Selected physiological responses of kudzu to different levels of induced water stress.ABSTRACT
This study was carried out to evaluate selected responses of kudzu [Pueraria montana var. lobata (Willd.)] to various levels of stress induced by polyethylene glycol (PEG) added to hydroponic growth media. Kudzu was grown hydroponically in dissolved concentrations of PEG (0.0, 52, 79.5, and 101 g l-1). The plants were grown under greenhouse conditions under the selected treatments. The objective of this study was to determine the level of stress under which kudzu could continue to grown and survive under. By determining the maximum level of osmotic stress that kudzu is able to withstand, the range of field areas to which kudzu could be adapted could then be determined. Kudzu was shown to have a significant decline in both growth and chlorophyll a and b concentration. However, caroteinoid was unchanged in all of the treatments. Kudzu response to drought resulted in an induced significant decrease in the water potential of the plant and an increase in the osmotic potential. Additionally, kudzu increased the total phenolic compounds as a defense response to drought stress. The results of this study support the drought tolerance of kudzu and indicate that the plant could be introduced successfully to areas with restricted water supplies.
The demand on food resources for both humans and livestock has increased worldwide due to the rapid increase in the human population and the destruction of the agricultural ecology of many areas. There are over 270,000 species of land plants, including 20,000 edible plants (Hammond, 1995). However, only about 20 species are utilized as the major sources of calories for human consumption. Certain species are excluded from many areas due to historical, physiological, or biotic factors. Kudzu [Pueraria montana var. lobata (Willd.)] is an underutilized plant species with a great potential to be used for multiple purposes such as for human and animal consumption and improving soil conditions.
Native to Japan, Kudzu is a leguminous, weedy vine with pubescent stems, trifoliate leaves, and a perennial deep root system (Forseth and Teramura, 1987). Kudzu has rapid shoot growth at a rate of 29 to 30 meters per growing season and a deep tap root (Sasek and Strain, 1988). Kudzu stems spread out in all directions, with new plants beginning at stem nodes every 30 to 60 cm. The dense packing of kudzu can result in tens of thousands of plants occupying a single acre of land. Furthermore, the extensive root biomass provides high potential of exploiting deeper sources of water (Schnoor et al, 1995). Moreover, kudzu can control soil erosion due to its large biomass and enhance soil fertility through nitrogen fixation (Tsugawa, 1986). Kudzu has adaptive capabilities to deal with adverse condition such as eroded soil with low fertility, low pH, and poor water holding capacity (Lynd and Ansman, 1990). These characteristics of kudzu make it an ideal candidate to be evaluated as an alternative species for use in restoration or to protect soil from erosion. In addition, Kudzu can be considered an excellent forage crop for a variety of livestock because of its nutritional value. Kudzu contains 17.43 percent protein and, 30.2 percent starch (Kidd, 2002). Kudzu has been used in Chinese herbal medicine for over 2000 years to combat acute febrile diseases (Fang, 1980). The significance of kudzu as a medicinal plant results from its relative abundance of antioxidants (Guerra et al., 2000 and Wenli, Yaping, and Bo, 2004).
Soil erosion is major problem in arid and semiarid regions of the world. There are many factors that aggravate this problem but none more important than the absence of vegetative cover. Due to limited water availability, most of the vegetative covers disappear soon after the end of the rain season. The exceptions are the very few species with the adaptive advantage to remain in the vegetative stage. However, most of these plants are small in size and provide limited soil cover. The ultimate solution is to find a plant with relative drought resistance and a large leave area index to be introduced to these areas to protect the soil from erosion. Kudzu's rapid growth rate and large leaf area index make it an excellent plant for containing soil erosion and preventing soil damage. Introducing kudzu might be beneficial in many ways, including ecologically through its potential to reduce soil erosion and its ability to enhance soil quality. Additionally, it will provide farmers with a suitable source of feed for livestock perhaps for human consumption as well. An experiment conducted within this study to evaluate antioxidant content in kudzu, spinach (Spinacia oleracea), shiitake (Lentinula edodes), and Nori seaweed (Porphyra including most notably P. yezoensis and P. tenera) showed that kudzu had the highest antioxidant concentrations, exceeding spinach by 2.5 fold.
One of the major factors that determine the successful establishment of any plant species in an arid environment is the ability of the plant to endure water stress. Kudzu has the potential to exploit water deep in the soil due to its tap root (Sasek & Strain, 1988.). In a separate article, Sasek and Strain (1990) report that an increase in atmospheric carbon dioxide shows a significant decline in water potential. They predict, using a climatic model, kudzu will spread northward over one hundred kilometers due to an increase in winter temperature in association with the increase in the atmospheric carbon dioxide. However, it is not clear how kudzu responds to drought stress at the early stages of growth and the actual affect of water stress on plant growth and physiology. In this study, hydroponically grown kudzu was exposed to different levels of water stress induced artificially by dissolving different concentrations (0, 52, 79.5, and 101 g l-1) of polyethylene glycol (PEG). PEG 8000 has been widely utilized in research for inducing artificial drought stress response in many plants (Steuter et al, 1981; Hardegree and Emmerich, 1990; Calamassi et al., 2001). Plant growth and photosynthetic pigments were used as indicators of kudzu's response to drought stress. Additionally, plant protective reaction was evaluated by determining the total phenols concentrations. The water potential and osmolality of the leaf were determined to evaluate the extent of the water stress in inducing these physiological responses.
METHODS AND MATERIALS
Kudzu plants were established for this experiment by germinating kudzu seeds that were collected from naturally growing kudzu in a field near Jacksonville, AL (USA). Kudzu seeds were scarified in concentrated sulfuric acid for 30 min. (Wechsler, 1977). The seeds were germinated in plastic pots containing a half-and-half mixture of vermiculite and potting soil. The plants were placed in a greenhouse and allowed to grow at 550 umol m-2 s-1 photon flux density, 45-50 % RH and air temperature of 25 [+ or -] 5 [degrees] C. Four weeks after emergence, uniform plants were individually transplanted into 900 ml glass containers, which contained half-strength Hoagland's solution (Hoagland and Arnon, 1938) at a pH of 6.5. Each container was wrapped with aluminum foil to prevent light penetration to prohibit algae growth. One week later, the plants were randomly assigned to water stress treatments. The water stress treatments were accomplished by the introduction of polyethylene glycol (PEG) to the growth media. The control was hydroponic media without polyethylene glycol. The selected concentrations of PEG were 52, 79.5, and 101 g 1-1. These selected PEG concentrations were calculated to induce water potential values of -0.5, -1.0, and -1.5 respectively using the following formula: [PSI]=1.22[PEG]2T-134[PEG]2-4.4[PEG], where T was 25[degrees]C (Michel, 1983). Each treatment in this study was replicated five times (5 samples). A continuous airflow through the hydroponic solution of each sample was achieved through a connection of a series of air tubes of glass containers. The plants were grown under the experimental conditions for fourteen days. At the conclusion of the experiment, individual samples were obtained from all the plants in the experiment to determine shoot water potential and osmolality. Additionally, plant growth and photosynthetic pigments were determined. The defense response of kudzu to water stress through antioxidant formation was evaluated by analyzing the plants under different treatments for anthocyanin and total phenolic compounds.
Photosynthetic pigment determination
Leaf tissue samples (0.05g) from each of the five samples of each treatment were individually placed in a vial containing 5 ml DMF (N, N-Dimethylformamide) and incubated in the dark for 36 h at 4[degrees] C to extract the photosynthetic pigments. Chlorophyll a and chlorophyll b concentrations of each sample from each treatment were determined spectrophotometrically by the method of Inskeep and Bloom (1985). Carotenoid concentrations of the DMF extract were determined spectrophotometrically at a wavelength of 470 nm and the concentrations calculated using the formula of Doong et al. (1993).
Samples of 0.10 g fresh weight were homogenized in 5 ml of methanol containing 1% HC1 (v/v). Homogenates were then filtered and anthocyanin and absorbance of the extract measured at 530 and 657 nm spectrophotometrically by the method of Mancinelli (1990).
Total Phenolic Content
The phenolic compound concentration in the leaf of kudzu was determined following a slightly modified procedure described by Singleton and Rossi (1965). Leaf tissue (3g) of each sample of each treatment was immediately frozen in liquid nitrogen and then crushed to a fine powder using a mortar and pestle. The individual samples were then extracted by stirring with 100 ml of methanol at 25 [degrees]C at 150 rpm for 24 h and filtered through Whatman No. 4 paper. The residue was then extracted with two additional 100 ml portions of methanol. The combined methanolic extracts were evaporated at 40 [degrees]C to dryness. The total phenolic content of each extract was determined using Folin-Ciocalteau reagent and gallic acid as standard. Subsamples from each extract (50 mg) were dissolved in 25 ml of extraction solvent (40 ml acetone: 40 ml methanol: 20 ml water: 0.1 ml acetic acid) and vortexed until the extract was dissolved. The samples were then heated at 60 C in a water bath for 1 hour. After cooling down to room temperature, the samples were centrifuged at 1640g for 20 minutes. The supernatant was placed in a test tube and 1.0 ml of Folin-Ciocalteau was added and allowed to stand at room temperature for 5 minutes; 1.0 ml of sodium bicarbonate solution (0.566 M) was added and the tubes were vortexed and stored in the dark at room temperature for two hours. The total phenolic determination was measured spectrophotometrically at absorption of 726 nm. Gallic acid was utilized as a standard with the concentration series from 0.03-0.2 mM.
Water potential measurement
Leaf water potential of the five samples from each treatment was measured as described by Al-Hamdani et al. (1990). Cell sap, 100 [mu]L, was extracted from each of the selected leaves with a French Press, loaded on a paper disc, and measured with a vapor pressure osmometer (model 5520, Wescor, Logan, UT).
The individual plants of each treatment were severed at the crown level, and the shoots and roots were separately oven dried at 85[degrees] C for 48 h. The shoot and root dry weights of each sample were recorded. These measurements were used to construct a shoot to root ratio for each treatment.
This study was repeated three times. Each replicate was represented by the different times the experiment was conducted. The data of each experiment were combined and variables were statistically analyzed as a randomized complete block design with each replicate as a block. Mean separations for the variables with significant F values, at 1 or 5 % level of probability, of the ANOVA analysis were based on the least significant difference (LSD) test (Steel and Torrie, 1980).
RESULTS AND DISCUSSION
The introduction of PEG to the growth media resulted in a gradual significant decrease in both osmotic potential and water potential of kudzu leaf with the increase of the PEG concentration (Table 1). Polyethylene glycol compounds have been extensively used to induce water stress in plants grown in hydroponic media (Asadi-kavan et al., 2009; Slama et al., 2007). Kaufmann and Eckard (1971) reported that PEG induced water stress in plants similar to those grown in soil in drought conditions. Similarly, the reduction in water potential that is associated with the increase in PEG concentration was reported due to the reduction in the free energy of the water proportionally influenced more by metric forces than osmotic forces (Steuter, 1981). In this study, the significant decreases of both osmotic potential and water potential were expected as a common response of the plants to drought stress. The observed decrease in osmotic potential was most likely due to the accumulation of ions, inorganic and organic molecules that commonly result in reduced osmotic potential as a part of a plant's adaptive response to drought stress (Hessini et al., 2009). Reduction of osmotic potential is considered as a driving force in inducing water movement from the soil into the plant to maintain turgor pressure (Blum et al., 1996). Additionally, osmotic adjustment is considered as a major adaptation criteria of the plant to combat water deficit (Oliver et al., 2010). A study was carried out using PEG to induce water stress in eight European provenances of Pinus halepensis to evaluate the water potential of the selected species to various levels of water stress (Calamassi et al., 2001). The water potential values of all the investigated pine plants were comparable to the values that were obtained in this study from kudzu leaf. The similarity in water potential response between these two studies can be used as an indicator that kudzu has the potential to osmotically adjust to combat water stress in a manner comparable to that of pine, which is considered highly adapted to arid conditions.
Table 1. Effect of different concentration of polyethylene glycol (PEG) ON WATER POTENTIAL AND osmotic potential. PEG (g/1) Water Potential (-MPa) Osmotic Potential (-MPa) 0.00 0.8313a 0.75477a 52.00 0.9495b 0.86215b 79.50 1.0697c 0.97133c 101.00 1.5147d 1.37539d Means followed by the same lowercase letter in each column are not significantly different based on the LSD test (P=0.01). The LSD values were 0.0052 and 0.00469 for water potential and osmotic potential respectively.
Water stress induced by the different PEG concentrations resulted in a significant reduction in both shoot and root growth (Table 2). However, the reduction in growth was similar at both PEG concentrations of 79.5 and 101 g 1-1. General observations of the plants grown at these two concentrations of PEG included the browning of the leaves, sthe stunting of stem growth, and rolling, which most likely resulted from the reduction in turgor pressure. The similarity in growth reduction between these two treatments could result from plants reaching the threshold of tolerance to drought at these concentration levels. The root of the plant appeared to be less sensitive to drought stress than the shoot as indicated by the lower value of shoot/root ratio of all water stress treatments in comparison to the control (Table 2). The significant reduction in the shoot/root ratio was similar at both 79.5 and 101 g/1 PEG. This again could result from the stress stunting the growth of kudzu at these concentrations. In many studies, root growth was shown to continue at the expense of both leaf and stem under water stress conditions (Kramer, 1983; Westgate and Boyer, 1985; Creelman, 1990). This common response of root growth at the expense of the shoot is considered an advantage for the plant to continue water uptake and enhance the water status of the plant (Caldwell, 1976). The observed reduction in plant growth under water stress treatments is considered a common response to water stress that has been observed in many related studies (Westgate and Boyer, 1985; Burnett et al., 2006; Jaleel et al., 2009). The impact of water stress on plant growth was interpreted as a result of a reduction in both cell enlargement and cell division (Sommer et al., 1999; Jaleel et al., 2009).
Table 2. Effect of different concentrations of glycol (PEG) on kuduz growth. PEG (g/1) Shoot DW (g) Root DW (g) Shoot/Root Ratio 0.00 14.672a 2.905a 5.084a 52.00 7.383b 1.992b 3.704b 79.50 3.797c 1.347c 2.819c 101.00 4.861c 1.581d 3.072c Means followed by the same lowercase letter in each column are not significantly different based on the LSD test (P=0.01). The LSD values were 2.652, 0.157, and 0.756 for Shoot DW, Root DW and the Shoot/Root Ratio respectively.
Kudzu plant subjected to water stress treatment showed a significant decline in both chlorophyll a and b in comparison to the control plants (Table 3). However, plants grown at 79.5 g/1 PEG showed the most significant decrease in both chlorophylls in comparison to other water stress treatments. The increase in chlorophyll a and b at 101 g/I PEG could be attributed to experimental errors or other unexplained reasons. However, this experiment was repeated twice with the same result. The decline in chlorophyll that was observed in this study is considered a common response of plants to water stress and has been reported in many similar studies (Sangtarash et al, 2009; Jung, 2004; Parida et al., 2007; Bacelar et al., 2006). The significantly higher ratio of chlorophyll a/b that was observed in plants growing at 79.5 and 101 g/1 PEG indicates that chlorophyll b was more susceptible to these water stress treatments than was chlorophyll a. However, the influence of 52 g l-1 PEG in the growth media on chlorophyll a/b was similar to that of both the control and the other water stress treatments. It appears from examining the literature that the effect of water stress on chlorophyll a/b ratio was mostly related to the severity of the drought stress and to the plant species. Sangtarash et al. (2009) showed higher chlorophyll a/b ratio in longstalk starwort plants (Stellaria longipes) growing in water stress conditions. Similarly, Efeoglu et al. (2009) reported that drought stress caused increases in chlorophyll a/b ratio in three cultivars of com (Zea mays) However, drought stress induced a decline in chlorophyll a/b ratio in Arabidopsis (Arabidopsis thaliana) (Jung, 2004). The significance of the chlorophyll a/b ratio is that it influences the efficiency of photosytem II of the thalakoid membrane and can higher chlorophyll a/b ratio can be used as an indicator of higher drought stress tolerance (Sangtarash et al., 2009). In this study, carotenoid in kudzu leaf was shown to have a similar concentration at all the treatments (Table 3). A similar result was obtained in arabiopsis in response to drought stress (Jung, 2004). However, another study that showed drought induced a significant reduction in carotein concentration in one of the examined wheat (Triticum durum) variety where as no significant difference was obtained in another variety (Loggini et al., 1999). Reduction in carotenoid concentration as influenced by water stress was shown in three varieties of corn (Efeoglu et al., 2009). These different studies support the conclusion that the response of carotenoid to water stress is dependent on plant species or the variety within the plant species in addition to the severity of the drought conditions.
Table 3. Effect of different concentrations of polyethylene glycol (PEG) on chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoid (Caro). PEG (g [l.sup.-1]) Chl a (mg [g.sup.-1] FW) CHl b (mg [g.sup.-1] FW) 0.00 32.232a 28.537a 52.00 29.899b 22.337b 79.50 21.004c 12.922c 101.00 24.94 ld 16.300d PEG (g [l.sup.-1]) Chl alb ratio Caro([mu]g [g.sup.-1] FW) 0.00 1.135a 165.376a 52.00 1.377ab 266.013a 79.50 1.625b 257.913a 101.00 1.533b 227.826a Means followed by the same lowercase letter in each column are not significantly different based on the LSD test (P=0.05). The LSD values were 2.703, 6.35, 0.278, and 103.133 for chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoid (Caro) respectively.
The response of kudzu to water stress in this study and the responses of the various plants in the examined literature can be attributed to the impact of free radicals induced by the water stress. This presumption is supported by the findings of several studies that showed that many physiological and biological responses, including those in this study, were negatively impacted by water stress induced free radicals (Smirnoff, 1993; Johnson et al., 2003; Nayyar and Gupta, 2006). The degree of tolerance of a plant species or a variety within a species is highly correlated with the ability of the plant to induce antioxidant response to combat the water stress induced free radicals (Smirnoff, 1993; Johnson et al., 2003). Polyphenolic compounds are considered a major antioxidant in combating free radical induced oxidative damage in the plant due to their high reactivity as hydrogen or electron donors (Manna et al, 2002; Blokhina et al., 2003). In this study, the total phenolic compound was significantly increased with the increase in water stress induced by the elevated PEG concentrations (Figure 1). This finding is supported by other studies with similar results obtained in different plant species (Parida et al., 2007; Sanchez-Rodriguez et al., 2010).
[FIGURE 1 OMITTED]
In conclusion, induced water stress on kudzu caused a reduction in plant growth and chlorophyll concentration, factors that were selected to evaluate the response of kudzu to the selected water stress. The damage most likely resulted from the impact of free radicals induced by water stress and the alteration of water relations within the plant. However, the extent to which kudzu can avoid drought stress can be determined by the formation of polyphenolic compounds and the levels of osmotic adjustment, both of which appeared significantly increased in response to drought. Additionally, the general observation of kudzu growing in its natural habitat appeared to show high drought tolerance due to the fact that our area (Jacksonville, AL USA) endures relatively high summer temperature and extended drought periods.
The authors of this study wish to acknowledge Samantha Davis and Ginnilee Feldtman for their significant contribution with technical assistance. In addition, we would like to acknowledge Jacksonville State University for financial support of this project.
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David M. Ponder and Safaa H. Al-Hamdani
Department of Biology, Jacksonville State University, 700 Pelham Road North, Jacksonville, Alabama, 36265, USA
Corresponding author: Safaa H. Al-Hamdani (email@example.com)