Process Biochemistry 42 (2007) 710714 www.elsevier.

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Physiological and ecological characters studies on Aloe vera under soil salinity and seawater irrigation
Zan Min Jin a, Chang Hai Wang a,*, Zhao Pu Liu b, Wei Jia Gong b
a

Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian 116024, China b College of Resource and Environment, Nanjing Agricultural University, Nanjing 210095, China Received 15 August 2006; received in revised form 31 October 2006; accepted 9 November 2006

Abstract Experiments were conducted in two Aloe vera cultivars (F0 and F50) to study the characters of physiology and ecology under salt stress. The results indicate decreases in tissue water, total soluble sugars and glucose, and increases in dry matter and membrane injury occurred both in F0 and F50 irrigated with 60% seawater. Less cell membrane injury were observed in F50. Moreover, total soluble sugars in F0 decreased obviously, however, no signicant change in F50, while sucrose in plants had no signicant change. Furthermore, F0 and F50 accumulated more inorganic cations in stems and roots. In addition, leaf K+ and Ca2+ contents were more in F50 than that in F0 to maintain normal plant growth though accumulation of Na+. F50 had a relative superiority in growth under salinity conditions due to higher K+/Na+ ratio and lower Na+/Ca2+ ratio than F0. # 2006 Elsevier Ltd. All rights reserved.
Keywords: Aloe vera; Cell membrane stability; Inorganic cations; Malondialdehyde; Shape parameters; Soluble sugars

1. Introduction Aloe vera L. is a perennial liliaceous plant with succulent green leaves joined at the stem in a whorled pattern. It is highly appreciated due to its short growth period and high economic value among all the aloe species, and is used in pharmaceuticals, folk medicine, healthcare, cosmetic products and food products [1]. Salt stress is a limiting factor of plant growth and yield, and becoming a serious problem in the world. An estimation shows that about one-thirds of irrigation sections are either saline or alkaline [2]. Salinity is more severe in China where only the area of tidal at is over 20,000 km2 (China Statistical Yearbook 22). Better understanding of the mechanisms that enable plants to adapt to salt stress and maintain growth would help in the selection of stress tolerant cultivars for exploiting tidal ats. The deleterious effects of salinity on plant growth are associated with low osmotic potential of soil solution, nutritional imbalance, specic ion effect, or a combination of these factors [3]. As a result, membrane disorganization, increase in activated oxygen species production and metabolic

toxicity occur. The degree to which each of these factors affects growth depends on the plant genotype and environmental conditions. Osmotic adjustment in plants subjected to salt stress can occur by the accumulation of high concentrations of either inorganic ions or low molecular weight organic solutes. At present most of research about salt stress focus on glycophyte under short-term salt stress in laboratory, however, there are no reports about A. vera, a CAM desert plant, under long-term salt stress in tidal at. Therefore, to explore the physiological and ecological characters of the plants adaptation to saline environment, two A. vera cultivars were used to assess the capability of osmotic adjustment by analysing the plant growth, soluble sugars and inorganic cations. Attempts were also made to study the effect of salinity on electrolyte leakage and lipid peroxidation in these two cultivars. The present investigation also offers a reference opinion and a theoretical basis on which the further study of salinity-resistant mechanism of desert plants can be directed and new salt-resist plant can be cultivated.
2. Materials and methods 2.1. Plant material and seawater treatment
Aloe (A. vera L.) cultivars F0 a general variety and F50, the sixth-generation irrigated with 50% seawater, were obtained from Hainan medium examination base of Beach Agriculture Academe of Nanjing Agricultural University of

* Corresponding author. E-mail address: chwang2001@sina.com (C.H. Wang). 1359-5113/$ see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.11.002

Z.M. Jin et al. / Process Biochemistry 42 (2007) 710714 China. Plants were cultivated in tidal at and irrigated with freshwater (pH 7.3, EC27.30.57 ds m1) or 60% seawater (pH 7.6, EC27.323.4 ds m1) for one and a half years. Basic ions in seawater main include 2.2mmol L1 HCO3, 40.3 mmol L1 SO42, 493.4 mmol L1 Cl, 19.6 mmol L1 Ca2+, 42.8 mmol L1 Mg2+, 15.3 mmol L1 K+ and 412.2 mmol L1 Na+. Each planting section area was 22 m2 (5.5 m 4.0 m) and they were separated by membranes to prevent inltration each other. Plant row spacing was 60 cm 50 cm and 54 plants each planting section. When the reading on moisture tonometer reached 38 kPa, each section was irrigated with 1 m3 water.

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acid) for 15 min at 97 8C. Absorbance was measured with spectrophotometer at 630 nm, glucose as a standard.

2.5. Extraction and measurement of Na+, K+, Ca2+ and Mg2+

After drying and milling all parts samples, the contents of Na+, K+, Ca2+ and Mg2+ in plant samples were measured as per the method described in QB/T 2489-2000 (Standard of Light Industry of the Peoples Republic of China).

2.6. Statistical analysis

A monofactor analysis of variance was performed with treatment level as main factor. Data for each parameter were subjected to one-way ANOVA and signicant differences between treatment means were determined by Duncan Multiple Range test (DMRT) using the SPSS software package (SPSS 11.5.0 for windows, standard Version 2002), and data were shown as means S.E. of three independent experiments with two replicates.

2.2. Determination of leaf dry matter and water content

The dry matter content of the samples was determined by drying a known fresh weight of homogenized samples at 50 8C followed by at 105 8C until there were no further changes in weight under these two temperatures. After cooling to room temperature, the samples were reweighed, and the dry matter and water content were calculated.

2.3. Cell membrane stability and malondialdehyde (MDA)

Cell membrane stability was determined by measuring an increase in electrical conductivity of the equilibrium solution [4]. The malondialdehyde content of plant material was determined by the thiobarbituric acid (TBA) reaction [4]. In order to eliminate the interference of the sugars to MDA-TBA reaction, the MDA concentration was determined by its experiential formula: C (mmol/L) = 6.45(A532 A600)0.56A450, from which the absolute concentration (mmol/g) of MDA was calculated.

3. Results and discussion 3.1. Leaf dry matter and water content The basic growth status of two A. vera varieties were shown in Table 1: there are no difference on leaves shapes of F0 and F50 irrigated with seawater, however, F50 grew taller than F0 under freshwater irrigation. Besides, roots fresh weight of F50 was heavier than that of F0 under seawater irrigation. The growth of plants, estimated as tissue water and dry matter contents, was inuenced by salinity. In plants treated with freshwater tissue water and dry matter contents were not signicantly different. However, when irrigation was done with 60% seawater, there were 0.75% and 1.69% loss in tissue water percentage in F0 and F50, respectively, and dry matter percentage increased by about 0.78 and 1.74 in F0 and F50, respectively. A. vera known as a xerophil can also continue to grow and reproduce in such stressed environments owing to characteristic adaptive physiologies [7]. In addition, F50 maintained its superiority over F0 at 60% seawater irrigation level. 3.2. Cell membrane stability and malondialdehyde (MDA) When irrigation was done with 60% seawater, MDA contents in F0 and F50 had no signicant changes (Fig. 1).

2.4. Determination of soluble sugars

Leaf sugar was extracted as the method described by Angelov et al. [5]. Weighted plant sample (about 5 g) was milled and extracted with 150 mL 80% (v/v) ethanol several times in boiling water bath until the extract was colorless. The ethanol soluble fractions from each sample were pooled, dried at 55 8C under vacuum, resolubilized in 10 mL of distilled water, and frozen (20 8C) until analyzed for sugars. Total soluble sugar (TSS), sucrose and glucose were determined using the methods of Riazi et al. [6]. TSS were analysed by reacting 0.1 mL of the alcoholic extract with 3.0 mL freshly prepared anthrone (150 mg anthrone + 100 mL 72% H2SO4) in a boiling water bath for 10 min and reading the cooled samples at 625 nm in a spectrophotometer, sucrose as a standard. Sucrose contents were determined by rst degrading reactive sugars present in 0.1 mL extracts with 0.1 mL 5.4 M KOH at 97 8C for 10 min. Three milliliters of freshly prepared anthrone reagent were then added to the cooled reaction product, and the mixture was heated at 97 8C for 5 min, cooled, and read at 620 nm, sucrose as a standard. Glucose estimation were performed in the hood, and a 1.0 mL aliquot of the alcoholic extract was heated with 5.0 mL o-toluidine reagent (60 mL o-toluidine and 2.0 g thiourea made to 1 L with glacial acetic

Table 1 Shape parameters in two Aloe vera cultivars irrigated with seawater for 1 year Shape parameters Seawater irrigation (%)/cultivar 0/F0 Plant tallness (cm) Leaf length (cm) Leaf extent (cm) Leaf thickness (cm) Plant leaves fresh weight (kg) Leaf tissue water (%) Leaf dry matter (%) Plant roots fresh weight (kg) Fresh weight radio of root/shoot 29.59 2.58b 15.07 1.24b 2.22 0.19a 0.80 0.07b 3.53 0.19b 97.14 0.27a 2.96 0.26c 0.169 0.009a 0.046 0.002b 0.05. 60/F0 15.86 0.62c 11.31 0.37c 2.74 0.06a 1.02 0.02a 0.55 0.02c 96.39 0.06b 3.74 0.07b 0.044 0.003c 0.080 0.005a 0/F50 39.25 0.44a 18.40 0.59a 2.62 0.14a 0.90 0.03ab 4.08 0.22a 97.39 0.16a 2.71 0.16c 0.166 0.003a 0.041 0.003b 60/F50 19.39 1.25c 11.96 0.85c 2.56 0.19a 0.92 0.07ab 0.79 0.04c 95.70 0.25c 4.45 0.63a 0.061 0.004b 0.079 0.008a

Means with common letters are not signicantly different at p

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Fig. 1. Cell membrane stability in two Aloe vera cultivars leaves. Means with common letters are not signicantly different at p

0.05.

F50 showed no increase in MDA content compared with F0 irrigated with freshwater. Moreover, it was observed that membrane injury increased obviously both in F0 and in F50, and membrane injury in F0 was worse than that in F50, which increased by 21% and 17% in F0 and F50 irrigated with 60% seawater, respectively. Cell membrane stability expressed in terms of the percentage of membrane injury has been used to assess tolerance of various plant species [810]. Therefore, less percentage of membrane injury in F50 than that in F0 supports the torlerance superiority of F50. 3.3. Soluble sugars The changes of soluble sugars of A. vera are shown in Table 2. Soluble sugars showed different patterns in plants. Under irrigation with 60% seawater TSS and glucose decreased by about 42% and 74% in F0, 29% and 67% in F50, respectively, when compared with F0 irrigated with freshwater. When irrigation was done with 60% seawater increases of sucrose were observed in F0 and F50. Other soluble sugars content in F50 was as about twice as that in F0 under same irrigation. Sucrose is the predominant transport and storage sugar at maturity [11]. Our results for accumulation of sucrose in F0 and in F50 under salt stress revealed that sucrose play a role in lowering osmotic potential. The accumulation of soluble sugars in plants has been widely reported as a response to salinity [1214]. However our results showed that salinity led to a decrease in TSS or glucose, consisted with the research that
Table 2 Soluble sugars contents in leaves of Aloe vera cultivars irrigated with seawater Sugar (mg g1 FW) Seawater irrigation (%)/cultivar 0/F0 Total soluble sugars Glucose Sucrose Other soluble sugars 3.74 0.56a 2.58 0.54a 1.01 0.18a 0.57 0.23ab 0.05.

consistently reduced levels of soluble sugars and starch were observed in leaves of 24-year-old trees salinized for several years [15]. Therefore, not all the plants accumulated soluble sugars under salt stress. These seemed to indicate that the osmotic adjustment in A. vera under salt stress is not dependent upon accumulation of organic solutes. 3.4. Na+, K+, Ca2+ and Mg2+ Inorganic cation contents had no signicant differences in various plant parts of two cultivars of A. vera when irrigation was done with freshwater (Fig. 2). While, these inorganic cation contents in A. vera were greatly affected by salinity. Na+ content was up to twice as many in all parts of plant under 60% seawater irrigation (Fig. 2A). But the accumulations in leaf and stem were more pronounced in F0, Na+ content of stem in F0 was one and half times higher than that in F50. K+ contents (Fig. 2B) of both stem and root greatly increased in F0 and F50, and only a slight difference was observed between them. However, the cultivars differed in their K+ contents of leaves; over half of K+ content of leaf in F0 reduced comparing with no change in F50. Ca2+ contents of leaf and stem signicantly decreased, and Ca2+ content of leaf in F50 was three times higher than that in F0 (Fig. 2C). But an increase in Ca2+ content of root observed at the 60% seawater level in both cultivars. Mg2+ contents of both stem and root greatly increased in F0 and F50, and leaf Mg2+ content had no obvious change (Fig. 2D). As a result of changes in Na+ and K+ contents, F50 contained signicantly higher K+/Na+

60/F0 2.17 0.09b 0.67 0.06b 1.29 0.05a 0.35 0.05b

0/F50 3.35 0.27a 1.84 0.11a 0.81 0.09a 0.91 0.22a

60/F50 2.65 0.38ab 0.85 0.17b 1.29 0.23a 0.73 0.25ab

Means with common letters are not signicantly different at p

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Fig. 2. Effect of seawater irrigation on inorganic cation contents in various plant parts of two cultivars of Aloe vera. Means with common letters are not signicantly different at p 0.05.

Table 3 The ratios of K+/Na+ and Na+/Ca2+ in leaves, stems and roots of Aloe vera cultivars irrigated with seawater Cation ratios Parts Seawater irrigation (%)/cultivar 0/F0 K /Na
+ +

60/F0 0.44 0.06c 1.05 0.12a 0.32 0.05b 2.55 0.54b 0.92 0.04c 0.72 0.01b

0/F50 3.94 0.65a 1.66 0.23a 0.63 0.03a 0.11 0.02a 0.08 0.01a 0.42 0.02a

60/F50 1.05 0.14c 1.44 0.33a 0.46 0.11ab 0.63 0.09a 0.44 0.07b 0.67 0.06b

Leaf Stem Root Leaf Stem Root

2.37 0.37b 1.11 0.26a 0.67 0.11a 0.20 0.01a 0.09 0.02a 0.38 0.02a 0.05.

Na+/Ca2+

Means with common letters are not signicantly different at p

ratios than F0 in all sections under the same irrigation levels (Table 3). In the presence of salinity F50 maintained signicantly lower Na+/Ca2+ ratios than F0 (Table 3). Regulation of transport and distribution of ions in various plant parts and within cells is an important feature of the mechanism of salt tolerance [16,17], inasmuch as the specic accumulation of Na+ in plant tissue is toxic and is found as one of the major causes of growth reduction under saline conditions [1720]. Similar to reed [21], the cations in A. vera were almost accumulated in stem and root, except for accumulation of Na+ was also observed in leaf. Such a distribution form of inorganic cations decreased the osmotic potential of the rooting environment and ensured the normal physiological function and metabolism of plant. High K+ content in leaf maintains steady-state photosynthetic rates and leaf stomatal conductance [22]. The maintenance of higher K+/Na+ ratio in F50 plants may have been one of the factors of their relative superiority in growth under saline conditions, since Wyn Jones et al. [23] suggested a

minimum value for K+/Na+ of 1 for normal growth of plants subjected to saline substrate. Our results proved the capability of osmotic adjustment of F50 was better than F0 under salinity. Ca2+ is a non-toxic inorganic nutrient and has a function of detoxication under saline medium [18]. Ca2+ enhanced salt tolerance in barley [24] and sorghum [25]. Salinity also restricted Ca2+ uptake and transport from root [26]. Thereby Ca2+ contents in all plant parts of the two cultivars decreased under salt stress. But F50 accumulated higher leaf Ca2+ and lower Na+/Ca2+ ratio in leaf and stem. Mg2+ deciency has been reported to accelerate the plant senescence process [27]. In present experiment the maintenance of leaf Mg2+ was observed in A. vera, contributing to the normal growth of plants against long-term salt stress. 4. Conclusion In spite of membrane injury in these two cultivars caused by salt stress, F0 and F50 may ensure the normal growth through

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the absorption and accumulation of inorganic cations in stems and roots. Therefore, A. vera L. can be planted in saline soil and irrigated with saline water. Moreover, lesser degree of membrane injury, higher K+/Na+ ratio, lower Na+/Ca2+ ratio, and the salt-induced enhancement of osmotic adjustment in F50 indicate that the relatively salt tolerant cultivar had a higher prevention capacity for a large and permanent efux of K+ and Ca2+. Our results also suggest that under salt stress the main osmotic adjustment form of A. vera is accumulating inorganic cations in root and not accumulating low molecular weight organic solutes such as soluble sugars. Acknowledgements This study was funded by a grant of the National High Technology Research and Development Program of China (863 Program), Marine Technology Projects (No. 2001AA627040). References
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