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Title: Hydrogen Sulfide Alleviates Aluminum Toxicity in Germinating Wheat Seedlings
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Running title: Hydrogen Sulfide Alleviates Aluminum Toxicity
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Article types: Research paper
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Authors: Hua Zhang
1, 2, *
, Zhu-Qin Tan 1, Lan-Ying Hu 1, Song-Hua Wang 3, Jian-Ping
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Luo 1, Russell L. Jones 2
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1
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Hefei, Anhui 230009, China;
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2
School of Biotechnology and Food Engineering, Hefei University of Technology,
Department of Plant and Microbial Biology, University of California, Berkeley,
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CA 94720, USA;
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3
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233100, China;
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*
Life Science College, Anhui Science and Technology University, Bengbu
Author for correspondence: Hua Zhang
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Tel: +86(0)-551 2901 506-8635, +1-510-280-4140;
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Fax: +86(0)551 2901 507;
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E-mail: .
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This work was supported by the Great Project of Natural Science Foundation from Anhui
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Provincial Education Department (ZD200910), the Natural Science Foundation of Anhui
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Province (070411009), and the innovation funding to undergraduate students at HFUT
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(XS08072, 0637).
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Hydrogen Sulfide Alleviates Aluminum Toxicity in Germinating Wheat
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Seedlings
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Abstract: Protective role of hydrogen sulfide (H2S) on seed germination and seedling
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growth was studied in wheat (Triticum) seeds subjected to aluminum (Al3+) stress. We
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show that germination and seedling growth of wheat is inhibited by high concentrations of
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AlCl3. At 30 mmol/L AlCl3 germination is reduced by about 50% and seedling growth is
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more dramatically inhibited by this treatment. Pre-incubation of wheat seeds in the H2S
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donor NaHS alleviates AlCl3-induced stress in a dose-dependant manner at an optimal
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concentration of 0.3 mmol/L. We verified that the role of NaHS in alleviating Al3+ stress
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could be attributed to H2S/HS- by showing that the level of endogenous H2S increased
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following NaHS treatment. Furthermore, other sodium salts containing sulfur were
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ineffective in alleviating Al3+ stress. NaHS pretreatment significantly increased the
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activities of amylases and esterases and sustained much lower levels of MDA and H2O2 in
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germinating seeds under Al3+ stress. Moreover, NaHS pretreatment increased the activities
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of guaiacol peroxidase, ascorbate peroxidase, superoxide dismutase and catalase and
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decreased that of lipoxygenase. NaHS pretreatment also decreased the uptake of Al3+ in
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AlCl3-treated seed. Taken together these results suggest that H2S could increase antioxidant
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capability in wheat seeds leading to the alleviation of Al3+ stress.
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Key words: aluminum stress; antioxidant enzymes; hydrogen sulfide; seed germination
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and seedling growth; wheat (Triticum).
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Hydrogen sulfide (H2S) has recently been identified as a third endogenous gaseous
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transmitter after nitric oxide (NO) and carbon monoxide (CO) in animals where it plays
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various roles ranging from regulation of the nervous and cardiovascular systems (Wang
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2002; Li et al. 2006; Yang et al. 2008). For example, Hosoki et al. (1997) demonstrated that
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H2S acts synergistically with NO to regulate smooth muscle relaxation. It is now well
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established that NO is involved in diverse physiological processes in plants (Delledonne
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2005). More recent evidence has been accumulated that CO is also involved in different
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biological process in plants such as root formation (Cao et al. 2007) and protection against
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oxidative damage induced by salinity (Huang et al. 2006; Xu et al. 2006) and mercury (Han
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et al. 2007). Plants are known to synthesize and release H2S. For example, H2S was proved
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to be released from leaves of cucumber (Cucumis sativus L.), sqush and pumpkin
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(Cucurbita pepo L.), cantaloupe (Cucumis melo L.), corn (Zea mays L.), soybean (Glycine
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max [L.] Merr.) and cotton (Gossypium hirsutum L.) (Wilson et al. 1978). H2S is also
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produced by cut branches, detached leaves, leaf discs, or tissue cultures, evidence that green
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cells of higher plants can release H2S into the atmosphere (Wilson et al. 1978; Winner et al.
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1981; Sekiya et al. 1982a, 1982b; Rennenberg 1983, 1984; Rennenberg et al. 1990). It is
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conceivable that H2S might serve as an informational signal to other parts of the plant, or to
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plants in the vicinity in a similar manner to NO and CO (Zhang et al. 2008a; Zhang et al.
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2009a, b).
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Recently, several types of specific desulfhydrases have been identified and
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functionally characterized in plants, confirming that H2S might be released by the action of
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desulfhydrases localized in different cellular compartments such as the cytosol,
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mitochondria and plastids (Leon et al. 2002; Riemenschneider et al. 2005b; Rausch and
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Wachter 2005). Although the individual roles of these enzymes are not yet understood,
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L-cysteine desulfhydrase expression and activity are induced upon pathogen attack,
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suggesting that released H2S has a role in plant defense (Bloem et al. 2004; Rausch and
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Wachter 2005). Riemenschneider et al. (2005a) also investigated that impact of elevated
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H2S on metabolite levels, the activity of enzymes and expression of genes involved in
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cysteine metabolism, confirming that H2S serves as a signal molecular to control thiol
3
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levels in Arabidopsis thaliana. Although H2S emission has been widely observed in many
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plant species its role as a signaling molecule in plants has yet not to be clearly defined.
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Nevertheless, H2S is known to play a role in various responses of plants against stresses.
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Roles of H2S in response to pathogen attack (Bloem et al. 2004), Cu2+ tolerance in
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germinating wheat seeds (Zhang et al. 2008a), osmotic stress tolerance in sweet potato
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seedlings (Zhang et al. 2009b), SO2 tolerance in pine trees (Hällgren and Fredriksson 1982;
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Sekiya et al. 1982b), and freezing tolerance in wheat shoots (Stuiver et al. 1992), all
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suggest that H2S is involved in mechanisms of plant resistance.
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Aluminum ions (Al3+) are ubiquitous in soil and are especially toxic in acidic soils.
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Furthermore, Al3+ is one of the major limiting factors affecting crop production in tropical
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regions having acid soils (Foy 1984; MacDonald and Martin 1988). It is estimated that 70%
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of tropical soil in the Americas are acidic and have toxic levels of Al3+ that are growth
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limiting for crops (Marschner 1995). One of the primary causes of Al3+ toxicity is oxidative
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stress due to accumulation of reactive oxygen species (ROS), such as the superoxide anion
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(O2·¯) and hydrogen peroxide (H2O2). These ROS have been shown to bring about lipid
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peroxidation in soybean root tips (Horst et al. 1992), detached rice leaves (Kuo and Kao
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2003), and in roots of Cassia tora L. (Wang and Yang 2005) and Melaleuca trees (Tahara et
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al. 2008). Our previous report on Al3+-induced oxidative stress in wheat seedlings
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confirmed that ROS are a key component of the response of wheat to Al3+ (Zhang et al.
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2008b). Production and removal of ROS generally involve non-enzymatic and enzymatic
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antioxidant systems and if the equilibrium between ROS production and breakdown fails,
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oxidative damage occurs (Apel and Hirt 2004). It is now becoming increasingly clear that
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plant resistance to Al3+-induced oxidative stress involves a wide range of signaling
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molecules, such as Ca2+/CaM, inositol 1,4,5-triphosphate (IP3), salicylic acid, H2O2 and NO
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(Jones and Kochian 1995; Wang et al. 2004; Wang and Yang 2005; Zheng and Yang 2005;
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Zhang et al. 2008b).
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In this paper we describe the effects of H2S pretreatment on the response of wheat
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seeds to Al3+ toxicity. In previous work, we demonstrated that H2S promotes seed
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germination and root formation, and acts as an antioxidant signal counteracting Cu2+ and
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osmotic stress in plants (Zhang et al. 2008a; Zhang et al. 2009a, b). We now show that H2S
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pretreatment offers significant protection against the toxic effects of Al3+. Our data show
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that there is a strong correlation between reduced ROS level and lowered lipid peroxidation
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and pretreatment of wheat seeds with H2S. We concluded that H2S acts as an antioxidative
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signaling molecule that participates in alleviation of Al3+ toxicity during wheat seed
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germination.
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Results
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Inhibition of germination and seedling growth in wheat by Al3+
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The inhibitory effects of Al3+ on wheat seed germination and wheat seedling growth and
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development were examined over a wide range of AlCl3 concentrations from 5 mmol/L to
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150 mmol/L. Wheat seeds are relatively insensitive to concentrations of AlCl3 of 5 mmol/L
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or below (Table 1 and data not shown). Wheat seed germination percentage was slightly
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higher in 5 mmol/L AlCl3 relative to controls germinated in H2O, and at 10 mmol/L AlCl3
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germination was identical to that in H2O (Table 1). Coleoptile length and radicle number
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were also unaffected by 5 mmol/L AlCl3, but radicle length was inhibited by 20%.
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To test the alleviating effects of H2S on wheat germination and growth we determined
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the concentration of AlCl3 which inhibited germination by 50%. As Table 1 and Figure 1
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show a concentration of 30 mmol/L AlCl3 inhibited germination by 51% and radicle and
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coleoptile length were reduced by 95% and 40% respectively. Radicle number in seeds
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incubated in 30 mmol/L was reduced to 25% of controls in H2O (Table 1). Concentrations
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of AlCl3 above 60 mmol/L inhibited radicle growth almost completely, but even at this
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concentration of AlCl3 coleoptile growth was still observed. Based on these data we chose a
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working concentration of 30 mmol/L AlCl3 to study the ameliorating effects of H2S.
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Amelioration of Al3+ stress in wheat by the H2S donor NaHS
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We first established whether the H2S donor NaHS had toxic effects on wheat seed
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germination and seedling growth. Wheat seeds were germinated for 36 h in increasing 5
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NaHS concentrations from 0.3 mmol/L to 1.5 mmol/L (Table 2 and Figure 2A). There was
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no statistically significant effect of NaHS relative to controls in H2O on any of the
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parameters that we measured. Germination, coleoptile and radicle elongation, and radicle
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number was essentially unchanged following incubation in NaHS relative to control seeds
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incubated in water for 36 h.
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We next tested the ability of NaHS to alleviate the toxic effects of AlCl3 by pre-treating
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wheat seeds with the H2S donor for 12 h prior to incubation with Al3+. To establish the
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effective concentration of NaHS in alleviating the effects of AlCl3, the H2S donor was
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applied to wheat seeds stressed with 30 mmol/L AlCl3 in the range of 0.3 to 1.5 mmol/L
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(Table 3 and Figure 2B). NaHS pretreatment was effective in alleviating the toxic effects of
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Al3+ at all concentrations that were tested, but the optimal NaHS concentration for
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alleviating germination and seedling growth was 0.6 mmol/L. At this concentration
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germination percentage was increased by 74%, radicle number by 80%, and radicle and
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coleoptile length by 202% and 255% respectively. Clearly, the H2S donor exerted a strong
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positive effect on wheat germination and seedling growth following exposure to Al3+.
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To verify the role of H2S in the promotion of seed germination induced by NaHS in
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Al3+ treated wheat we tested the effect of a range of sodium salts including those that
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contained sulfur. Seeds were pre-incubated in 0.6 mmol/L Na2S, Na2SO4, Na2SO3, NaHSO4,
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NaHSO3, and NaAC for 12 h and incubated for a further 48 h in 30 mmol/L AlCl3. As the
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data in Figure 2C show, only NaHS was able to overcome the toxic effects of Al3+. There
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was no significant effect of the other sodium salts on germination of wheat seeds. From
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these results it is concluded that either H2S or HS-, rather than other compounds derived
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from NaHS, are responsible for the alleviating Al3+ stress in wheat seedlings.
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At millimolar concentrations, NaHS solutions are neutral to basic, the pH of NaHS
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solutions increase from pH 6.85 at 0.01 mmol/L to pH 8.69 at 4 mmol/L. We chose a
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working concentration of 0.6 mmol/L NaHS for our experiments. In order to prove that the
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promotive role of NaHS pretreatment on seeds germination against Al3+ stress is
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contributed to NaHS rather than the pH value due to NaHS added in the pretreatment
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solutions, 0.6 mmol/L PBS (Na2HPO4-NaH2PO4 buffer) at different pH value (5.8, 6.0, 6.2,
6
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6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.8, 8.0) were used as the controls of NaHS with different
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concentration (0, 0.3, 0.6, 0.9, 1.2, 1.5 mmol/L). Wheat seeds were pretreated with water
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control (CK), 0.6 mmol/L NaHS, and 0.6 mmol/L Na2HPO4-NaH2PO4 buffer solutions at
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different pH value 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.8, and 8.0, respectively. After
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12 h of pretreatment, seeds were washed with water and then subsequently subjected to
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30.0 mmol/L AlCl3 stress for a further 48 h. Figure 2D showed that the strong positive
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effect of H2S donor on seed germination and seedling growth against Al3+ stress is
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independent on pH value, because pretreatments with PBS buffer (pH 5.8~8.0) did not
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promote seed germination under Al3+ stress as NaHS pretreatments did (Figure 2D).
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Effect of NaHS pretreatment on the activities of amylase and esterase
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Whereas hydrolytic enzymes such as α-amylases and esterases are unlikely to be required
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for germination of what seeds, seedling growth and development are known to depend on
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the mobilization of stored endosperm reserves. We therefore examined the activities of
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amylases and esterase in wheat seeds exposed to AlCl3 following pretreatment with and
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without NaHS (Figure 3 and 4). Hydrolases were extracted from dry wheat seeds (CK0’),
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from seeds pretreated in water or NaHS for 12 h (12’/0) and from seeds incubated for up to
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48 h in 30 mmol/L AlCl3 (12~48). Amylase activity was determined following
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electrophoresis (Figure 3A) or by measurement of enzyme activity colorimetrically (Figure
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3B). Amylase activity was not detected following electrohporesis of extracts from dry
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seeds and activity was low when it was measured colorimetrically. Pretreatment of seeds
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with H2O or NaHS for 12 h caused an increase in amylase activity but the increase was
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much greater in the presence of NaHS. The H2S donor was effective in ameliorating the
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effect of AlCl3 on the activity of amylase and its activity increased linearly for the first 36 h
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of incubation. There was a much smaller increase in amylase activity in response to AlCl3
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in seeds pretreated in H2O.
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There was a much less pronounced effect of NaHS on esterase activity (Figure 4A
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and B). Esterase activity was high in dry seeds and was relatively unchanged by incubation
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in H2O for 12 h or following treatment with AlCl3. NaHS pretreatment for 12 h did not
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increase esterase activity, but there was a small and statistically significant effect of NaHS
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on esterase activity when seeds were incubated for up to 48 h in 30 mmol/L AlCl3. This
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stimulation of esterase activity by the H2S donor was observed following eletrophoresis
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(Figure 4A) and by colorimetric measurement of esterase activity (Figure 4B).
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Effect of NaHS pretreatment on contents of H2O2, O2·¯ and MDA
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Malondialdehyde (MDA) has been widely used to estimate the extent of lipid peroxidation
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in plant tissues and we used this assay to determine the effect of NaHS treatment in wheat
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seeds exposed to AlCl3 (Figure 5A). MDA-reactive lipids were low in dry seeds and
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increased almost two-fold following incubation for 12 h in either H2O or NaHS. There is a
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dramatic increase in MDA-reactive lipids following 12 h incubation in AlCl3 in seeds
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pretreated with H2O, but the increase was dramatically lower in seeds pre-incubated in
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NaHS (Figure 5A). The amount of MDA-reactive lipids increased in seeds exposed to
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AlCl3 for 24 h, 36 h and 48 h, but the H2S donor dramatically reduced their amount relative
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to seeds pre-incubated in H2O.
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Next, we measured the accumulation of H2O2 and O2- in wheat seeds pretreated with
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the H2S donor then exposed to Al3+ stress (Figure 5B, C). The concentrations of H2O2 and
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O2- were both low in dry wheat seeds and they increased about two-fold following
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incubation in either H2O or NaHS for 12 h. Exposure of H2O-pretreated seeds to AlCl3
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brought about more than a three-fold increase in ROS species, but following pretreatment
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with NaHS for 12 h the increase in H2O2 and O2- was greatly reduced. Thus H2O2 levels
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increased about 50% and O2- increased 60% in AlCl3 following NaHS pretreatment,
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whereas
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(Figure 5B, C).
H2O pretreatment increased H2O2 and O2- content by more than 200% after 12 h
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Effects of NaHS pretreatment on SOD, CAT, APX, POD and LOX activities
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We next investigated whether reduced levels of ROS in seeds pretreated with NaHS might
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result from an increase in the activity of ROS-scavenging enzymes in AlCl3 pretreated
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seeds (Figure 6). The activities of all ROS scavenging enzymes were low in dry wheat
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seeds and they increased following incubation for 12 h in either H2O or NaHS. The
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activities of SOD and APX fell significantly during the first 12 h of incubation of H2O
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pretreated seeds in AlCl3. By contrast SOD activity did not change following Al3+
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treatment of NAHS-pretreated seeds (Figure 6A), but the activity of APX increased by
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about 50% in these seeds (Figure 6C).
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Catalase and peroxidase activities were not markedly affected by AlCl3 treatment in
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seeds pretreated with H2O, but these two enzymes showed an increase in their activities
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during 36 h of incubation in NaHS. Thus CAT activity increased almost two-fold whereas
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POD actvity increased about three-fold in AlCl3 treated seeds pretreated with NaHS
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(Figure 6B, D).
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We also assayed the activity of lipoxygenase, the enzyme that gives rise to molecules
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such as jasmonic acid. LOX activity was low in dry seeds and increased only slightly on
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further incubation in H2O or NaHS for 12 h (Figure. 6E). LOX activity continued to
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increase in H2O pretreated seeds incubated in AlCl3 and by 24 h after exposure to Al3+
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LOX activity was more than 300% higher than in seeds pretreated with H2O for 12 h but
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there was a dramatic decline in LOX activity in these seeds when incubated in AlCl3 for 36
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h and 48 h. By 48 h after AlCl3 treatment the activity of LOX returned to the level found in
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seeds pretreated in H2O for 12 h (Figure 6E). LOX activity in NaHS pretreated seeds also
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increased following Al3+ exposure but the increase was much less than that observed in
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seeds pretreated with H2O and there was a similar decline in LOX activity after 36 h of
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incubation.
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NaHS pretreatment reduces Al accumulation in wheat seedlings
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We measured the accumulation of Al in wheat seeds and seedlings under the conditions of
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our germination experiments. As expected the Al content in dry seeds and 12 h imbibed
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seeds was low, but following incubation in AlCl3 Al concentrations increased (Figure 7).
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The increase in Al was much more dramatic in seeds pre-incubated for 12h in H2O
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compared to those pre-incubated in NaHS. After 48 h incubation in AlCl3, the Al
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concentration in H2O pretreated seeds was more than twice that found in seeds
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pre-incubated in NaHS (Figure 7).
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Endogenous H2S content is elevated in wheat seedlings treated with NaHS
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We measured H2S concentration in wheat seeds under the various conditions of incubation
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(Figure 8). Endogenous H2S was low in dry seeds, but it increased following incubation for
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12 h. As expected, the increase in H2S was much greater in NaHS-treated seeds. AlCl3
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treated seeds showed a sharp increase in H2S during the first 12 h and thereafter there was
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no change in its concentration. There was also an increase in H2S levels in H2O-pretreated
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seeds after 12 h incubation in AlCl3, but thereafter the amount of H2S declined reaching the
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level found in seeds pre-incubated in H2O for 12 h. These results show convincingly that
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the H2S donor NAHS donor contributes to an increase in endogenous H2S concentrations in
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wheat seedling tissues.
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Discussion
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Aluminum is abundant in soils and in acidic soils it is often present at concentrations that
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are inhibitory to plant growth and development. In this paper we show that the inhibitory
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effects of AlCl3 on germination and seedling growth in wheat can be alleviated by
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pretreatment with the H2S donor NaHS. Our data show that AlCl3 at 30 mmol/L inhibits
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wheat seed germination by about 50% and coleoptile and radicle growth is even more
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severely inhibited at this concentration of Al3+. Preincubation of wheat seeds in NaHS for
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12 h alleviates the toxic effects of AlCl3. Pretreatment of wheat seeds with NaHS also
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increases the production of ROS scavenging enzymes and reduces the oxidation of lipids.
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NaHS is a commonly used H2S donor in biological systems (Hosoki et al. 1997).
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NaHS dissociates to Na+ and HS- in solution and HS- associates with H+ to produce H2S.
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That NaHS functions as an H2S donor, alleviating the effects of Al3+ in wheat is supported
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by two lines of evidence. First, among the solutions that we tested, only NaHS effectively
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reverses the toxic effects of Al3+ in wheat and its ameliorating role was independent on the
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pretreatment of pH value changes. Solutions of Na2S, Na2SO4, Na2SO3 NaHSO4, and
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NaHSO3 were largely ineffective in stimulating germination or seedling growth in 10
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AlCl3-treated wheat. Second, wheat seeds exposed to NaHS show a dramatic increase in
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extractable H2S levels after incubation in this H2S donor (Figure 8). Seeds incubated in
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H2O also show a significant increase in endogenous H2S but this is likely to be derived
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from the mobilization of sulfur containing storage proteins in the wheat grain (Shewry
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1995).
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In accord with previous observations (Zheng and Yang 2005) our data show that Al3+
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stress in plants results in overproduction of ROS. We show that AlCl3 brings about a
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dramatic increase in H2O2 and O2- levels and that these increases are mitigated by
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pretreatment with NaHS. Furthermore, NaHS pretreatment increases the activities of
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enzymes that can scavenge ROS including APX, CAT, POD and SOD. The net result of the
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increase in ROS scavenging enzymes is a dramatic reduction on lipid oxidation as shown
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by much lower amounts of MDA-reactive lipids in NaHS pretreated wheat. Taken together
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this data strongly supports the idea that NaHS alleviates Al3+ stress by reducing ROS levels.
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It is well established in plants that the reactive nature of ROS makes them potentially
288
harmful to all cellular components. Plants have evolved the capacity to eliminate ROS with
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an efficient scavenging system and enzymes such as APX, CAT and SOD are among the
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most effective ROS scavengers (Van Breusegem et al. 2001; Mittler 2002, 2006). In our
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study, overproduction of O2·¯, H2O2 in Al3+ stressed wheat (Figure 5) might contribute to
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the lipid peroxidation and cellular membrane damage, which in turn results in inhibition of
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seed germination and seedling growth (Table 1 and Figure 1). H2O2 is produced in response
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to various stimuli in plants (Bowler and Fluhr 2000) and acts as a signal for the activation
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of stress-response and defense pathways (Bartosz 1997; Blokhina et al. 2003). Thus, H2O2
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can be viewed as cellular indicator of stress and as a second messenger involved in signal
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transduction pathways linked to stress (Knight and Knight 2001). Whether H2S works in
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concert with H2O2 as a second messenger in stress responses such as that brought about by
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Al3+ remains to be elucidated.
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In summary, our data strongly support the hypothesis that the H2S donor NaHS
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alleviates Al3+ toxicity in wheat by suppressing the production of ROS. The H2S donor
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increases the activities of ROS scavenging enzymes that bring about a reduction in H2O2
11
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and O2- leading to a reduction in lipid peroxidation and membrane damage. These data
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have implications for studies of metal and other stresses in plants and our goals are to
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understand the details underlying the H2S signal transduction pathway.
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Our data also provide evidence that pretreatment with exogenous H2S donor leads to
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higher levels of endogenous H2S signal and that this higher H2S concentration alleviates
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Al3+-induced oxidative damage to germinating seed. This phenomenon was correlated with
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the suppression of H2O2 and MDA overproduction in NaHS-pretreated seeds under Al3+
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stress. The H2S donor-induced lowering of H2O2 and MDA levels could be attributed to the
311
increased activities of ROS-scavenging enzymes and the decreased activity of LOX. Many
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phenomena such as expression of L-cysteine desulfhydrase upon pathogen attack (Bloem et
313
al. 2004), freezing tolerance affected by H2S fumigation (Stuiver et al. 1992), and emission
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of H2S from plant against SO2 injury (Hällgren and Fredriksson 1982; Sekiya et al. 1982b),
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indirectly support our conclusions that H2S might activate an H2O2-mediated antioxidant
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signaling pathway and play a protective role in plant defense. Our evidence adds to the
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concept that H2S can be another important signal molecule for abiotic stress tolerance.
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Genetic analysis and further physiological studies will help establish H2S as a player in
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signal in transduction cascades in plants and improve our understanding of the mechanisms
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of H2S perception and how it is transduced into specific downstream responses.
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Materials and methods
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Materials and treatments
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Wheat (Triticum aestivum L., Yangmai 158) seeds were supplied by the Jiangsu Academy
325
of Agricultural Sciences, Jiangsu Province, China. Sodium hydrosulfide (NaHS, Sigma)
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was used as hydrogen sulfide (H2S) donor according to Hosoki et al. (1997). Wheat seeds
327
were sterilized with 0.1% HgCl2 for 3 min, washed extensively with dH2O, and dried with
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filter papers. To establish the inhibitory effect of AlCl3 on germination and seedling growth,
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sterilized wheat seeds of approximately equal size were selected and allocated randomly in
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Petri dishes (9 cm diameter × 1.2 cm depth, 50 seeds per dish) and germinated in the dark at
12
℃ with 0, 5, 10, 15, 20, 25, 30, 60, 90, 120, 150 mmol/L AlCl , for 48 h. Seeds were
331
25
332
recorded as germinated when the length of the coleoptile or radicle reached 50% of the
333
length of seed. The length of coleoptiles and radicles and radicle number were also
334
recorded. The protective roles of H2S on the Al3+-induced inhibition of seed germination
335
and seedling growth was examined by pre-treating seeds with 0, 0.3, 0.6, 0.9, 1.2, 1.5
336
mmol/L NaHS respectively for 12 h, and subsequently subjecting them to a semi-inhibitory
337
AlCl3 concentration. To verify that NaHS alleviated Al3+ stress via the production of H2S or
338
HS-, various sodium salts were tested including Na2S, Na2SO4, Na2SO3 NaHSO4, NaHSO3,
339
and NaAC. Seeds were pretreated for 12 h with water (CK), optimal concentration of NaHS
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obtained from the above experiment or with Na2S, Na2SO4, Na2SO3 NaHSO4, NaHSO3, or
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NaAC at the same concentration as NaHS, and then subjected to the semi-inhibitory Al3+
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stress for a further 48 h and germination percentage and growth measured. To test whether
343
the possible positive effect of H2S donor on seed germination against Al3+ is dependent on
344
the pretreatment of pH value or not, which is resulted from NaHS pretreatment with
345
different concentration (0, 0.3, 0.6, 0.9, 1.2, 1.5 mmol/L) added in the solutions,
346
Na2HPO4-NaH2PO4 buffer solutions at different pH value 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2,
347
7.4, 7.8, 8.0 were use as the controls of different concentration of NaHS (0, 0.3, 0.6, 0.9,
348
1.2, 1.5 mmol/L). Wheat seeds were pretreated with water (CK), 0.6 mmol/L NaHS, and
349
0.6 mmol/L Na2HPO4-NaH2PO4 buffer solutions at different pH value 5.8, 6.0, 6.2, 6.4, 6.6,
350
6.8, 7.0, 7.2, 7.4, 7.8, and 8.0, respectively. After 12 h of pretreatment with 0.6 mmol/L
351
NaHS and and 0.6 mmol/L Na2HPO4-NaH2PO4 buffer solutions at different pH value, seeds
352
were washed with water and then subsequently subjected to 30.0 mmol/L buffered AlCl3
353
stress for a further 48 h; and germination percentage and growth are measured. The AlCl3
354
treatment solution was replaced by a fresh batch every 12 h and geminating seeds (0.5±0.05
355
g) were randomly selected every 12 h and sampled for analysis.
3
356 357
Enzyme assays and electrophoretic analysis
358
The activity and electrophoretic analysis of amylase were preformed as described by Zhang
359
et al. (2005). Wheat seeds (0.5±0.05 g) were homogenized in 5 mL ice-cold phosphate
13
360
buffer (200 mmol/L, pH 8.3, containing 1% PVP), the homogenate was centrifuged at
361
12,000 g for 30 min and the supernatant was used as amylase preparations for further assay.
362
Total amylase activity was determined using the starch-iodine method and one unit of
363
activity (U) was calculated as the amount of the enzyme required to reach 50% of initial
364
color intensity. Electrophoresis was performed in 10% vertical polyacrylamide gels. Fifteen
365
μl of amylase preparation was applied per well. To visualize the bands of amylase, the gel
366
was incubated in 50 mmol/L PBS (pH 7.0) containing 1% boiled soluble starch at 25
367
30 min. After being washed with distilled water for 3 times, the gel was stained with 0.6%
368
I2 and 6% KI solution.
℃ for
369
Native PAGE and activity determination of esterases (EC 3.1.1.3) were assayed
370
following the methods of Deising et al. (1992). To visualize the bands of esterase activities,
371
gel was washed twice for 20 min in 100 mmol/L Tris-HCl, pH 8.0. The indoxyl acetate
372
substrate (35 mg) was dissolved in 1 mL of acetone and added to 49 mL of 100 mmol/L
373
Tris-HCl, pH 8.0. Gels were incubated with the solution by constant agitation at room
374
temperature until bands of desired intensity appeared. Esterase activity was assayed at 30
375
by measuring the hydrolysis of p-nitrophenyl butyrate at 400 nm. Reaction mixtures
376
consisted of 600 μl of Tris-HCl buffer (100 mmol/L, pH 8.0), 200 μl of an enzyme
377
preparation, and 200 μl of a stock solution of 37.5 mmol/L p-nitrophenyl butyrate in the
378
same buffer.
℃
379 380
Determination of MDA, H2O2 and O2·¯
381
The contents of MDA-reactive lipids, H2O2 and O2·¯ were determined by the procedures
382
described by Wang et al. (2004). Wheat seeds were ground in 3 mL of 0.1% trichloroacetic
383
acid (TCA) solution. The homogenate was centrifuged at 15 000 g for 10 min and 0.5 mL
384
of the supernatant fraction was mixed with 2 mL 20% TCA containing 0.5% thiobarbituric
385
acid (TBA). The mixture was heated at 90
386
10,000 g for 5 min. The absorbance was recorded at 532 nm and the value for non-specific
387
absorption at 600 nm was subtracted. The extinction coefficient of 155 mmol L-1 cm-1 was
388
used to calculate the content of MDA.
℃ for 20 min, cooled and then centrifuged at
14
389
For determination of H2O2, wheat seeds were ground and extracted in 3 mL cold
℃ for 20 min. Then, 0.5 mL of
390
acetone. The homogenate was centrifuged at 10,000 g at 4
391
the supernatant fraction was mixed with 1.5 mL of CHCl3 and CCl4 (1:3, V/V) mixture.
392
Subsequently 2.5 mL of distilled water was added and the mixture centrifuged at 1,000 g
393
for 1 min and the aqueous phase collected for H2O2 determination. The reaction system
394
included 0.5 mL sample, 0.5 mL of buffer (PBS, 200 mmol/L, pH 7.8), and 20 μL 0.5 unit
395
of catalase (as controls) or inactive catalase protein (catalase was inactivated by incubating
396
in boiled water for 5 min,). After the mixture was incubated at 37
397
200 mmol/L Ti-4-(2-pyridylazo) resorcinol (Ti-PAR) was added. Then the reaction
398
mixtures were incubated at 45
399
measured.
℃ for 10 min, 0.5 mL of
℃ for another 20 min. The absorbance at 508 nm was
400
O2·¯ content was calculated by an extinction coefficient of 2.16×104 M–1 cm–1. Seeds
401
(0.5±0.05 g) were ground with 3 mL of 50 mmol/L Tris–HCl buffer (pH 7.5). The
402
homogenate was centrifuged at 5,000 g at 4°C for 10 min. The reaction mixture (1 mL)
403
contained
404
3’-(1-(phenylamino-carbonyl)-3,4-tetrazolium-
405
acid hydrate) and 50 μL of sample extracts. Corrections were made for the background
406
absorbance in the presence of 50 U of SOD.
50
mmol/L
Tris–HCl
buffer
(pH
7.5),
0.5
mM
bis(4-methoxy-6-nitro)
XTT
(sodium,
benzenesulfonic
407 408
Assays of LOX, SOD, CAT, APX, POD activities
409
Activity of lipoxygenase (LOX, EC 1.13.11.12) was determined following the description
410
by Surrey (1964) and those of superoxide dismutase (SOD, EC 1.1.5.1.1), catalase (CAT,
411
EC1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), guaiacol-dependent peroxidase
412
(POD, EC 1.11.1.7) were assayed according to García-Limones et al. (2002). Wheat seeds
413
were homogenized in ice-cold 50 mmol/L phosphate buffer (pH 7.8) containing 1.0
414
mmol/L EDTA. The homogenate was centrifuged at 15,000 g at 4
415
supernatant was used for activity measurement. The activities of APX were determined in
416
the presence of 0.5 mmol/L ascorbic acid and 0.5 mmol/L H2O2 by monitoring the decrease
417
in absorbance at 290 nm. Activities of CAT were determined spectrophotometrically by
℃ for 10 min. The
15
418
monitoring the decrease in absorbance at 240 nm. Activities of SOD were assayed by
419
measuring its capacity of inhibiting the photochemical reduction of nitro-blue tetrazolium.
420
Analysis of guaiacol POD capacity was based on the oxidation of guaiacol using hydrogen
421
peroxide. The reaction mixture contained 2.5 mL of 50 mmol/L potassium phosphate buffer
422
(pH 6.1), 1 mL of 1% hydrogen peroxide, 1 mL of 1% guaiacol and 10–20 μL of enzyme
423
extract. The increase in absorbance at 420 nm was read.
424 425
Al content determination
426
Seed samples (0.5 g) were ground in mortars with 5 mL deionized water, then the
427
homogenate was digested at 80
428
(1:1:3). After evaporation, the residue was mixed with 2% (v/v) nitric acid and the volume
429
was increased to 5 mL with deionized H2O and Al content was quantified by atomic
430
absorption spectrophotometer using a Hitachi Model 180/80 flame (180–80 Hitachi, Tokyo,
431
Japan) and graphite furnace (Hitachi 180/078) atomic absorption spectrophotometer, and an
432
automatic data processor were used for measurement.
℃ with a mixture of HNO , H O , and deionized H O 3
2
2
2
433 434
Measurement of endogenous H2S in seeds
435
H2S was determined by formation of methylene blue from dimethyl-p-phenylenediamine in
436
H2SO4 as described previously (Zhang et al. 2008a).
437 438
Statistical analysis
439
Significances were tested by one-way or two-way ANOVA, and the results are expressed as
440
the mean values ± SD of three independent experiments. Each experiment was repeated at
441
least three times.
442 443
Acknowledgments
444
This work was supported by the Great Project of Natural Science Foundation from Anhui
445
Provincial Education Department (ZD200910), the Natural Science Foundation of Anhui
16
446
Province (070411009), and the innovation funding to undergraduate students at HFUT
447
(XS08072, 0637).
448 449
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569
Tables
570
Table 1. Inhibitory effect of Al3+ on the germination of wheat seeds. Concentration 3+
of Al
(mmol/L) 0 5
Germination
Length of radicle
Length of
Radicle number
percentage (%)
(cm)
coleoptile (cm)
(50 seeds)
96.4±2.1
2.7±0.9
1.6±0.4
153±9.3
98.2±1.1
1.5±0.3 1.4±0.1 1.0±0.2 0.7±0.2 0.8±0.2
148±6.9 142±5.7 126±7.3 68±4.6 48±5.8
10
96.5±2.5
15 20
85±4.2 73.7±3.6
25
60.9±4.7
2.1±0.3 1.1±0.2 0.7±0.2 0.4±0.3 0.3±0.1
30 60
51.1±4.3 42.3±5.1
0.16±0.1 0.1±0.02
0.6±0.2 0.3±0.1
38±6.5 16±4.3
90 120 150
30.2±6.1 21.3±6.3 9.7±5.6
0 0 0
0.3±0.2 0.2±0.1 0.1±0.02
0 0 0
571
Wheat seeds were exposed to 0, 5, 10, 15, 20, 25, 30, 60, 90, 120, 150 mmol/L AlCl3 for 48
572
h. Values are the means±S.D. (n = 6).
22
573
Table 2. Effects of NaHS treatment on wheat seed germination under normal condition. Concentration of NaHS treatment (mmol/L) Germination percentage (%) Length
of
radicle (cm) Length of coleoptile (cm) Radicle number (50 seeds)
0.0
0.3
0.6
0.9
1.2
1.5
92.7±3.2 a
95.2±6.4 a
90.1±5.7 a
91.2±4.8 a
91.6±5.7 a
89±3.8 a
1.23±0.17a
1.32±0.24a
1.15±0.26a
1.33±0.41a
1.36±0.51a
1.18±0.33a
0.96±0.23a
0.98±0.17a
1.03±0.47a
0.92±0.25a
0.98±0.25a
1.07±0.23a
146.7±8.8a
153±6.7 a
149.6±4.8a
158.6±9.7 a
141.3±11.4a
158.3±7.1 a
574
Wheat seeds were cultured in 0, 0.3, 0.6, 0.9, 1.2, 1.5 mmol/L NaHS respectively for 36 h,
575
and then the germination percentage was investigated. Values are the means±S.D. (n = 6).
576
Different letters mean significance of difference between the treatments (P<0.01, ANOVA;
577
P-test LSD).
23
578
Table 3. Effects of NaHS pretreatment on wheat seed germination under 30 mmol/L Al3+
579
stress. Concentration of NaHS pretreatment (mmol/L) Germination percentage (%) Length
of
radicle (cm) Length of coleoptile (cm) Radicle number (50 seeds)
0.0
0.3
0.6
0.9
1.2
1.5
45.7±4.6 a
52.3±3.3 b
79.7±6.5 c
60.3±7.6 d
56.3±4.3 d
52±5.6 b
0.18±0.09a
0.36±0.14b
0.64±0.12c
0.45±0.16b
0.38±0.11b
0.41±0.21b
0.45±0.31a
0.68±0.22b
1.36±0.21c
0.85±0.32d
0.92±0.31d
0.79±0.12d
25.2±3.3 a
32±2.4 b
45.3±6.7 c
38.6±4.3 c
33.1±1.7 b
34.2±3.2 b
580
Wheat seeds were pretreated with 0, 0.3, 0.6, 0.9, 1.2, 1.5 mmol/L NaHS respectively for
581
12 h, and subsequently subjected to 30.0 mmol/L AlCl3 for further 48 h, and then the
582
germination percentage was investigated. Values are the means±S.D. (n = 6). Different
583
letters mean significance of difference between the treatments (P<0.01, ANOVA; P-test
584
LSD).
24
585
Legends of Figures
586
Figure 1 Inhibitory effect of aluminum on the germination of wheat seeds for 48 h. Wheat
587
seeds were exposed to 0, 30, 60, 90, 120, 150 mmol/L AlCl3 for 48 h, then were
588
photographed.
589
Figure 2 Effects of H2S donor NaHS on the germination of wheat seeds.
590
(A) No negative effect of H2S donor NaHS on the germination of wheat seeds under
591
normal condition. Wheat seeds were cultured in 0, 0.3, 0.6, 0.9, 1.2, 1.5 mmol/L NaHS
592
respectively for 48 h, then photographed.
593
(B) H2S donor NaHS promoted the germination of wheat seed under Al3+ stress. Wheat
594
seeds were pretreated with 0, 0.3, 0.6, 0.9, 1.2, 1.5 mmol/L NaHS respectively for 12 h, and
595
then subsequently subjected to 30 mmol/L Al3+ stress for further 48 h, then photographed.
596
(C) The protective role of NaHS on seeds germination against Al3+ stress could be
597
attributed to H2S or HS-. Seeds were pretreated with H2O (CK), NaHS, Na2S, Na2SO4,
598
Na2SO3, NaHSO4, NaHSO3, and NaAC with 0.6 mmol/L, respectively for 12 h, and
599
subsequently subjected to 30 mmol/L Al3+ stress for further 48 h, then the germination
600
percentage was investigated.
601
(D) Effects of different pH values pretreatment on wheat seed germination under 30
602
mmol/L Al3+ stress. Wheat seeds were pretreated with water control (CK), 0.6 mmol/L
603
NaHS, and 0.6 mmol/L Na2HPO4-NaH2PO4 buffer solutions at different pH value 5.8, 6.0,
604
6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.8, 8.0 respectively. After 12 h of pretreatment, seeds were
605
subjected to 30.0 mmol/L AlCl3 stress for further 48 h, and then the germination percentage
606
was investigated. For each treatment 50 seeds were used and the germination percentage
607
represented the mean number of germinated seeds from three independent experiments at
608
least. One-way ANOVA was used for comparisons between the means. Vertical bars
609
represent the SD of the mean (n = 4). Different letters mean significance of difference
610
between the treatments (P<0.01).
611
Figure 3 Native PAGE (A) and activity determination (B) of amylase in wheat seeds
612
pretreated with water as control (CK) or 0.6 mmol/L NaHS (S) for 12 h (shown as from 0’
613
to 12’/0 h of the pretreatment times) prior to exposing to 30 mmol/L Al3+ stress for further 25
614
48 h (shown as 12’/0, 12, 24, 36, 48 h, respectively). Within the 12 h of pretreatment time
615
plus 48 h of Al3+-stressed time, 0’ represented dry intact seeds samples; 12’/0 represented
616
the samples from the seeds pretreated with water (Control, shown CK in Figure) or NaHS
617
(shown T or NaHS in Figure) after 12 h, and at the point time, subjected to 30 mmol/L Al3+
618
stress; and 12, 24, 36, 48 as samples of the seeds exposed to Al3+ stress for further 12, 24,
619
36, 48 h respectively. Asterisks show significant differences between the NaHS treatments
620
and controls at P<0.01.
621
Figure 4 Changes of esterase activities in wheat seeds. (A) Native PAGE of esterase in
622
wheat seeds. (B) Activity determination of esterase in wheat seeds. Samples of seeds were
623
prepared and the numbers or letters presented as the same mentioned in Figure 3. Asterisks
624
show significant differences between the NaHS treatments and controls at P<0.01.
625
Figure 5 Effect of NaHS pretreatment on contents of H2O2 (A), O2·¯(B) and MDA (C) in
626
germinating seeds. The numbers (0’, 12’/0, 12, 24, 36, 48) or letters (CK or NaHS)
627
presented as the same mentioned in Figure 3.
628
Figure 6 Effect of pretreatment with NaHS on the activities of SOD (A), CAT (B), APX
629
(C), POD (D) and LOX (E) in germinating wheat seeds under 30 mmol/L Al3+ stress.
630
Seeds were treated and the numbers or letters presented as the same mentioned in Figure 3.
631
Figure 7 The content of Al in germinating wheat seeds. Seeds were pretreated with water
632
(shown CK) or 0.6 mmol/L NaHS (shown NaHS) for 12 h and subsequently exposed to 30
633
mmol/L Al3+ for further 48 h. For designations see Figure 3.
634
Figure 8 Effect of NaHS pretreatment on endogenous H2S content in wheat seeds exposed
635
to Al3+ stress. For designations see Figure 3.
26
Figure 1
636
0.0
90.0
30.0
60.0
120.0
150.0
637
27
Figure 2
638
A
0.0
0. 3
0. 6
0. 9
1. 2
1. 5
0.0
0. 3
0. 6
0. 9
1. 2
1. 5
B
639
28
90
C
a
80 Germination percentage (%)
70 60
b
50
b
b
b
b
bc
40
bc
30 20 10
6.8
aA
C
3 N
SO N
6.6
aH
SO
4
O3
aH N
a2 S
O4 N
N
a2 S
a2 S N
S aH N
H
2O
0
100
D Germination percentage (%)
90 80 70 60 50 40 30 20 10 0 CK NaHS 5.8
6
6.2
6.4
7
7.2
7.4
7.6
7.8
8
pH value 640
29
Figure 3
641
A
CK 0’
CK T 12’/ 0
CK T 12
CK T 24
CK T 36
CK T 48
642
80
CK NaHS
70
*
*
60
-1
-1
(U min g DW)
Amylase activities
B
·
·
*
50 40
*
30
*
20 10 0 0'
12'/0 12 24 36 Treatment time (h)
48
643
30
Figure 4
644
A
-1
-1
(U min g DW)
B
Esterase actvities
CK 0’
·
·
CK T 12’/ 0
8 7 6 5
CK T 12
CK T 24
CK NaHS
*
*
12
24
CK T 36
CK T 48
*
*
36
48
4 3 2 1 0 0'
12'/0
Treatment time (h) 645
31
Figure 5
646
60
A
CK NaHS
-1
(nmol g DW)
Content of MDA
50
·
40
* 30
*
*
*
20 10 0
0'
12'/0
12
24
36
48
Treatment time (h)
70
CK NaHS
60 50
(nmol g DW)
40
-1
Content of H2 O2
B ·
30
*
*
*
*
12
24
36
48
20 10 0 0'
12'/0
Treatment time (h)
(nmol g DW)
-1
Content of O2
.-
C
·
8 7 6 5 4 3
CK NaHS
*
*
*
*
2 1 0 0'
12'/0
12 24 36 Treatment time (h)
48
32
Figure 6
647
-1
(U mg protein)
SOD activities
A
·
16 14 12 10 8 6 4 2 0
0.35
-1
(U mg protein)
CAT activities
*
12'/0 12 24 36 Treatment time (h)
CK NaHS
0.3
·
* *
0'
B
*
CK NaHS
48
* *
0.25
*
0.2 0.15 0.1 0.05 0 0'
12'/0
12
24
36
48
Treatment time (h) 0.6
C
CK NaHS
(U mg protein)
-1
APX activities
0.5
·
*
* *
0.4 0.3 0.2 0.1 0 0'
12'/0
12
24
36
48
Treatment time (h) 648 649
33
-1
(U mg protein)
POD activities
D
·
0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0
*
CK NaHS
*
* *
0'
12'/0
12
24
36
48
Treatment time (h)
-1
(U mg protein)
LOX activities
E
·
8 7 6 5 4 3 2 1 0
CK NaHS
* *
*
0'
12'/0
12
*
24
36
48
Treatment time (h) 650
34
Figure 7
651
350
CK NaHS
-1
( g g DW)
Content of Al
300 250 200
· μ 150
*
100
*
*
50 0 0'
12'/0
12
24
36
48
Treatment time (h) 652
35
Figure 8
653
1.6
CK NaHS
-1
( mol g DW)
Content of H2S
1.4 1.2
*
*
* *
1
*
· 0.8 μ 0.6
0.4 0.2 0 0'
12'/0
12
24
36
48
Treatment time (h)
36
