Ca2+ and Mg2+ were determined by atomic absorption spectrophotometer. Chloride Samples (0.5–1.0 g) were extracted with 1 N HNO3 as described by Storey (1995) and Cl was determined by a modified method of silver titration. Abundant AgNO3 solution (0.025 N) was used to precipitate chloride of aqueous extracts and excess Ag+ was estimated by 0.02564 N NH4SCN titration. NH4Fe(SO4)2 was used as a colour indicator for the isoionic point determination (Chen et al. 2001a).

177 Soil analysis

X-ray analysis

Volume of polymer in hydrogel-amended soil

Sections were analysed in a Philips EM 420 electron microscope with the energy dispersive system EDAX 9100, using a low background holder. The operating parameters were as follows: accelerating voltage 120 kV; take-off angle 25 and the time for collecting X-rays was 30 s. Tissue compartments like cell wall, cytoplasm, vacuole, nucleus or plastids were discerned easily and analyzed separately. The diameter of the electron beam was adjusted to the size of the structure: cell walls were normally measured with a beam diameter of 250 nm, very fine cell walls were measured with a beam diameter of 100 nm; for analyzing vacuoles, the electron beam was widened to cover the whole diameter of the vacuole. In the present study, the following tissue compartments were examined: cortical cell wall, cortical cytoplasm and vacuole, endodermal cell wall and xylem vessel. Ten to 20 measurements of each compartment were taken from each section. The X-ray spectra were processed with the SQ program of EDAX9100 software after manual fitting of the background. Quantitative data were obtained by comparing the peak integrals of the elements with the K+ peaks of standards containing known K+ amounts (Fritz and Jentschke 1994), taking into account the calibration coefficients of the elements relative to K+ (Cliff and Lorimer 1975). All steps of tissue processing were carried out under water-free conditions, water-soluble elements are kept in situ by this procedure (Fritz 1989). This means that the quantitative data obtained by the described method include mobile as well as nonmobile fractions of the elements. Therefore the data are expressed as mmol content per dm3 analyzed volume, considering the fact that many of the detected elements do not exist in a real solution in most of the cell compartments.

Soil samples were taken at 20–30 cm depth in pots in two replicates and soil water content by weight (WC) was measured. Additional soil samples were taken from hydrogel amended soil for polymer volume estimates. Gel fragments were separated from the soil and put into a volumetric flask after being washed free of soil particles (there was a negligible swelling of gel fragments upon washing). Total volume of polymer was estimated by water displacement, and the volume of swelling polymer was 11.8€1.1% of amended soil. Soil water content, pH and ion concentrations Following soil water content measurement, extracts of soil samples (dried soil: deionized water = 1:2, weight/volume) were used for pH, cation and chloride measurements. Na+ and K+ were measured by atomic absorption spectrophotometer (Perkin-Elmer 2280, Norwalk, Connecticut, USA), Ca2+ and Mg2+ by atomic absorption spectrophotometer (Hitachi 180–80, Hitachi, Tokyo, Japan). Cl was estimated by silver titration. Mean concentration of ions in soil water was calculated using the following formula: Y (mM) = ion content of soil (mmol kg-1 dried soil) / soil water content (kg H2O kg–1 dried soil). Sample preparation and X-ray analysis Standard procedures required for sample preparation were followed (Fritz 1989). A brief description is given below.

Data analysis Freeze-drying Additional fine root samples (1-year-old lateral roots that were largely suberised except for the distal region of root, 10–20 cm long, diameter <1 mm) were taken from long roots (extensively branched with laterals) of three replicate plants in the three treatments. Roots, covered with or without polymer fragments were collected and immediately placed into aluminium sample holders and rapidly frozen in a 2:1 mixture of propane: isopentane at the temperature of liquid nitrogen. Samples were vacuum freeze-dried at 45C for 48 h and then slowly allowed to equilibrate to room temperature (ca. 26C) over a period of 24 h under vacuum. Samples were stored over silica gel until infiltration in plastic. Infiltration and polymerisation Freeze-dried root samples, cut with a razor blade into pieces not longer than 1 mm were transferred into vacuum-pressure-chambers, and infiltrated in ether at 27C overnight before infiltrating with plastic. Plastic used was a 1:1 mixture of styrene and butyl methacrylate containing 1% benzoylperoxide stabilized with 50% phthalate. Infiltration with plastic was carried out in three steps: 1:1 ether: plastic for 24 h, 1:3 ether: plastic for 24 h, and finally 100% plastic for 24 h. Following infiltration, samples were transferred into gelatine capsules and polymerised at 60C for at least 7 days.

All data were subjected to ANOVA and significant differences between means were determined by Duncan’s multiple-range test. Unless otherwise stated, differences were considered statistically significant when P <0.05.

Results Biomass Compared with no salt controls, biomass of plants grown in saline soil was lower (Fig. 1). It is noteworthy that hydrogel-treated plants (salt + hydrogel) had a 2.7-fold higher biomass than those grown in unamended soil (salt) (Fig. 1). Dry weight of leaf, stem (new twig and old branch) and root (fine root and main root) were all significantly improved by the incorporation of polymer into saline soil (Fig. 1).

Cutting After polymerisation, samples were cut into 1 m thick sections using a dry glass knife with a ultramicrotome (Ultracut E, ReichertJung, Vienna, Austria). The sections were mounted on adhesivecoated mesh-100 fine-bar copper grids (Fritz 1991), coated with carbon and stored over silica gel until analysis.

Fig. 1 Biomass of P. euphratica plants grown in nursery soil (no salt), saline soil (salt) and polymer-amended saline soil (salt + hydrogel). Each value is the mean of three plants and bars represent the standard error of the mean

178

and total root surface was covered by gel fragments (Table 1). Most of the hydrogel-covered roots were fine roots with diameters smaller than 1 mm (Table 1). Effect of hydrogel amendment on soil water content, pH and ion concentration

Fig. 2 Hydrogel-covering root of P. euphratica plants grown in saline soil amended with 0.6% Stockosorb K410

Root length and surface area Fine roots (ranging from 0 to 1 mm in diameter), occupied approximately 95% of total root length and 80% of total root surface area regardless of treatments (Table 1). Amendment of saline substrate with 0.6% polymer greatly enhanced root growth compared to salt, even though salinity reduced root growth of plants grown in saline soil (Fig. 1, Table 1). Plants in polymer-amended soil had about 3.5-fold higher root length and root surface area than those grown in unamended soil (Table 1). Root aggregation in hydrogel was clearly seen in the present study (Fig. 2), and more than 6% of the total root length

Table 1 Root length and root surface area of P. euphratica plants grown in nursery soil (No salt), saline soil (Salt) and polymer-amended saline soil (Salt + hydrogel). Each value (SE) is the mean of three plants and values in the same column followed by different letters are significantly different

Treatment

Water content (WC) of nursery soil (no salt) was much higher than that of saline soil (Table 2). Compared with salt, hydrogel incorporation significantly increased soil WC by 41%, resulting from the high water absorbing capacity of polymer, which took up 15 times its own weight (Table 2). Hydrogel application did not affect pH of soil extracts, which varied between 6.55 and 6.71 (Table 2). Concentration of Cl and cation in saline soil were consistently higher than that of nursery soil, with the exception of K+ concentration which was lower (Table 2). Hydrogel amendment drastically changed the ion composition of soil water. Mean concentration of Na+ and Cl was significantly decreased by hydrogel incorporation compared to salt (Table 2). This is mainly the result of dilution (Table 2). Compared with salt, soil Ca2+ of amended saline soil was markedly enhanced due to the presence of gel fragments where Ca2+ was greatly enriched (Table 2). Soil K+ and Mg2+ were not significantly affected by hydrogel treatment, being in the range of 4.06–5.10 mM and 16.47–21.08 mM, respectively (Table 2).

Root diameter (mm) 0–0.5

Root length (cm) No salt 3,327.4a Salt 541.4c Salt + hydrogel 2,520.2b Hydrogel-covered 146.1(36.2) 2 Root surface area (cm ) No salt 288.7a Salt 49.7c Salt + hydrogel 222.1b Hydrogel-covered 14.3(3.3)

Table 2 Water content ( WC, kg H2O kg1 dried soil or hydrogel), pH and ion concentrations (mM) of nursery soil (No salt), saline soil (Salt), polymer-amended saline soil (Salt + hydrogel) and Hydrogel. Mean concentration of ions in soil or hydrogel was calculated using the following formula: Y (mM) = ion content

Total

0.5–1.0

1.0–1.5

1.5–2.0

>2.0

639.3a 135.9c 508.4b 41.2(9.7)

139.6a 23.5b 112.2a 6.2(1.3)

46.6a 5.4b 37.8a 0.4(0.07)

16.5a 1.7b 14.0a 0.3(0.01)

185.2a 37.1c 142.6b 11.3(3.1)

70.4a 35.3a 12.3b 3.8b 58.4a 27.8a 3.0(0.65) 0.3(0.05)

24.8a 2.4b 19.8a 0.04(0.01)

4,169.4a 707.9c 3,192.6b 194.2 604.4a 105.3c 470.7b 29.0

(mmol kg1 dried soil or hydrogel) / water content (kg H2O kg1 dried soil or hydrogel). Each value is the mean of three replicates and values in the same column followed by different letters are significantly different

Treatment

WC

pH

Cl

Na+

K+

Ca2+

Mg2+

No salt Salt Salt + hydrogel Hydrogel

1.49b 0.22d 0.31c 15.06a

7.07a 6.71b 6.55b 6.64b

4.20c 60.49a 42.52b 1.60d

2.25c 56.23a 40.96b 1.50d

6.09a 5.10b 4.06b 1.58c

10.00d 45.24c 68.72b 133.91a

3.24c 21.08a 16.47a 8.46b

179 Table 3 Tissue element contents (mmol/Kg) of P. euphratica plants grown in nursery soil (No salt), saline soil (Salt) and polymer-amended saline soil (Salt + hydrogel). Each value is the mean of three plants and values in the same column followed by different letters are significantly different Treatment Leaf No salt Salt Salt + hydrogel New twig No salt Salt Salt + hydrogel Fine root No salt Salt Salt + hydrogel

Cl–

Na+

K+

Ca2+

Mg2+

151.7c 509.6a 272.8b

8.1b 20.3a 12.7b

849.8a 556.4b 604.4b

405.8c 543.5b 604.1a

194.6a 129.0b 137.8b

56.9c 189.1a 88.0b

11.6b 24.5a 9.4b

571.7a 355.2b 425.0b

126.4b 216.5a 203.2a

108.4a 78.5b 78.7b

73.5c 235.9a 110.1b

27.0c 78.4a 45.7b

284.6a 182.4b 214.7b

213.4c 487.8b 756.1a

100.9a 120.4a 137.6a

Ion relations in plant tissues Cl
Compared with no salt plants, Cl levels in all tissues (leaf, new twig and fine root) were markedly increased by external salinity (Table 3). However, the presence of polymer significantly reduced Cl accumulation in all tissues compared to salt (Table 3). Cation

salinity except for increased concentrations in fine roots (Table 3). K+ was also depressed in these salinized plants relative to no salt (nursery plants) (Table 3). Hydrogel application did not affect tissue K+ concentrations (Table 3). Salinized plants accumulated higher Ca2+ than those in nursery soil (Table 3). Hydrogel-treated plants had 55% and 11% higher Ca2+ in fine roots and leaves respectively, relative to salt soils (Table 3). Unlike Ca2+, shoot Mg2+ was reduced by salinity irrespective of the presence or absence of polymer (Table 3). However, root Mg2+ was at a similar level relative to no salt plants (Table 3). Ion distribution within cells in roots Na+ Compared with no salt, salinity caused a significant rise in Na+ in cortical cell walls, endodermal cell walls and xylem vessels of young roots, and Na+ levels declined from outer to inner roots (Table 4). Hydrogel amendment significantly restricted Na+ accumulation in the apoplast (referring to cell walls and xylem vessels) irrespective of roots covered with or without gel fragments (Table 4). In cortex, cytoplastic Na+ and vacuolar Na+ tended to rise in response to external salinity, but no significant difference was observed between salt and hydrogel-treated plants (Table 4). A similar pattern was observed in old roots except for lower levels of Na+ in apoplast, cytoplasm and vacuole (Table 4).

Compared with no salt controls, salt treatment significantly caused build up of Na+ in all tissues of plants grown in unamended saline soil (Table 3). However, the Na+ level of hydrogel-treated plants was not affected by

Compared with no salt plants, external salinity significantly increased Cl levels in apoplast and symplast of

Table 4 Cellular Na+ content (mmol/dm3) in young roots (the distal region, white) and old roots (the proximal region, suberised) of plants grown in nursery soil (No salt), saline soil (Salt) and hydrogel-amended saline soil (Salt + hydrogel). Each value is the mean of three individual plants. For each plant, three young and old

root samples were analyzed by X-ray microanalysis and 10–20 measurements of each compartment (cortical cell wall, endodermal cell wall, xylem vessel, cortical cytoplasm and vacuole) were taken from each section. Values in the same column followed by different letters are significantly different

Treatment

Young roots No salt Salt Salt + hydrogel Hydrogel-covered Non-hydrogel-covered Old roots No salt Salt Salt + hydrogel Hydrogel-covered Non-hydrogel-covered

Cl


Compartment Cortical cell wall

Endodermal cell wall

Xylem vessel

Cortical cytoplasm

Cortical vacuole

135c 316a

34c 103a

2c 38a

33b 55a

23b 38a

219b 177b

58b 58b

19b 22b

56a 52a

43a 41a

50b 161a

25c 99a

11b 25a

27a 32a

18b 38a

58b 69b

45b 41b

18ab 24a

25a 34a

35a 37a

180 Table 5 Cellular Cl content (mmol/dm3) in young roots (the distal region, white) and old roots (the proximal region, suberised) of plants grown in nursery soil (No salt), saline soil (Salt) and hydrogel-amended saline soil (Salt + hydrogel). Each value is the mean of three individual plants. For each plant, three young and old Treatment

Compartment Cortical cell wall

Young roots No salt Salt Salt + hydrogel Hydrogel-covered Non-hydrogel-covered Old roots No salt Salt Salt + hydrogel Hydrogel-covered Non-hydrogel-covered

Endodermal cell wall

Xylem vessel

Cortical cytoplasm

Cortical vacuole

367b 802a

228b 360a

135b 269a

22b 60a

27c 45b

1,033a 915a

373a 386a

121b 217a

67a 57a

73a 88a

185b 333a

150b 296a

23c 116a

17b 56a

29b 51a

77c 189b

92b 137b

62b 101a

10b 20b

42a 46a

Table 6 Cellular Ca2+ content (mmol/dm3) in young roots (the distal region, white) and old roots (the proximal region, suberised) of plants grown in nursery soil (No salt), saline soil (Salt) and hydrogel-amended saline soil (Salt + hydrogel). Each value is the mean of three individual plants. For each plant, three young and old Treatment

Young roots No salt Salt Salt + hydrogel Hydrogel-covered Non-hydrogel-covered Old roots No salt Salt Salt + hydrogel Hydrogel-covered Non-hydrogel-covered

root samples were analyzed by X-ray microanalysis and 10–20 measurements of each compartment (cortical cell wall, endodermal cell wall, xylem vessel, cortical cytoplasm and vacuole) were taken from each section. Values in the same column followed by different letters are significantly different

root samples were analyzed by X-ray microanalysis and 10–20 measurements of each compartment (cortical cell wall, endodermal cell wall, xylem vessel, cortical cytoplasm and vacuole) were taken from each section. Values in the same column followed by different letters are significantly different

Compartment Cortical cell wall

Endodermal cell wall

Xylem vessel

Cortical cytoplasm

144b 188b

97b 115b

21b 25b

10b 32a

2b 4a

296a 373a

193a 77b

33a 35a

29a 30a

6a 4a

294b 458a

129b 197ab

10b 15b

23a 21a

3b 6ab

642a 536a

232a 162ab

43a 15b

28a 32a

young roots except for xylem vessels of hydrogel-covered roots (Table 5). Hydrogel-treated roots had 60–90% higher Cl in vacuoles than salt (Table 5). Unlike young roots, the presence of polymer significantly restricted Cl accumulation in both apoplast and symplast of old roots except for vacuole and xylem of non-hydrogel-covered roots (Table 5). Restriction of Cl– accumulation was more pronounced in roots covered by gel fragments (Table 5). It is noteworthy that hydrogel-treated roots (young and old roots) accumulated more Cl– in vacuole than in cytoplasm, contrasting with salt treatment where vacuolar fraction of Cl– remained lower than that of cytoplasm (Table 5).

Cortical vacuole

9a 13a

Ca2+ Compared with plants grown in no salt medium, Ca2+ level was often increased in root cells of salinized plants (Table 6). Hydrogel-treated plants accumulated higher Ca2+ in cortical cell walls of young roots than salt (Table 6). However, the increase of Ca2+ in endodermal cell walls caused by polymer amendment was only observed in hydrogel-covered roots (Table 6). Xylem Ca2+ in young roots increased responding to hydrogel treatment, but the Ca2+ rise in the xylem was only observed in old roots which were covered by polymer (Table 6). Compared with salt, vacuolar Ca2+ in old roots was increased by polymer amendment; however, cyto-

181 2+

plasm Ca was similar regardless of the presence or absence of hydrogel (Table 6).

Discussion Effects of hydrogel on plant growth Although salinity reduced plant growth, growth of P. euphratica plants in saline soil was markedly improved by the incorporation of cross-linked polyacrylamide, and biomass of hydrogel-treated seedlings was 2.7-fold higher than salt controls (Fig. 1). Hydrogel-treated plants had an approximately 3.5-fold higher root length and root surface area than those grown in unamended saline soil (Table 1). In the present study, incorporation of polymer in saline soil resulted in beneficial effects on tree growth, which was consistent with other investigations conducted on crops using poly(ethylene oxide) hydrogel (Szmidt and Graham 1990), polyacrylamide hydrogel (Awad et al. 1986) and cross-linked poly(ethylene oxide)-co-polyurethane hydrogel (El-Sayed et al. 1991; El-Sayed and Kirkwood 1992) under saline conditions. Effects of hydrogel on tissue Na+ and Cl Salt treatment steadily increased Na+ and Cl levels in all tissues of plants grown in unamended saline soil. The build up of these salt ions in roots and shoots was significantly restricted by hydrogel amendment (Table 3). Similarly, El-Sayed and Kirkwood (1992) have reported that polymer incorporation in sand caused a reduction of salt ions in quiescent maize pollen. Thus it may be inferred that hydrogel amendment may minimise the adverse effects of salinity by reducing the levels of salt ions in plant tissues. The effects of hydrogel on reducing uptake of Na+ and Cl– were presumably due, in part, to salt-buffering capacities of the polymer. Polymers were capable of absorbing large quantities of water (15 times their own weight) in the presence of salts (Table 2) and expanding to form gel fragments which occupied approximately 12% of soil volume. Na+ and Cl concentration in the gel matrix were markedly diluted due to the amount of water retained in the polymer (Table 2). Root aggregation in hydrogel was clearly seen in the present study and over 6% of total roots was covered by polymer (Fig. 2, Table 1). Beneficial effects of root aggregation around gel fragments have been noted in other studies, where water use efficiency was improved through increased root contact with a source of moisture (Woodhouse and Johnson 1991). In this study, root aggregation in hydrogel allows good contact with a source of lower salinity, accordingly, uptake of Na+ and Cl was subsequently reduced. Our previous studies have shown that salt tolerance of P. euphratica mainly results from its higher capacity to exclude salt (Chen et al. 2001a, 2002a). In the present study, the salt exclusion capacity of P. euphratica was increased by hydrogel amendment in the saline soil;

as a result, the adverse effects of salinity were reduced and plant growth was ultimately improved. Although the hydrogel was estimated to occupy 12% by volume of the soil, only 6% of the root length or surface area was found within the polymer (Table 1). This value was likely an underestimation since the roots that aggregated around the surface of gel fragments were not measured, due to the difficulty of estimation. Effects of hydrogel on Na+ and Cl– distribution within roots Data from X-ray microanalysis revealed possible effects of hydrogel treatments on intracellular ion compartmentation within root cells. External salinity significantly increased Na+ and Cl– levels in apoplast and a decreasing gradient of salt ions was observed from the cortex to the xylem (Tables 4, 5). However, amendment with hydrogel reduced Na+ levels in apoplast (cell walls and xylem vessels) of both young and old roots (including roots covered with or without gel fragments) compared to salt (Table 4). Similarly, hydrogel application limited apoplastic Cl levels in old roots and the effects were more pronounced in hydrogel-covered roots (Table 5). Collectively, these results suggest that hydrogel incorporation reduced apoplasmic ion transport into the inner root. This contributed to the restriction of the subsequent root-to-shoot salt transport, enhancing the salt exclusion capacity of P. euphratica (Table 3). A significant increase of Na+ and Cl was observed in the cytoplasm and vacuole of salinized plants (Tables 4, 5). Hydrogel treatment played a vital role in compartmentizing salt ions in vacuoles, especially Cl–, as compared to salt. As shown in Table 5, the Cl– concentration in hydrogel-covered roots was higher in the vacuole than in the cytoplasm and the tendency was observed more in old roots than young roots. More Cl ions were sequestered in the vacuole when these ions passed through the cortex to the xylem during radial transport and thus, as a result, ion loading into the xylem was less and subsequent axial transport was consequently limited (Table 3). This is consistent with our previous report that salt compartmentation in root cortical vacuoles contributed to the salt exclusion of P. euphratica (Chen et al. 2002a). Therefore, hydrogel-treated P. euphratica trees exhibited a higher capacity to exclude salt, which was due, at least in part, to the restriction of symplasmic salt transport. Effects of hydrogel on tissue and cellular Ca2+ Plants grown in hydrogel-amended soils contained higher Ca2+ in both root and leaf compared to those grown in unamended saline soil (Table 3). Apoplast-Ca2+ within root cells was generally improved by hydrogel amendment, but the enhancement was more pronounced in hydrogel-covered roots (Table 6). This obviously resulted

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from the enrichment of Ca2+ in polymer fragments that covered roots (Table 2). Since calcium is required for the maintenance of membrane integrity and for the control of permeability (Hanson 1984), elevated Ca2+ presumably alleviates the adverse effects of NaCl on plant tissues. Furthermore, Ca2+ may inhibit Na+ influx by mediating Na+-permeable channels (Allen et al. 1995; Roberts and Tester 1997). Enrichment of Ca2+ in gel fragments might be attributed to cation-exchange on the polymer. Some investigators proposed that polyacrylamide gels might improve plant growth under saline conditions by absorbing and sequestering cation salts into the gel granule and that, as a result, the damage of toxic ions (Na+) to plants is reduced (Martin et al. 1993). There has been evidence indicating that the accumulation of Na+ in polyamide gels (in sodium form) resulted in lower Na+ in barley shoots (Awad and Doering 1994). Similarly, in our study, ion analysis of hydrogel revealed the presence of sodium (Table 2), indicating that Na+ can be absorbed by the polymer and migrates into the gel matrix. However, concentration of Na+ was much less than that of Ca2+ in the gel matrix (Table 2). This difference is accounted for by the polymer’s cation-exchange character since amide groups are able to form coordination bonds, especially with polyvalent cations. Theng (1974) verified that the strength of this bond decreases in the order, Ca2+>Na+. Moreover, the polymer used in our study is a highly crosslinked polyacrylamide with about 40% of the amide group hydrolysed to carboxylic groups; this provides more oxygen atoms for the formation of stable bonds with Ca2+, instead of Na+. Therefore, root aggregation in hydrogel allows good contact of roots with a Ca2+ source and the restriction of Na+ uptake was mainly the result of competition between Na+ and Ca2+. In summary, we conclude that hydrogel amendment in the saline soil (potassium mine refuse) enhanced Ca2+ uptake and increased salt exclusion capacity of P. euphratica. As a result, the adverse effects of salinity were reduced and plant growth was ultimately improved. Ca2+ enhancement and the increased salt exclusion of P. euphratica presumably resulted from an improved Ca2+/ Na+ ratio in soil solution. The addition of hydrogel is advantageous to the quality of soil solution available to the plant. Polymer application (1) lowered salt concentration in soil water resulting from its salt-buffering capacity, and (2) enriched Ca2+, which is accounted for by the polymer’s cation-exchange character. The beneficial effects of polymer amendment likely result from hydrogel covering roots since root aggregation allows good contact of roots with Ca2+ and reduces contact with Na+ and Cl, which presumably plays a major role in improving salt tolerance. The experiment represents a short-term study. The application of hydrogel Stockosorb K 410 increased survival and growth of a salt resistant woody species, P. euphratica , over a period of 2 years. The survival and early growth of young seedlings after being established in the field is a key process for afforestation under saline

conditions. Surviving trees increase their salt resistance capacity with increasing plant size in subsequent years. Our results suggest that Stockosorb K 410 can be used to aid plant establishment and growth in saline soil although the beneficial effect of the hydrogel Stockosorb K 410 normally lasts 3–5 years (personal observations). Acknowledgements The research was supported in part by Stockhausen GmbH Co. KG, Krefeld (Germany), the Foundation for the author of National Excellent Doctoral Dissertation of PR China (Grant No. 200152), the Teaching and Research Award Program for Outstanding Young Teachers in Higher Education Institution of MOE, PR China, and the National Natural Science Foundation of China (Grant No. 30070613 and 39830320). We thank Konrad Wehr for valuable help with operating the microscope, Karin Lange and Andrea Franz for technical assistance with sample embedding and sectioning. Dr. Genben Bai is acknowledged for kindly providing plant material.

References Allen EG, Wyn Jones RG, Leigh RA (1995) Sodium transport in plasma membrane vesicles isolated from wheat genotypes with differing K+/Na+ discrimination traits. Plant Cell Environ 18:105–115 Awad F, Doering HW (1994) Mobilization of nutrients from slowrelease fertilizer as influenced by hydrogel and water quality. Agrochimica 39:123–132 Awad F, Abd El Rahman M, Farag F (1986) The combined effect of some soil conditioners and saline irrigation water. Agrochimica 30:427–438 Azzam RA (1983) Polymeric conditioner gels for desert soils. Commun Soil Sci Plant Anal 14:739–760 Azzam RA (1985) Tailoring polymeric gels for soil reclamation and hydroponics. Commun Soil Sci Plant Anal 16:1123–1138 Baasiri M, Ryan J, Mucheik M, Harik SN (1986) Soil application of hydrophilic conditioner in relation to moisture, irrigation frequency and crop growth. Commun Soil Sci Plant Anal 17:573–589 Buoranis DL, Theodoropoulus AG, Drossopoulus JB (1995) Designing synthetic polymers as soil conditioners. Commun Soil Sci Plant Anal 26:1455–1480 Callaghan TV, Abd El Nour H, Lindley DK (1988) The environmental crisis in the Sudan: the effect of water-absorbing synthetic polymers on tree germination and early survival. J Arid Environ 14:301–317 Chen S, Li J, Wang S, Httermann A, Altman A (2001a) Salt, nutrient uptake and transport and ABA of Populus euphratica; a hybrid in response to increasing soil NaCl. Trees 15:186–194 Chen S, Li J, Bi W, Wang S (2001b) Genotypic variation in accumulation of salt ions, glycinebetaine and sugars in poplar under conditions of salt stress (in Chinese). Chin Bull Bot 18: 587–596 Chen S, Li J, Fritz E, Wang S, Httermann A (2002a) Sodium and chloride distribution in roots and transport in three poplar genotypes under increasing NaCl stress. For Ecol Manage 168:217–230 Chen S, Li J, Yin W, Wang S, Fritz E, Polle A, Httermann A (2002b): Tissue and cellular K+, Ca2+ and Mg2+ of poplar under saline conditions (in Chinese). J Beijing For Univ 24: 84–88 Cliff G, Lorimer GW (1975) The quantitative analysis of thin specimens. J Microsc 103:203–207 Conover CA, Poole RT (1979) Influence on activity of Viterra and effects on growth and shelf life of Marantha and Pilea. Proc Fla State Hortic Soc 92:332–333 El-Sayed H, Kirkwood RC (1992) Effects of NaCl salinity and hydrogel polymer treatments on viability, germination and solute contents in Maize ( Zea mays) pollen. Phyton (Horn, Austria) 32:143–157

183 El-Sayed H, Kirkwood RC, Graham NB (1991) The effects of a hydrogel polymer on the growth of certain horticultural crops under saline conditions. J Exp Bot 42:891–899 Fritz E (1989) X-ray microanalysis of diffusible elements in plant cells after freeze-drying, pressure-infiltration with ether and embedding in plastic. Scanning Microsc 3:517–526 Fritz E (1991) The use of adhesive-coated grids for the X-ray microanalysis of dry-cut sections in the TEM. J Microsc 161:501–504 Fritz E, Jentschke G (1994) Agar standard for quantitative X-ray microanalysis of resin-embedded plant tissues. J Microsc 174:47–50 Gehring JM, Lewis AJ (1980) Effect of hydrogel on wilting and moisture stress of bedding plants. J Am Soc Hortic Sci 105:511–513 Hanson JB (1984) The function of calcium in nutrition. In: Tinker PB, Luchli A (eds) Advances in plant nutrition, vol A. Praeger, New York, pp 159–208 Httermann A, Leise K, Zommorodi M, Wang S (1997) The use of hydrogels for afforestation of difficult stands: water and salt stress. In: Zhou H, Weisgerber H (eds) Afforestation in semiarid regions. Datong, China, pp 167–177 Jeschke WD (1984) K+-Na+ exchange at cellular membranes, intracellular compartmentation of cations, and salt tolerance. In Staples RC (ed) Salinity tolerance in plants: Strategies for Crop Improvement. Wiley, New York, pp 37–65 Ma H, Fung L, Wang S, Altman A, Httermann A (1996) Preliminary study on the salt resistance mechanisms of Populus euphratica. J Beijing For Univ 5:31–40

Ma H, Fung L, Wang S, Altman A, Htermann A (1997) Photosynthetic response of Populus euphratica to salt stress. For Ecol Manage 93:55–61 Martin CA, Ruter JM, Roberson RW, Sharp WP (1993) Element absorption and hydration potential of polyacrylamide gels. Commun Soil Sci Plant Anal 24:539–548 Roberts SK, Tester M (1997) A patch clamp study of Na+ transport in maize roots. J Exp Bot 48:431–440 Saleh HH, Hussein AH (1987) Effect of polymeric material (Aquastore) on wheat seedlings emergence, soil salinity and available phosphorus in sandy soil. Tenth Symposium on the Biological Aspects of Saudi Arabia, Jedda, 20–24 April 1987 Still SM (1976) Growth of ‘Sunny Mandalay’ chrysanthemums in hardwood-bark-amended media as affected by insolubilized poly(ethylene oxide). HortScience 11:483–484 Storey R (1995) Salt tolerance, ion relations and the effects of root medium on the response of citrus to salinity. Aust J Plant Physiol 22:101–114 Szmidt RAK, Graham NB (1990) The effect of poly (ethylene oxide) hydrogel on crop growth under saline conditions. Acta Hortic 287:211–218 Theng BKG (1974) The chemistry of clay-organic reaction. Hilger, London, UK Woodhouse J, Johnson MS (1991) Effect of superabsorbent polymers on survival and growth of crop seedlings. Agric Water Manage 20:63–70