Plasma membrane NADPH oxidase and apoplastic POD can be responsible for accumulation of H 2 O 2 in mesophyll cells of Elsholtzia haichowensis under copper stress

Under copper (Cu) stress, the activities of some enzymes involved in the antioxidant defense system in leave cells of Cu accumulator Elsholtzia haichowensis were investigated. Our results showed that Cu treatment significantly increased the activities of plasma membrane-bound nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, apoplastic peroxidase (POD), apoplastic superoxide dismutase (SOD), symplastic SOD and symplastic ascorbate peroxidase (APX) in leaves of E. haichowensis, when additional treatment with N-N-diethyldithiocarbamate as an inhibitor of SOD, NaN3 as an inhibitor of peroxidase, diphenyleneiodonium as an inhibitor of NADPH oxidase and 1, 2dihydroxybenzene-3, 5-disulphonic acid as an O2 scavenger and Cu-induced activities of antioxidant enzymes in leave cells of E. haichowensis were significantly inhibited. The results suggested that plasma membrane NADPH oxidase and apoplastic POD can be responsible for the accumulation of H2O2 in mesophyll cells of E. haichowensis under copper stress. In the leaf symplast, however, Cuinduced activities of SOD and APX may be important in avoiding the build-up of toxic H2O2 concentrations.

Elsholtzia haichowensis, of the family Lamiaceae, is a Cu-accumulator plant widely distributed in Cu-mining wastes and Cu-contaminated soils in China (Lou et al., 2004;Song et al., 2004).Previous studies showed that E. haichowensis was able to tolerate Cu in shoots at 160 µg g -1 dry weight from plants grown under hydroponic conditions when supplied with 500 µM exogenous Cu (Lou et al., 2004;Qian et al., 2005), which was much higher than the toxicity threshold (20 to 30 µg g -1 DW) of most other crop species (Marschner, 1995).Therefore, E. haichowensis must employ some internal mechanism in order to tolerate the elevated levels of Cu in shoot tissues.
Cytochemical analysis demonstrated that the accumulation of H 2 O 2 was primarily localized in the cell walls and extracellular space of mesophyll cells of E. haichowensis, as a consequence of Cu treatment (Zhang et al., 2010).In this paper, we studied the changes in the activities of some of the enzymes involved in the antioxidant defense system in leave cells of E. haichowensis under Cu stress.We also investigated the effect of additional treatment with some inhibitors, such as N-N-diethyldithiocarbamate (DDC) as an inhibitor of SOD, NaN 3 as an inhibitor of peroxidase, diphenyleneiodonium (DPI) as an inhibitor of NADPH oxidase and 1, 2-dihydroxybenzene-3, 5-disulphonic acid (Tiron) as an O 2 -scavenger on the antioxidant enzymes, which may help us to assess the possible link between these subcellular antioxidant enzymes in leaves of E. haichowensis.

Plant materials and treatment with inhibitors
Seeds of E. haichowensis were collected from a coppercontaminated site at Tongling (Anhui Province), China, in December 2009 and were germinated at room temperature (20 to 30°C) in plastic dishes filled with vermiculite.After seven days, the seedlings were selected for uniformity and transferred to vessels containing Hoagland nutrient solution.The 30 day-old seedlings were treated for 6 days with 100 µM Cu supplied in the nutrient solution as CuSO4•5H2O.The experimental design was completely randomized, with three replicate vessels each containing 20 seedlings.The solution pH was adjusted to 5.3 with NaOH or HCl upon renewal of the nutrient solution every 2 days.Shoots from control and Cu-treated plants for six days were additionally treated with distilled water or inhibitors: 10 mM DDC, 1 mM NaN3, 50 µM DPI, or 10 mM Tiron.The root was excised at the base of the stem before inhibitors treatment to avoid its influence.After 8 h incubation, the leaves were homogenized and assayed for detection enzyme activities by spectrophotometry.

Isolation of plasma membranes and determination of NADPH oxidase activity
Leaf plasma membranes were isolated using the two-phase aqueous polymer partition system according to the method of Larsson et al. (1987), with some modifications.Briefly, fresh leaves were homogenized in four volumes of extraction buffer containing 50 mM Tris-HCl (pH 7.5), 0.25 M sucrose, 1 mM ASC, 1 mM EDTA, 0.6% polyvinylpyrrolidone, and 1 mM PMSF.The homogenate was filtered through four layers of cheesecloth and then centrifuged at 10,000 × g for 20 min at 4°C.The supernatant was recovered and centrifuged at 100,000 × g for 40 min at 4°C to obtain a microsomal membrane pellet.The pellet was suspended in 0.33 M sucrose, 3 mM KCl, and 5 mM potassium phosphate (pH 7.8).The plasma membrane fraction was isolated by adding the microsomal suspension to an aqueous two-phase polymer system to give a final composition of 6.2% (w/w) Dextran T500, 6.2% (w/w) PEG 3350, 0.33 M sucrose, 3 mM KCl, and 5 mM potassium phosphate (pH 7.8).Three successive rounds of partitioning followed.The resulting upper phase was diluted five-fold in Tris-HCl dilution buffer (10 mM, pH 7.4) containing 0.25 M sucrose, 1 mM EDTA, 1 mM DTT, 1 mM ASC, and 1 mM PMSF.All procedures were carried out at 4°C.Protein content of plasma membranes was determined according to the method of Bradford (1976), with bovine serum albumin (BSA) as a standard.

Isolation of apoplastic and symplastic fractions and assays of antioxidant enzyme activities
Apoplastic fluid was extracted by a vacuum infiltrationcentrifugation technique essentially as reported by Vanacker et al. (1998) but with some modifications (Zhang et al., 2010).About 2 g of fresh leaves were quickly washed in distilled water, placed in petri dishes in 10 mM sodium phosphate buffer (pH 6) containing 1% (w/v) polyvinylpolypyrrolidone (PVPP), 1 mM EDTA, and 0.5 mM phenylmethylsulphonyl fluoride (PMSF), and then submitted to a vacuum for 10 min at 4°C.Afterwards, leaves were carefully dried with filter paper and placed in syringes, which were then placed in centrifuge tubes and centrifuged at 1500 × g for 5 min and the apoplastic fluid recovered at the bottom of the tubes.The remaining tissue was used to obtain the soluble symplastic fraction after homogenization in the same buffer and centrifugation at 15000 × g for 20 min.According to the activity of the cellular marker enzyme glucose-6-phosphate dehydrogenase (G6PDH) in the apoplastic fluid, the contamination of the apoplastic fluid by cytosolic protein was less than 1%.Soluble proteins were extracted with ice-cold extraction buffer (50 mM potassium phosphate + 1 mM EDTA, pH 7.0) with 1% PVPP (w/v).APX activity was measured by monitoring the decrease in the absorbance at 290 nm as ascorbate was oxidized, as described by Nakano and Asada (1981).POD was determined according to Zheng and Van Huystee (1992), the reaction was followed at A470.SOD activity was determined by the ability of SOD to inhibit the formation of nitroblue formazan from nitroblue tetrazolium (NBT), according to the method of Giannopolitis and Ries (1977).In this assay, SOD activity was expressed in units per milligram of protein.One unit of SOD is defined as the amount required to inhibit photoreduction of NBT by 50%.Protein content was estimated according to the method of Bradford (1976), using BSA as a standard.

Effect of Cu and inhibitors on the activity of plasma membrane NADPH oxidase
Under biotic and abiotic stress conditions, plasma membrane-bound NADPH oxidases have been accepted as being responsible for O 2 -production, and this enzyme can use cytosolic NADPH to produce O 2 -, which is quickly dismutated to H 2 O 2 by SOD (Mittler, 2002).Compared with control, 100 µM Cu treatment significantly increased the activity of plasma membrane NADPH oxidase.Treatment with DPI, an inhibitor of NADPH oxidase, significantly decreased the activity of plasma membrane NADPH oxidase, but NaN 3 as an inhibitor of peroxidase and Tiron as an O 2 -scavenger significantly also decreased the activities of Cu-induced NADPH oxidase (Figure 1).

Effect of Cu and inhibitors on the activity of apoplastic and symplastic antioxidant enzymes
The activities of the antioxidant enzymes SOD, POD and APX, were analyzed in the apoplastic and symplastic compartments of E. haichowensis leaves.The total activities of SOD, POD, and APX in the leaf symplast were significantly higher than those in the apoplast when calculated on a fresh-weight basis (Figure 2).In the apoplastic space of E. haichowensis leaves, the activity of SOD accounted for about 2.0% of the total foliar SOD activity, APX for 1.3 to 2.0%, and POD for 10.1 to 12.2% (Table 1).Compared with the control, treatment with 100 µM Cu significantly increased the total activities of apoplastic and symplastic SOD, apoplastic POD, and symplastic APX (Figure 2).Treatment with DDC, NaN 3 , DPI and Tiron significantly inhibited the activities of Cuinduced apoplastic SOD and symplastic APX, treatment with NaN 3 , DPI and Tiron significantly inhibited the activities of Cu-induced apoplastic POD and treatment with DDC, NaN 3 and Tiron significantly inhibited the activities of Cu-induced symplastic SOD (Figure 2).In previous experiments, cytochemical analysis demonstrated that the H 2 O 2 -dependent CeCl 3 precipitates were primarily localized in the cell walls of mesophyll cells of E. haichowensis, whereas the O 2 -dependent DAB precipitates were mainly localized in the chloroplasts of mesophyll cells after Cu treatment (Zhang et al., 2010).Accumulation of H 2 O 2 in Cu-stressed plants may be the consequence of a distribution of balance between its production and scavenging.H 2 O 2 can be generated by specific enzymes such as SOD, NADPH oxidase, amine oxidase, and a cell-wall peroxidase (Neill et al., 2002).It has been reported that plasma membrane NADPH oxidase and apoplastic POD may be involved in H 2 O 2 production under some stresses (Romero-Puertas et al., 2004;Hsu and Kao, 2007;Yeh et al., 2007;Fecht-Christoffers et al., 2006).In this study, we observed that Cu treatment significantly increased the activity of plasma membrane NADPH oxidase and apoplastic POD (Figures 1 and 2).Furthermore, the inhibition of NADPH oxidase by DPI, NaN 3 as an inhibitor of peroxidase and the scavenging of O 2 -by Tiron, significantly decreased Cu-induced activities of NADPH oxidase and apoplastic POD in the leaves of E. haichowensis.These results showed that the source of H 2 O 2 in the cell walls of E. haichowensis leaves could be partly attributed to the activity of NADPH oxidase, and the enzyme can use cytosolic NADPH to produce O 2 -which quickly dismutates to H 2 O 2 in a reaction catalyzed by apoplastic SOD.The activity of apoplastic POD can also be partly responsible for the accumulation of H 2 O 2 in the cell walls of mesophyll cells of E. Haichowensis.
The accumulation of H 2 O 2 is prevented by CAT, GR, POD, and APX.It is interesting to note that induction of the apoplastic SOD activity coincided with changes in the activity of plasma membrane NADPH oxidase and the activity of apoplastic POD in plants exposed to 100 µM Cu, whereas the total symplastic SOD activity coincided with that of APX.SODs, which catalyze the dismutation of O 2 -to H 2 O 2 and O 2 , are another key group of enzymes involved in protection of cells against oxidative stress.This result is of some significance, because the expected increase in H 2 O 2 as a result of the SOD reaction was accompanied by an increased enzymatic capacity to decompose it.Results also suggested that SOD and APX are major scavengers of ROS in the symplastic spaces of E. haichowensis leaves.
Treatment with NaN 3 and Tiron significantly inhibited the activities of all Cu-induced antioxidant enzymes while treatment with DDC only inhibited the activities of Cuinduced SOD and symplastic APX, Treatment with DPI had not significantly effect on the activities of Cu-induced symplastic APX (Figure 2).These results showed that Cu-induced increase in the activities of various antioxidant enzymes in different cell compartments may represent a defense mechanism against oxidative stress.In conclusion, our results suggest that the activity of plasma membrane-bound NADPH oxidase, apoplastic SOD and apoplastic POD may play an important role in the antioxidant defense system in the leaves of apoplast of E. haichowensis.In the symplast, SOD and APX can eliminate H 2 O 2 accumulation as a result of Cu treatment.

Figure 1 .
Figure 1.Cu treatment and additional treatment with DDC, NaN3, DPI and Tiron induced changes in plasma membrane-bound NADPH oxidase activities in the leaves of E. haichowensis.Plants were exposed to control treatment (Con) or 100 µM Cu treatment for six days and their shoots were subsequently treated with distilled water or 10 mM DDC, or 1 mM NaN3, or 50 µM DPI or 10 mM Tiron.Excised leaves were homogenized and enzyme activity assayed by spectrophotometry.Values are means ± SE (n = 3) of three separate experiments.Means denoted by the same letter did not significantly differ at < 0.05 according to Duncan's multiple range test.

Figure 2 .
Figure 2. Effects of Cu treatment and additional treatment with DDC, NaN3, DPI and Tiron on apoplastic and symplastic APX, POD, and SOD activities in the leaves of E. haichowensis.Plants were exposed to control treatment (Con) or 100 µM Cu treatment for six days and their shoots were subsequently treated with distilled water or r 10 mM DDC, or 1 mM NaN3, or 50 µM DPI, or 10 mM Tiron.Excised leaves were homogenized and enzyme activity assayed by spectrophotometry.Values are means ± SE (n = 3) of three separate experiments.Means denoted by the same letter did not significantly differ at P < 0.05 according to Duncan's multiple range test.

Table 1 .
The percent of POD, APX and SOD activities in the apoplast, with respect to the total present in the whole leaves of E. haichowensis.