Plant high tolerance to excess manganese related with root growth, manganese distribution and antioxidative enzyme activity in three grape cultivars

Ecotoxicology and Environmental Safety 74 (2011) 776–786

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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Plant high tolerance to excess manganese related with root growth, manganese distribution and antioxidative enzyme activity in three grape cultivars Dongling Mou a,b, Yinan Yao a,b,n, Yongqing Yang c, Yuanming Zhang a, Changyan Tian a, Varenyam Achal a a

Key Laboratory of Biogeography and Bioresources, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, 818 South Beijing Road, Urumqi 830011, PR China b College of Life Sciences, Guizhou University, Guiyang 550025, PR China c College of Life Sciences, Chongqing Normal University, Chongqing 400047, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 July 2010 Received in revised form 25 October 2010 Accepted 27 October 2010 Available online 13 November 2010

The cuttings of grape (Vitis vinifera Linn.) were exposed to Hoagland’s solution containing five different manganese (Mn) concentrations to investigate Mn toxicity and the possible detoxifying responses. Three genotypes (i.e. cultivars Combiner, Jingshou and Shuijing) were used in present study. The results showed that grape species is highly tolerant to excess Mn. The plant growth is stimulated by as high as 15 or 30 mM Mn, and then depressed by higher Mn levels. The grape tolerance to excess Mn is related with plant capacity to keep constant or increased root growth as well as to keep high root activity. Also, the grape could employ some effective but intraspecific strategies to detoxify cellular Mn stress by excluding excess Mn out of leaf tissues or by enhancing antioxidative capacity. On the other hand, the present study showed that there existed different (or contrast) distribution pattern for excess Mn in grape. Majority of Mn was transferred and accumulated in the above-ground part in Combiner while Jingshou stored most Mn in root systems. For the first time our result showed the extreme tolerance and contrast performance at Mn translocation in an important fruit species with revealed genomic information. & 2010 Elsevier Inc. All rights reserved.

Keywords: Vitis vinifera Hypertolerance Antioxidative enzyme Manganese transfer Root growth

1. Introduction Manganese (Mn) is an essential element necessary for enzymatic activities in all subcellular compartments (Marschner, 1995). However, Mn is toxic when in excess. The high concentration of Mn interferes with the absorption and utilization of other mineral elements (Clark, 1982), affects energy metabolism, decreases photosynthetic rates (Nable et al., 1988) and causes oxidative stress (Fecht-Christoffers et al., 2003). In the field ecosystem, there are many factors inducing Mn excess for plant. One is Mncontaminated soils derive from mine tailings (Xue et al., 2004). Another is acid soil with a very lower pH value (pHo5.0), as in some places of subtropical and tropical area, because soil Mn bioavailability is related with pH change, with 100-fold increment by decrease of one unit in pH (Kabata-Pendias and Pendias, 2001). Foy (1984) suggested that Mn toxicity is probably the most important limiting factor after aluminum for plants in acid soils. Phytoremediation has attracted people’s attentions by using hyperaccumulators or tolerant plant species (Raskin and Ensley, n Corresponding author at: Key Laboratory of Biogeography and Bioresources, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, 818 South Beijing Road, Urumqi 830011, PR China. Fax: +86 991 7885300. E-mail address: [email protected] (Y. Yao).

0147-6513/$ - see front matter & 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2010.10.040

2000). The majority of the reported Mn hyperacculators are native to the ultramafic substrates. Recently Fernando et al. (2009) reported that Macadamia integrifolia could be used for remediating Mn-affected soils, because no toxic symptom occurred even under 10 mM Mn treatment. Due to acid soil characteristics and the need of specific plant characteristics for phytoremediation, such as rapid growth, high biomass and strong root systems, many authors suggest that it is necessary to select other Mn-tolerant, fastgrowing woody species as good candidates for phytoremediation (Lei et al. 2007; Fernando et al., 2009). Generally Mn-tolerant genotypes employ a serial of detoxifying strategy to detoxify excess Mn, such as sequestration and translocation of Mn into vacuole and endoplasmic reticulum, chelation in the cytosol and evoking the antioxidant enzymes induction (Pittman, 2005; Boojar and Goodarzi, 2008). The extent of such tolerance and degree of adaptation are highly variable. Interspecific and intraspecific differences in Mn tolerance have been reported in crops and grass such as maize, ryegrass, pokeweed, various cereals species and some woody species (Pulford and Watson, 2003; Doncheva et al., 2009; Mora et al. 2009; Dou et al. 2009) In recent years, grape trees were introduced and widely cultivated in the subtropical and tropical area in south China, and many grape cultivars exhibited good performance for plant growth in the acidic soil. But to the best of our knowledge, studies

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and then dehydrated in an ethanol series according to Maricle et al. (2009). Following this, leaves were cryofractured under liquid nitrogen with forceps. Specimens were then immersed in steps of increasing hexamethyldisilazane (HMDS) concentration. Samples were mounted on a brass plate and were sputter-coated with gold and platinum for 3 min in a SPI Sputter (SPI Supplies, West Chester, PA, USA). Also part of root hair zone was sampled and prepared like the leaf abaxial parts showed above. Specimens were examined in an ISI-SX30 scanning electron microscope (International Scientific Instruments, Prahran, Victoria, Australia).

on grape responses to excess Mn are limited. Until now, the identified plant species exhibiting high tolerate to excess Mn are wild plant species without available genome information, and also those plants are difficult to grow in controlled environment (Fernando et al., 2008). So it is necessary to identify some other Mn-tolerant plant species with revealed genome information, which would help us to identify molecular mechanism that confer Mn tolerance. In this study, we examined the effects of high concentrations, in comparison with adequate doses, of Mn on genotypic responses in growth parameters, Mn distribution in different organs and plant detoxification responses in three grape cultivars which are widely grown in China. The specific aims of the study are (1) to determine the level of Mn toxic to grape growth and accumulation pattern of Mn in different plant parts, and (2) to compare the genotypic differences of grape responses to elevated Mn levels.

The element of secreted or crystal material in the leaf surface in three cultivar was analyzed by energy dispersive (X-ray) spectroscopy (EDS) in an FEI Tecnai F20 FEG according to Ducˇic´ and Polle (2007). The microscope was operated at a low FEG emission current of 38 KV in SEM mode with a serial of condenser apertures. Both bright-field (BF) and dark-field (DF) images were obtained in the SEM mode configuration with the sample tilted at 25 1C towards the EDS detector. Count rates of a few hundred counts per second were acquired for the EDS spectra for 60 live seconds.

2. Materials and methods

2.6. Superoxide radical generation (O 2 ) and membrane permeability (MP)

2.1. Plant materials and experimental setup

Plant oxidative stress was measured by leaf O 2 production rate, which was quantified according to Johnson et al. (2003). The freeze-dried top 3rd–4th leaf tissues (0.5 g) were incubated at 20 1C for 20 min in 3 mL 0.12 mM XTT in 50 mM phosphate buffer, pH 8.2. The assay solution was centrifuged (13,000g) for 5 min. The A450 of the supernatant was measured and expressed as micromoles of superoxide generated per minute using the molar extinction coefficient for the XTT formazan product. Determination of leaf membrane permeability (MP) was performed according to Zheng et al. (2008), transferring 2.0 g of thoroughly washed leaves to 10 mL distilled water and shaking it every 20 min. After 3 h, the absorbance of the supernatant at 264 nm was measured using a UV spectrophotometer, for non-electrolytes (such as amino acids and polysaccharides) of most plant material have an absorption peak at 264 nm, and the MP was expressed as increase of absorbance in 264 nm (unit: OD264 nm g  1 h  1).

Three grape (Vitis vinifera Linn.) genotypes extensively cultivated in China, Combier (Cultivar Combier), Jinshou (Cultivar Jinshou) and Shuijin (Cultivar Shuijin), were chosen as plant materials. During dormancy, cuttings from three cultivars were rooted in humid sand crates and placed in a controlled greenhouse room. Young grape plantlets (about 0.5 cm in shoot base diameter) were further grown in 46-L plastic containers containing sand for 10 cuttings per container in a semi-controlled environmental condition in a naturally lit greenhouse with a temperature range 17.0–28.0 1C, relative humidity range of 45–90%, and supplied with 3000 mL Hoagland’s solution every other day (Hoagland and Arnon, 1950). The chemical composition of Hoagland’s solution was as follows: macroelement (g L  1) Ca, 0.201; K, 0.566; N, 0.280; P, 0.155; Mg, 0.048; S, 0.131; microelement (mg L  1) Fe, 6.2; Mn, 0.55; B, 0.50; Zn, 0.05; Cu, 0.031; Ni, 0.011; Mo, 0.007; Co, 0.001. Before the sterilization of the medium, the pH was adjusted to 5.7 with 0.1 N NaOH. After 40 days, the seedlings were exposed to solutions with Mn stress or control. Totally, there were five treatments with basic Hoagland’s solution separately containing Mn2 + concentration as follow: CK [0.00055 g L  1 (0.01 mM), i.e., basic Mn concentration of Hoagland solution as above], 0.82 g L  1 (15.0 mM), 1.65 g L  1(30.0 mM), 2.47 g L  1 (45.0 mM), 3.30 g L  1 (60.0 mM). The pH of the solution was adjusted to 5.770.2 with NaOH or HCl as required. The experimental setup was completely randomized with two factors (three cultivars and five Mn concentrations), three replications in each treatment, with each replicate including ten cuttings. 150 cuttings of each cultivars were randomly allocated to different Mn treatments for 105 days. 2.2. Root morphological traits and plant biomass All seedlings were harvested at the end of the experiment and divided into leaves, stems and roots. The root morphological parameters, such as total root length, root surface area, root volume and average root diameter were determined with a root scanner at 400 dpi (Epson Perfection 4990 scanner, model J131B, Epson Inc.) according to Fae´ et al. (2009). Root hair zone was detected by scanning electron microscopy (SEM) to observe the morphological change of root hares under Mn stress. Root activity was determined using the triphenyl tetrazolium chloride (TTC) method as described by Clemensson-Lindell (1994). Roots, stems and leaves were dried at 80 1C to constant weight in an oven, and weighed. Root/shoot ratio was also determined. 2.3. Manganese element The dried tissue of different plant parts was ground, weighed and then digested using a microwave sample-preparation system. One gram of sample powder was digested with a solution containing 4 mL HNO3 (71% w/w) and 1 mL of HCl (32% w/ w), and then Mn element was analyzed by atomic absorption spectroscopy according to Rashed (1995). EC10, the Mn concentration in nutrient solution corresponding to a 10% reduction of plant biomass, were used to determine the threshold concentration of Mn supplement causing plant toxicity (Paschke, 2002), EC10 was calculated using linear regression. Biological transfer coefficient (BTC), the ratio of above ground Mn content to root Mn content, was also calculated. 2.4. Leaf surface and root hair zone observed by scanning electron microscopy (SEM) The top 3rd–4th leaves were sampled in each cultivar. Abaxial parts of leaves were cut down and fixed in 5% (v:v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.0)

2.5. Element of the secreted or crystal substance in the leaf surface

2.7. Antioxidant enzyme activity The fully expanded young leaves (top 3rd–4th leaves) were used for measurement of antioxidant enzyme activity. The superoxide dismutase (SOD, EC 1.15.1.1) activity was assayed by monitoring the inhibition of photochemical reduction of nitro blue tetrazolium (NBT) as described by Lei et al. (2007). One unit of SOD activity (EU g  1) was defined as the amount of enzyme required to cause 50% inhibition of the reduction of NBT as monitored at 560 nm. The catalase (CAT, EC 1.11.1.6) activity was measured according to Jin et al. (2008). The reaction mixture (1.5 mL) consisted of 100 mM phosphate buffer (pH 7.0), 0.1 mM EDTA, 20 mM H2O2 and 50 mL enzyme extract. The reaction was started by addition of the enzyme extract. The decrease of H2O2 was monitored at 240 nm, and one unit of CAT enzyme was defined as the amount of enzyme which degraded 1 mmol H2O2 per min for 1 g fresh leaf (EU g  1 min  1). The peroxidase (POD, EC 1.11.1.7) activity was determined at 25 1C with guaiacol (Thongsook and Barrett, 2005). In the presence of H2O2, POD catalyzes the transformation of guaiacol to tetraguaiacol (brown product). The oxidation of guaiacol was measured by the increase in absorbance at 470 nm. The reaction mixture contained 50 mL of 20 mM guaiacol, 2.8 mL of 10 mM phosphate buffer (pH 7.0) and 0.1 mL enzyme extract. The reaction was started with 20 mL of 40 mM H2O2. POD activity was expressed as the increase in absorbance at 470 nm per minute for 1 g fresh leaves (EU g  1 min  1). For polyphenol oxidase (PPO, EC 1.10.3.2) extraction, 1 g leaves were homogenized in 15 mM ß-mercaptoethanol, 20 mM Tris–HCl (pH 7.8), 20% glycerol, 1 mM phenylmethyl sulfonyl fluoride (PMSF) and 1% (v/v) Triton X-100. PPO activity was measured using 30 mM catecol in sodium acetate buffer (pH 4.5) and the reaction was initiated by the addition of the enzyme extract containing 50 mM phosphate buffer (pH 7.0) at 420 nm, and assessed by the enzymatic oxidation of catecol (Sa´nchez-Ferrer et al., 1988). One unit of PPO activity was expressed as a change of absorbance of 0.01 per minute per gram (EU g  1 min  1). 2.8. Statistical analyzes Statistical analyzes were conducted using SPSS 11.5 for Windows. Data were log-transformed if necessary to ensure assumptions of normality and homogeneity of variances. The effects of the treatments (Mn treatment and cultivars difference) on the parameters were tested using two-way ANOVA as described by Lei et al. (2007). Within each cultivar, pairwise comparisons between different Mn levels were conducted using LSD (least significance difference) test at P o0.05 levels. Pearson’s correlation coefficients were calculated to determine the relationships between variables using individual data according to Luo et al. (2006).

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3. Results

3.2. Element secretion in the leaf surface

3.1. Biomass and Mn partitioning

In our study, different leaf epidermal surfaces existed according to SEM image (Fig. 3), the Combier and Shuijin had plenty of epidermal hairs, while Jinshou had no hairs. As demonstrated by the EDS spectra on leaf surface of the three cultivars, Mn elements were excreted onto leaf surface with high concentration (2–4%, Fig. 3i) after Mn treatment, but it was not detected in the control (Fig. 3h; Appendix figure). On the other hand, crystallized materials due to the excretion of some compounds were detected in leaf surfaces in Jinshou and Shuijin under excess Mn treatments (Fig. 3d, f, g), but was not detected in the control (Fig. 3c, e), and S, Si, K, Ca element were detected in these crystallized compounds (Fig. 3j).

Combier

CK 15 30 45 60

CK 15 30 45 60

20 Root Stems leaves EC 10 B C=13.7mM 18 A J=12.1 mM A 16 A S=8.5 mM A 14 B C B 12 A C 10 C D C D 8 a D a a c a a b d b 6 b c a c 4 b c 2 c a b c d b a c d e c b a d d 0 CK 15 30 45 60

-1

Biomass (g plant )

As compared to the control, the total plant biomass, shoot biomass and root biomass were significantly decreased by 45.0 and 60.0 mM Mn levels in all cultivars (Fig. 1a). On the contrary, these parameters were found to be increased by 15 mM Mn treatment in all cultivars, in particular the root biomass, and in Combier it was further increased by 30 mM Mn. The change of root/shoot ratio was consistent with biomass performance (Fig. 1b), increased by 15 or 15–30 mM (cultivar Combier). The EC10 values of three cultivars followed the order: Combier4Jinshou4Shuijin, with 13.7, 12.1 and 8.5 mM separately. In increasing Mn treatments, Mn concentrations in the different plant parts were increased in all cultivars (Fig. 2a–c), reaching 8000 mg Kg  1 in the root (Jinshou) or shoot (Shuijin) under 60 mM Mn level. Cultivars-specific performances on Mn uptake and translocation were observed. More Mn content was shifted to shoot in Combier as showed by pronounced higher leaf Mn concentration than that in root (Fig. 2a). On the contrary, Jinshou showed pronouncedly higher Mn concentration in the root than that in the leaf (Fig. 2b), and Shuijin exhibited intermediate performance between the above two cultivars (Fig. 2b). On the other hand, Combier performed highest BTC values (3.0–9.0), but Jinshou showed lowest BTC values (only 1.0–2.0) under elevated Mn2 + treatment (Fig. 2d).

Jinshou

Shuijin

Excess Mn (mM) treatment in different grape cultivars 0.7

a a a

Root / shoot ratio

0.6 a

0.5 0.4

a

b b

b

c

c c c

c

0.3 0.2

d d

0.1 CK 15 30 45 60

Combier

Jinshou

CK 15 30 45 60

CK 15 30 45 60

0 Shuijin

Excess Mn (mM) treatments in different grape cultivars Fig. 1. The effects of excess Mn treatments on the biomass (dry weight) of different plant parts and root/shoot ratio in three grape cultivars. Values shown are mean 7 SD. (a) Biomass of different plant parts and (b) root/shoot ratio. Different letters besides the column indicate significant differences at P o0.05 for roots, stems and leaves, respectively, by normal, italic and bold letters in (a).

3.3. Root growth In present study, root parameters were variously affected by Mn treatment in different cultivars (Table 1). In Combier, little difference in root length was found between 0 and 30 mM Mn levels, root area and root volume increased with the increasing of Mn levels (ranged from 0 to 30 mM), but all these three root parameters were decreased under higher Mn2 + levels (45–60 mM). In Jinshou and Shuijin, except for the root volume increased by 15 mM Mn treatment, root length and root area were decreased with increase in Mn concentration. Root diameter increased with increase in Mn levels in all grape cultivars except for Shuijin at extremely high Mn level (60 mM). For root hairs, Jinshou and Shuijin were much more affected by Mn than Combier (Fig. 4). At the concentration of 30 mM Mn treatment, pronouncedly decreased root hair elongation and hair density in Jinshou and Shuijin was found although it had little effect in cultivar Combier; while under 60 mM Mn treatment, root hairs were torn down and little hair remained in root surface in all cultivars. Root activity kept higher values or was increased by Mn range 0–30 mM in three grape cultivars (Table 1), it was decreased by 45– 60 mM Mn in Jinshou and Shuijin while only by highest level (60 mM) in Combier. 3.4. Oxidative stress and antioxidant enzyme activity The Mn treatments caused oxidative stress, as indicated by significant increases of leaf O 2 production rate and membrane permeability (MP) value in three cultivars (Po0.001, Fig. 5a, c). PPO activity, another indicator of oxidative stress, showed a hump response to Mn levels, peaking at 30 mM Mn in three cultivars (Fig. 5b). Among three cultivars, Shuijin had highest values of leaf O 2 production rate, MP and PPO activity under Mn stress (Fig. 5a–c). The SOD activity was little affected by Mn2 + stress except for the decrease under highest Mn level (60 mM) in three grape cultivars (Fig. 6a). The CAT and POD activity were increased under 15 mM (Jinshou and Shuijin) or 15–30 mM Mn level (Combier) and then decreased under higher Mn levels compared with the control (Fig. 6b, c). These two parameters were pronouncedly higher in Combier compared to other two cultivars (Fig. 6a–c). 3.5. Correlation between different parameters As showed in Table 2, biomass and root activity was positive related (Po0.01), and those parameters exhibited similar correlation with other parameters. They were positively correlated with root growth parameters like root length, root area and root volume, and were also positively related with plant antioxidative activity such as SOD, CAT and POD activity but negative correlated with O 2 production rate. Root/shoot ratio (R/S ratio) exhibited little

leaves A

B a a

c

d

b b

c c

d e D

CK

15 30 45 Excess Mn (mM) treatment

a C

b b D

b

b b

c c E

CK

15 30 45 Excess Mn (mM) treatments

A a B

a

b C c c D d d E

15 30 45 Excess Mn (mM) treatment

60

A

B b

b

b

CK

60

a

Shuijin

a

Jinshou

60

10 a 9 8 7 6 5 c c c 4 3 2 1 0

b

Combier

c e

d

e

e

d e

Jinshou Excess Mn (mM) treatment

f e

d

CK 15 30 45 60

B C

9000 8000 7000 6000 5000 4000 3000 2000 1000 0

779

CK 15 30 45 60

Stems

CK 15 30 45 60

9000 8000 7000 6000 5000 4000 3000 2000 1000 0

Root Combier

Mn concentration (µg g-1)

9000 8000 7000 6000 5000 4000 3000 2000 1000 0

Biological transfer coefficient

Mn concentration(µg g-1)

Mn concentration (µg g-1)

D. Mou et al. / Ecotoxicology and Environmental Safety 74 (2011) 776–786

Shuijin

Fig. 2. The effects of excess Mn treatments on the Mn concentrations of different plant parts and biological transfer coefficient for Mn in three grape cultivars. Values shown are mean 7 SD. (a) Mn concentration for Combier cultivars; (b) Mn concentration for Jinshou cultivars; (c) Mn concentration for Shuijin cultivars and (d) Biological transfer coefficient. Different letters above the column indicate significant differences at Po 0.05 for roots, stems and leaves, respectively, by normal, italic and bold letters in (a)–(c). Different letters above the column in (d) indicated significant differences at Po 0.05 across three cultivars.

correlation with other parameters except membrane permeability (MP) (r ¼  0.664, Po0.01) and with SOD activity (r ¼0.554, Po0.05). The root growth parameters were closely related with each other as showed in root length, root area and root volume. But root diameter exhibited a negative indicator for plant growth, it was negatively related with other root growth parameters and positively related with O 2 production rate. On the other hand, the CAT activity was closely related with POD activity (Po0.01).

4. Discussion Mn concentrations on Mn toxicity were often below 5 mM in previous studies (Sarkar et al., 2004). In our study, plant growth in three grape genotypes was stimulated by as high as 15 or 30 mM Mn (Fig. 1a). Considering the stimulated effect on plant growth by 15–30 mM Mn and high EC10 value, we conclude that the grape species is hypertolerant (or extremely tolerant) to Mn toxicity according to the definition by Ernst et al. (2008). Until now only one Mn-hypertolerant species, Schima superba, exhibited as highly tolerant as or more tolerant than grape examined in this study (Yang et al. 2008). Paschke et al. (2005) observed that the growth of Agrostis gigantean was stimulated by as high as 4 g L  1 Mn, and they ascribed this to hormetic effects. However, according to results of present study there are other factors for this phenomenon. First, root biomass and root/shoot ratio were significantly increased by 15–30 mM Mn (Fig. 1a, b), and root activity and root growth traits, such as root length, root area and root volume were closely related with biomass (Tables 1 and 2). The stimulated root

growth necessarily helped nutrition absorption. Second, grape leaf could exclude the toxic Mn element out of cytoplasm as detected by EDS spectra (Fig. 3), and many reports ascribed this as an important mechanism for the detoxification of heavy metal (Bidwell et al., 2002; Pittman, 2005). Recent studies on sunflower plants, one Mntolerant species, showed that the entire trichome in leaf hair had been blackened for accumulation of Mn (Hajiboland et al. 2008). Third, grape has a good mechanism to avoid damaging from as high as 4000–5000 mg kg  1 of tissue Mn concentration in leaves (Combier) and roots (Jinshou) under 15–30 mM Mn (Fig. 2a–c), as few reports documented that plant species could keep high growth rate with such tissue Mn concentration (Paschke et al., 2005). Further exploration on the detoxifying mechanism for grape species is currently conducted in our lab, and we have observed that the grape could keep Mn at a limited level in grape cytoplasm but has a high capacity for Mn binding to the cell walls and Mn compartmentalization to the cell vacuoles (unpublished data). Due to high Mn transfer coefficient (Fig. 2d), rapid growth and high above-ground biomass, Combier cultivar could be used for Mn extraction and clearance in Mn-contaminated area (Pulford and Watson, 2003; Pilon-Smits, 2005). In the contrast, it was very interesting that Jinshou cultivar retained majority of Mn in the roots, because almost all the previous studies reported that Mn could be easily transferred to above-ground and kept lower concentration in the roots (Horst, 1983; Loneragan, 1988). Due to extreme Mn-tolerance in the root and constitutively high root/ shoot ratio, cultivar Jinshou could be used as the pioneering plant for Mn Phytostabilisation in the tailing area of Mn mine (Padmavathiamma and Li, 2009). Generally the translocation from

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Fig. 3. The leaf surfaces and element secretion pattern in the leaf surface under excess Mn treatments separately detected by scanning electron microscopy (SEM) and energy dispersive X-ray spectrometric microanalysis (EDS). (a), (c), (e) showed SEM image in the controls of C, J and S cultivars; (b), (d), (f) showed SEM image in the 30 mM Mn treatment; (g) showed the crystallized compounds in J or S cultivars under 30 mM Mn treatment and (h), (i), (j) showed the EDS spectra of the detected elements in control (for (a), (c), (e)), 30 mM Mn treatment (for (b), (d), (f)) and crystallized compounds (g).

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Table 1 The effects of excess Mn treatments on root growth and root activity in three grape cultivars. Values shown are mean 7SD. Values in each cultivars within a column followed by the same letter do not differ significantly at P o 0.05 level by LSD pairwise comparisons. Cultivar

Mn (mM)

Length (m)

Area (dm2)

Volume (cm3)

Root diameter (mm)

Root activity mg TTF g  1 h  1

Combier

CK 15 30 45 60

38.047 0.6ab 39.77 7 0.5a 37.77 7 0.5b 8.567 0.09d 6.447 0.04e

6.55 7 0.1b 8.107 0.5a 8.52 7 0.5a 1.78 7 0.11d 1.45 7 0.10e

8.97 70.06c 13.12 70.14b 15.34 70.11a 2.93 70.08d 2.54 70.41e

0.55 7 0.004c 0.65 7 0.002b 0.66 7 0.011b 0.707 0.035a 0.72 7 0.001a

7.917 0.20ab 8.077 0.09a 7.767 0.20b 7.787 0.05b 5.497 0.07c

Jinshou

CK 15 30 45 60

25.017 0.09a 15.18 7 0.09b 14.53 7 0.13c 7.017 0.10d 5.377 0.13e

4.89 7 0.06a 3.92 7 0.09b 3.057 0.10c 2.17 7 0.06d 1.57 7 0.06e

7.64 70.05b 8.05 70.10a 5.32 70.06c 5.16 70.03d 3.68 70.08e

0.62 7 0.001c 0.69 7 0.082c 0.82 7 0.009b 0.99 7 0.019a 0.93 7 0.032a

7.357 0.09ab 7.597 0.13a 7.017 0.27bc 6.697 0.15c 3.847 0.35d

Shuijin

CK 15 30 45 60

34.93 7 0.09a 30.857 0.33b 14.017 0.81c 6.087 0.10d 4.417 0.13e

6.45 7 0.10a 6.62 7 0.10a 3.027 0.04c 1.29 7 0.01d 1.27 7 0.03e

9.46 70.06b 11.49 70.02a 5.19 70.05c 3.00 70.08d 2.10 70.01e

0.59 7 0.096b 0.67 7 0.044b 0.69 7 0.006b 0.91 7 0.053a 0.63 7 0.062b

6.457 0.156b 6.687 0.18a 6.587 0.09ab 5.437 0.07c 3.977 0.23d

*** ** *

*** ** **

*** ** ns

*** ** ns

*** * ns

Anova significance Mn2 + Cultivars Mn2 +  Cultivars

ns, not significant; *, **, *** indicate significant difference between treatments at P o 0.05, P o 0.01, Po 0.001, respectively.

Fig. 4. The effects of excess Mn treatments on root hairs detected by scanning electron microscopy (SEM). (a), (d), (g) separately showed the controls for C, J, S cultivars; (b), (e), (h) separately showed the 30 mM Mn treatments for of C, J, S cultivars and (c), (f), (i) separately showed the 60 mM Mn treatments for of C, J, S cultivars.

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a

10 B

B

8 C

b

B

a

b

c

A

b c

6 4 2

Combier

d

d

Jinshou Shuijin

e

0

CK

15 30 45 Excess Mn (mM) treatment

SOD activity (EU g-1 FW)

Leaf O2·- production rate (µM·g-1·min-1)

12

60

200 190 ab 180 a 170 160 A 150 140 130 120 110 100 CK

A

a a

b

c

c

100

b

b

50

E d b

CAT activity (EU g-1 min-1)

PPO activity (EU g-1 min-1)

B

150

A

Combier Jinshou Shuijin 15

15 30 45 Excess Mn (mM) treatment

60

60

a

12

b

10 A

8 6

B

d

4 b

c

BC

CK

C

a

15

c

c

30

45

D c

60

Excess Mn (mM) treatment

A

12

B

B

10 C

8

D c b

b

b a

a

ab a

a

a

2

0.9 POD activity (EU g-1 min-1)

(OD264 g h )

-1 -1

Leaf membrane permeability

45

1

14

4

14

0

16

6

30

a

2

c

0 CK

A b b

A

16

C D

a

Excess Mn (mM) treatment

A

200

a

a

a

18

250

a

a

CK

15 30 45 Excess Mn (mM) treatment

60

b

0.7 0.6

a

0.5

a

c

0.4 0.3 0.2 0.1

0

a

0.8

A

d

A

c

b B

B

30

45

c

e

0 CK

C

15

60

Excess Mn (mM) treatment Fig. 5. The effects of excess Mn treatments on the superoxide radical generation (O 2 ), PPO enzyme activity and membrane permeability in three grape cultivars. Values shown are mean 7SD. (a) O 2 production rate; (b) PPO activity and (c) membrane permeability. Different letters besides the lines indicate significant differences in each cultivars at P o 0.05 level for C, J and S cultivars, respectively, by normal, italic and bold letters.

Fig. 6. The effects of excess Mn treatments on leaf SOD, CAT and POD enzyme activity in three grape cultivars. Values shown are mean 7 SD. (a) SOD activity; (b) CAT activity and (c) POD activity. Different letters besides the lines indicate significant differences in each cultivars at P o 0.05 level for C, J and S cultivars, respectively, by normal, italic and bold letters.

roots to shoots is dependent on several steps: (1) Mn uptake and sequestration in roots; (2) loading into xylem and (3) transport with the transpiration stream to the shoots (Ducˇic´ et al., 2006). Assuming that Mn is freely mobile in the xylem as other metal species, we suggested that the difference of root-to-shoot translocation of Mn in three grape cultivars could be due to xylem loading. Recent studies showed that xylem loading process is the critical factor for different Cd concentrations in the shoots between both Solanum melongena and Solanum torvum (Moria et al., 2009). Uraguchi et al. (2010) firmly demonstrated that root-to-shoot translocation via the xylem is the major process determining the

Cd accumulation level in shoots across the comparison of 69 accessions of rice. Our studies provided good genotypes to clarify the mechanism for Mn translocation from root to shoot. On the other hand, besides the efflux of Mn, calcium (Ca), sulfur (S), potassium (K), silicon (Si) and some other elements were also detected in the excreted or crystallized compound in grape leaf surface (Fig. 3), but this phenomenon was not observed in cultivar Combier. Although the internal mechanism needed to be further investigated, the results suggested that those elements might be also involve in metal homeostasis and involved in the alleviation of cellular Mn toxicity in grape. Many previous studies had testified

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Table 2 Correlation coefficients among plant growth and physiological properties of three grape cultivars as affected by excess Mn treatments.

Biomass R/S ratio Root length Root area Root volume Root diameter Root activity O 2 rate MP PPO SOD CAT

R/S ratio

Root length

Root area

Root volume

Root diameter

Root activity

O 2 rate

MP

PPO

SOD

CAT

POD

0.334

0.818** 0.270

0.860** 0.362 0.982**

0.879** 0.473 0.917** 0.975**

 0.488 0.161  0.651**  0.592*  0.458

0.828** 0.127 0.645** 0.647** 0.630*  0.407

 0.558*  0.035  0.620*  0.542*  0.433 0.633*  0.689**

0.005  0.664**  0.098  0.081  0.084  0.128  0.023 0.272

0.074 0.343 0.035 0.096 0.178 0.113  0.005 0.294  0.191

0.607* 0.554* 0.469 0.510 0.547*  0.151 0.692**  0.413  0.290 0.252

0.684**  0.204 0.522* 0.583* 0.611*  0.464 0.550*  0.149 0.582* 0.178 0.199

0.728** 0.232 0.415 0.531* 0.637*  0.059 0.579*  0.089 0.256 0.104 0.404 0.707**

 R/S ratio, root/shoot ratio; O 2 rate, leaf O2 production rate; MP, membrane permeability. n

P o 0.05. P o0.01.

nn

that sulfur metabolism play an important role in reduction of heavy metal toxicity (Ernst et al., 2008). Recently, Doncheva et al. (2009) observed that silicon supplement could alleviate Mn toxicity in maize. Recent study on Douglas fir showed that root growth relied on hydroponic Mn concentration (Ducˇic´ and Polle, 2007). Our results indicated that there was a restricted value of root Mn concentration for the stimulation of root growth, that is less than 2200 mg kg  1 (i.e. 15–30 mM Mn for Combier and 15 mM for Jinshou and Shuijin), and the root growth were shrunk when exceeding that dose (Table 1, Fig. 2a–c). Lidon (2002) observed that excess Mn would damage root hairs, we also found that root hairs were sparsed and torn down above 30 mM (Fig. 4). In our results, cultivar Combier exhibited higher Mn-tolerance in root growth and root hair growth, which could be related with lower root Mn concentration. On the other hand, the characteristics for the increase of root diameter under excess Mn were not reported before (Table 1). These findings also could be related with IAA oxidation caused by excess Mn (Fecht-Christoffers et al., 2007), which was validated by its positive relationship with O 2 production rate in our results (Table 2). Another possible reason could be involved with formation of dead cell layer for restriction of Mn access, which might be an important adaptation mechanism for Mn tolerance in grape species. In higher plants, heavy metal induced oxidation stress by generation of reactive oxygen species (ROS) including superoxide radical (O 2 ) (Li et al., 2010). The non-quenched ROS would ultimately lead to cellular damage, which is expressed by increase of plasma membrane permeability (MP) (Fig. 5a, c). Polyphenol oxidase (PPO) function in oxidation of poly-phenolic compounds and is thought as an oxidation indicator in plant cell (Mayer, 2006). Chatzistathis et al. (2010) observed that periodic measurements of PPO activity in the olive leaves can be used for the early detection of Mn toxicity. Profoundly enhancement of O1 2 production rate, MP and PPO activity indicated that plant suffered high oxidation stress with increasing Mn levels (Fig. 5b, c). This study showed that O1 2 production rate could serve as an indicator in grape for excess Mn stress, due to its negative relationships with biomass, root growth and root activity (Table 2). Under extremely high Mn ( 430 mM) condition, the PPO activity was decreased possibly due to the damage of enzyme function. The higher values of O 2 , MP and PPO activity in Shuijin indicated that it suffered more oxidation and was more sensitive to excess Mn, which ultimately led to more reductions of plant growth and biomass accumulation compared with other two genotypes (Figs. 1 and 5). One mechanism involved

in the tolerance to oxidative stress is the activation of antioxidant enzymes including superoxide desmutase (SOD), peroxidase (POD) and catalase (CAT) as showed by our results (Fig. 6). Boojar and Goodarzi (2008) observed that the plants growing on Mn mine had higher antioxidative enzyme activities than the plants growing outside the Mn mine. It is well known that SOD quench O 2 to H2O2, and H2O2 could be further reduced to H2O by CAT or POD (Shi and Zhu, 2008). In our results, the closely positive relation between CAT or POD activity showed that those two enzyme cooperatively ¨ functioned in detoxifying cellular H2O2 (Table 2). Fuhrs et al. (2009) also demonstrated that apoplastic class III peroxidases (PODs) are decisive in the avoidance of manganese (Mn) toxicity in cowpea. The higher levels of POD and CAT activity in Combier could result in lower oxidative stress and higher tolerance, expressed by lower values of O 2 , MP and PPO activity in comparison with other cultivars. Recently Mora et al. (2009) also reported that Mntolerant genotypes in perennial ryegrass had high activity of antioxidative enzymes and relatively low lipid peroxidation than the sensitive genotypes under excess Mn. In conclusion, our results showed that grape species is extremely tolerant to excess Mn, which is expressed by plant stimulation under 15–30 mM Mn and extremely high EC10 values. Although the underlying mechanism for extreme tolerance to excess Mn need to be further explored, several aspects could be accounted for that. The first, it is propitious to plant nutrition uptake and ionic balance for the grape to keep constant or increased root growth as well as to keep high root activity. The second, the grape employs some effective but intraspecific strategies to detoxify cellular Mn stress by excluding excess Mn out of leaf tissues as Jinshou and Shuijin, or by enhancing antioxidative capacity as Combier cultivar. We propose some highly Mn-tolerant cultivars could be developed by integrating the two traits into one genotype through breeding program. On the other hand, the present study showed that there existed different (or contrast) distribution pattern for excess Mn in grape. For fruit safety purpose, people select the cultivars (like ‘‘Jinshou’’) which store majority of excess Mn in root system for cultivation in acid soil with high Mn level to limit Mn concentration in aerial parts. The present study also suggests the possibility of application of some grape (like ‘‘Combier’’) on phytoremediation of Mn mining area due to its high biomass and high values in concentration and biological transfer coefficient for excess Mn in aerial parts. As the genome sequence information of grape have been revealed (Jaillon et al. 2007), it warrants further exploration of the underlying mechanism for the extreme tolerance and genotypic responses to excess Mn.

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Acknowledgments

Appendix A

The research was supported by the Program of ‘‘100 Distinguished Young Scientists’’ and National Basic Research Program (2009CB825104) and the program of ‘‘Guizhou outstanding youth talent in science and technology’’.

The leaf surfaces element secretion pattern under excess Mn treatments separated detected by energy dispersive X-ray spectrometric microanalysis (EDS). See Figs. A1–A3.

Fig. A1. Leaf surfaces element secretion pattern in Combier cultivars.

Fig. A2. Leaf surfaces element secretion pattern in Jinshou cultivars. (a) CK and (b) 30 mM Mn treatment.

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Fig. A3. Leaf surfaces element secretion pattern in Shuijin cultivars.

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