Characterization of the tolerance to excess manganese in four maize varieties

Abstract

Manganese (Mn) is an essential micronutrient in all organisms, but may become toxic when present in excess. Four maize (Zea mays L.) varieties, Kneja 605, Kneja 434, Kneja 509 and Kneja 537, were studied with respect to their responses to excess Mn in hydroponic solution. In the varieties Kneja 605, Kneja 509 and Kneja 537, increasing Mn concentrations in the nutrient solution negatively affected biomass accumulation, photosynthetic rate, transpiration, stomatal conductance and chlorophyll content. In addition, these varieties showed increased electrolyte leakage and lipid peroxidation (malondialdehyde [MDA] content). Increased Mn leaf concentrations, higher contents of chlorophyll a and chlorophyll b, higher photosynthetic rate and transpiration, lower concentrations of MDA and insignificant changes in the electrolyte leakage in the leaves were found in var. Kneja 434 compared with the other maize varieties studied. This variety appeared to possess a stronger ability to cope with Mn phytotoxicity, suggesting high potential for Mn detoxification and var. Kneja 434 could be a good candidate for improving maize productivity on acid soils under non-tropical conditions.

Key words:

• electrolyte leakage
• lipid peroxidation
• maize production
• manganese tolerance
• manganese toxicity

Introduction

Maize (Zea mays L.) is one of the world’s most important cereal crops. As a result of increasing demand, a shift to maize production has been observed, especially in tropical and subtropical regions (Morris 2001). Soil acidity can be an important constraint in these areas and the adaptation of maize to acid soils in the tropics is an important research issue (The et al. 2006a). Phosphorus efficiency and tolerance to aluminum or manganese (Mn) toxicity have been identified as relevant traits for plants to be productive on acid tropical soils (Foy et al. 1978; Kochian et al. 2004). Different mechanisms are responsible for tolerance to these major constraints of acid soils and tolerance to these factors appears to be inherited in an independent way (The et al. 2006b).

The limitation of maize production as a result of soil acidity is not restricted to the tropics. In Bulgaria almost 40% of the arable land is dedicated to cereal production and corn production ranges second behind winter wheat. Maize productivity, however, is relatively low with an average of approximately 3 t ha⁻¹ (Balabanova 2004). The main constraint is drought as a result of insufficient rainfall or irrigation. In addition, soil-derived stress factors can play a role. Approximately 40% (4.3 million ha) of Bulgarian soils have a pH value below 5 and have been classified as non-resistant to acidification (Kolchakov et al. 2005). Soil acidification (pH <5.5) enhances the solubility of Mn, increasing its bioavailability and creates Mn toxicity in many natural and agricultural systems (Foy 1988).

Manganese is an essential trace element throughout all stages of plant development. For optimal growth and development plants need to accumulate at least 30 mg Mn kg⁻¹ dry weight in tissues, regardless of the plant species (Marschner 1995). The most important role of Mn is associated with oxygen evolution in plants. Manganese participates in both oxygen radical production via the O₂-evolving four-atom Mn cluster located on the luminal surface of the D₁ protein in PSII and in oxygen radical detoxification by its role in superoxide dismutase activity in mitochondria (Shenker et al. 2004). Manganese is required as a co-factor in more than 30 enzymes (Burnell 1988) or as an activator for an array of enzymes, such as carboxylases and phosphatases in the cytosol and glycosyl transferases in the Golgi apparatus (Marschner 1995). The availability of Mn and its uptake by plants depend on soil properties. Plants exposed to excess Mn exhibit strong inhibition of chloroplast structure and functions (Doncheva et al. 2009). It has been proposed that the accumulation of phenolic substances in the apoplast, an early symptom of Mn toxicity in legumes, could be triggered by Mn toxicity mechanisms in the symplast (Führs et al. 2008).

Plant species and genotypes may differ in their tolerance to excess Mn (Horst et al. 1999). The mechanisms of Mn tolerance are still not clearly established. In some species, resistance to Mn toxicity results from a tolerance of higher internal Mn concentrations in the leaf tissues (Horst 1983; Moroni et al. 2003). Investigations with Arabidopsis thaliana mtp11 mutants defective in a cation diffusive facilitator were found to be hypersensitive to excess Mn and to accumulate higher root and shoot Mn concentrations than wild-type plants (Peiter et al. 2007). Manganese exclusion as a result of Golgi-mediated exocytosis was proposed as an operating mechanism in the more Mn-tolerant wild type. Internal detoxification as a result of storage in either or both a non-toxic form and in compartments with low metabolic activity, such as vacuoles or trichomes, can play a role.

The aim of the present study was to characterize the differences in tolerance to excess Mn in four Bulgarian varieties of maize (Zea mays L). A better understanding of the strategies that underlie Mn tolerance in this important crop species will help to improve maize productivity on acid soils in non-tropical regions.

Materials and methods

Plant materials

Seeds of maize (Zea mays L.) plants (var. Kneja 605, var. Kneja 434, var. Kneja 509 and var. Kneja 537) were germinated on wet filter paper. After 5 days the seedlings were transferred to aerated nutrient solution in a greenhouse under natural light. The basic nutrient solution contained (μmol L⁻¹): 200 CaSO₄.2H₂O, 100 MgSO₄.7H₂O, 400 KNO₃, 300 NH₄NO₃, 5 MnSO₄.H₂O, 0.38 ZnSO₄.7H₂O, 0.16 CuSO₄.H₂O, 10 Fe-ethylenediaminetetraacetic acid, 5 NaH₂PO₄.H₂O, 8 H₃BO₃, 0.06 (NH₄)Mo₇O₂₄.H₂O. After 72 h the seedlings were transferred to a new fresh nutrient solution (two plants per beaker; beaker volume 1.2 L) with the same composition as above supplemented with 50, 200 and 500 μmol L⁻¹ Mn as MnSO₄. Control plants received the basic nutrient solution containing 5 μmol L⁻¹ Mn throughout the experiment. The nutrient solution was changed twice per week. Plants were harvested for analyses 5 days after the Mn treatment. Twelve plants were used for each treatment and three independent experiments were carried out.

Plant growth parameters

Plant growth was determined as an increase in the dry weights of both root (RW) and shoot (SW) on day 5 after the onset of the Mn treatment.

Determination of leaf Mn content

Detached leaves were carefully washed under a continuous stream of deionized water and dried at 60°C for several days. Samples of dry leaves (0.1 g) were ashed at 550°C. The ash was dissolved and brought to a standard volume with 20% HCl and the concentration of Mn in the solution was determined using an inductively coupled-plasma atomic emission spectrometer ([ICP-AES] Liberty-II; Varian, Middelburg, The Netherlands).

Leaf pigments

Chlorophyll a, chlorophyll b and the total carotenoids were extracted from leaf disks with 80% acetone. The pigment content was determined spectrophotometrically according to Arnon (1949). The chlorophyll values were calculated following Wang et al. (2001) according to Porra (1991).

Measurement of CO₂ gas exchange rates

Measurements of the net photosynthetic rate (P_N), stomatal conductance (g _s ) and transpiration rate (E) were done with an infrared gas analyzer (Li 6400; Li Cor, Lincoln, NE, USA). Gas-exchange measurements were done under a saturating photosynthetic photon flux density (PPFD) of 500 μmol m⁻² s⁻¹, 380 μmol CO₂ mol⁻¹, and a leaf temperature of 34 ± 2°C. The data were statistically processed using Systat 7.0 (Chicago, IL, USA).

Lipid peroxidation

The peroxidation of lipids in the leaves was assessed by the thiobarbituric acid (TBA) reaction as described by Heath and Packer (1968). Thiobarbituric acid (i.e. the reactive substance) was quantified as malondialdehyde (MDA), which is an end product of lipid peroxidation. The TBA reagent consisted of 20% (w/v) trichloroacetic acid (TCA) and 0.65% (w/v) TBA. The specific absorbance of the TBA-reactive substances was determined as a TBA–MDA complex at 532 nm. After subtracting the non-specific absorption at 600 nm, the MDA amount was calculated using its molar extinction coefficient (155 m[mol L⁻¹]⁻¹ cm⁻¹) and the results are expressed as MDA g⁻¹ fresh weight (FW).

Electrolyte leakage

The electrolyte leakage (EL) test was conducted according to Yamori et al. (2005) with modifications. One gram of tissue was cut into 2-cm segments, rinsed in deionised water to remove the contents of the cut cells and placed in test tubes containing 15 mL of bidistilled water for 36 h at 4°C. The conductivity of the supernatant (C ₁) was measured with an electro-conductivity meter. The tubes were placed in a boiling-water bath for 60 min, and the electrical conductivity was obtained after attaining equilibrium at 25°C (C ₂). The EL was calculated using the following equation: EL (%) = C₁/C₂ × 100.

Statistical analyses

The results are the means of three independent experiments. A comparison of the means was made using Fisher’s least significant difference test (P ≤ 0.05) after a multifactor anova analysis.

Results

Manganese induced a reduction in both the dry shoot weight (SDW) and the dry root weight (RDW) in var. Kneja 605, var. Kneja 509 and var. Kneja 537 (Fig. 1). With increasing Mn concentrations in the nutrient solution, the SDW and RDW were significantly reduced in var. Kneja 605 compared with var. Kneja 509 and var. Kneja 537. The highest Mn concentration (500 μmol L⁻¹) caused a reduction in SDW and RDW in var. Kneja 605, var. Kneja 509 and var. Kneja 537 by 44 and 52%, 28 and 34%, and 27 and 29%, respectively. In contrast, excess Mn did not cause a significant decrease in the shoot and root dry biomass in var. Kneja 434.

As the accumulation of Mn in the leaves could be responsible for Mn tolerance, Mn concentrations in the leaves were estimated. Exposure to excess Mn led to increased leaf Mn concentrations in all varieties studied. The Mn treatment caused higher Mn concentrations in the leaves of var. Kneja 434 compared with var. Kneja 509, var. Kneja 537 and var. Kneja 605 (Fig. 2). The differences in the Mn leaf concentrations were most pronounced at the highest Mn concentration (500 μmol L⁻¹) in the nutrient solution. The net CO₂ assimilation rate (P_N), transpiration rate (E) and stomatal conductance (g_s) in var. Kneja 509, var. Kneja 537 and var. Kneja 605 decreased with increasing leaf Mn content (Fig. 3). In contrast, increasing Mn leaf concentrations significantly increased the transpiration rate and stomatal conductance in var. Kneja 434, whereas the net photosynthetic rate was not affected. Pigment content showed changes in all varieties, but no correlation was found with increasing Mn leaf concentrations (Fig. 4). The chlorophyll content decreased to a higher extent in var. Kneja 605, var. Kneja 509 and var. Kneja 537 compared with var. Kneja 434. Our results showed a loss of chlorophyll a and b by 46 and 40%, respectively, in the leaves of var. Kneja 605, whereas in var. Kneja 434 the decrease in chlorophyll a and b was 22 and 17%, respectively. In addition, the total carotenoid levels in var. Kneja 605 were much higher than the levels in var. Kneja 434, var. Kneja 509 and var. Kneja 537 (Fig. 4).

Figure 1 Shoot dry weight and root dry weight of var. Kneja 434, var. Kneja 605, var. Kneja 509 and var. Kneja 537 grown in nutrient solutions with different Mn concentrations (μmol L−1): 5 (control), 50, 200 and 500. Values represent the means of three independent experiments. Different letters indicate significant differences assessed by Fisher’s least significant difference test (P < 0.05) using anova multifactor analysis.

Figure 2 Manganese concentrations in the leaves of var. Kneja 434, var. Kneja 605, var. Kneja 509 and var. Kneja 537 after exposure to different Mn concentrations in nutrient solution. FW, fresh weight.

Figure 3 Effects of leaf Mn concentrations on (a) net CO2 assimilation (PN), (b) transpiration rate (E) and (c) stomatal conductance (gS) in the leaves of var. Kneja 434, var. Kneja 605, var. Kneja 509 and var. Kneja 537. Measurements were carried out at 500 μmol m−2 s−1 photosynthetically active radiation. FW, fresh weight.

Figure 4 Effects of leaf Mn concentrations on chlorophyll a, chlorophyll b and carotenoids in the leaves of var. Kneja 434, var. Kneja 605, var. Kneja 509 and var. Kneja 537. FW, fresh weigt.

The membrane damage caused by Mn excess was assessed indirectly by the conductivity of solute leakage from the cells (Fig. 5a). Data on EL indicated that the destruction of cell membranes in var. Kneja 605 plants was more severe than the destruction observed in the other varieties. On the contrary, the exposure of var. Kneja 434 plants to excess Mn did not change significantly the EL in the leaves, suggesting that the membrane integrity was not destroyed. Thus, var. Kneja 537 and var. Kneja 509 were less able to cope with excess Mn compared with var. Kneja 434. The MDA level as the final product of membrane lipid peroxidation in the leaves is given in Fig. 5b. Significant lipid peroxidation was found in the leaves of var. Kneja 605, var. Kneja 537 and var. Kneja 509. In contrast, no significant changes were detected in the TBA-reactive substances in the leaves of var. Kneja 434.

Discussion

The present study reveals clear differences in Mn tolerance among maize varieties adapted to the non-tropical growth conditions in Bulgaria. In all tested varieties the Mn content in the leaves increased with increasing Mn supply (Fig. 2). Plants possess a range of potential internal tissue mechanisms that may be involved in detoxification of heavy metals, thus directly contributing to their metal tolerance capacity (Hall 2002). In our study, a Mn supply of 50 μmol L⁻¹ or higher caused significantly higher Mn concentrations in the leaves of var. Kneja 434 compared with of the concentrations in var. Kneja 605, var. Kneja 509 and var. Kneja 537. However, despite the lower Mn accumulation in their leaves, var. Kneja 605, var. Kneja 509 and var. Kneja 537 exhibited a stronger Mn-induced growth reduction both in the shoots and roots than var. Kneja 434 (Fig. 1). These results confirm our previous observation about Mn-tolerant var. Kneja 434 based on an insignificant callose accumulation, slight alterations in the chloroplast structure, mainly in stomatal but not in granal thylacoids, unchanged maximum quantum efficiency of PSII photochemistry estimated by the ratio F_v/F_m and the actual efficiency of PSII electron transport, non-photochemical quenching qN of chlorophyll fluorescence, and a significant increase in the thickness of the epidermal layers, suggesting that non-photosynthetically active epidermal cells may be implicated in the sequestration of excess Mn (Dikova et al. 2005; Doncheva et al. 2009; Stoyanova et al. 2008).

Figure 5 Changes in (a) electrolyte leakage (EL) and (b) the level of lipid peroxidation (MDA content) in the leaves of var. Kneja 434, var. Kneja 605, var. Kneja 509 and var. Kneja 537 grown in nutrient solutions with different Mn concentrations (μmol L−1): 5 (control), 50, 200 and 500. Values represent the means of three independent experiments. Different letters indicate significant differences assessed by Fisher’s least significant difference test (P < 0.05) using anova multifactor analysis. FW, fresh weight.

Tolerance to high shoot or leaf Mn concentrations rather than restriction of uptake has also been described in other crop species, such as rice (Horiguchi 1987), cotton (Foy et al. 1995), rapeseed (Moroni et al. 2003) and Douglas fir (Ducic and Polle 2007). The determination of the Mn leaf concentration in younger leaves could be useful in assessing soil Mn status (Kitao et al. 2001). The inherent tolerance mechanisms that give plants the ability to withstand high concentrations of Mn in photosynthetically active tissues are still not clearly established. Our results show that increased Mn leaf concentrations were positively correlated with transpiration rate, stomatal conductance and photosynthesis in var. Kneja 434 (Fig. 3). Higher Mn concentrations accompanied by an increase in tolerance and a lack of a decline in photosynthesis with Mn stress have been reported for deciduous broad-leaved plants in northern Japan (Kitao et al. 1997). In contrast, in var. Kneja 605, var. Kneja 509 and var. Kneja 537 excess Mn negatively affected photosynthesis and transpiration by closure of the stomata and inhibition of CO₂ fixation. Manganese is considered to move towards the leaves by the transpiration stream in the xylem and it is mobile only in the xylem (Page and Feller 2005; Xu et al. 2006). Transport in the xylem is directed from the roots to the above-ground plant parts in the transpiration stream (Marschner 1995). Maintenance of high stomatal conductance and transpiration under high Mn exposure in the leaves of Mn-tolerant var. Kneja 434 may, at least in part, be responsible for its higher leaf Mn accumulation. An increase in the Mn concentration from the midrib to the marginal part was observed within a leaf of Phytolacca acinosa, suggesting that Mn was transported along the transpiration stream. Therefore, more Mn was accumulated at the end of the transpiration stream, indicating a transpiration-driven distribution pattern of Mn in the leaf (Xu et al. 2006). A similar distribution pattern was also observed for Cd (Cosio et al. 2005), Ni and Zn (Mizuno et al. 2003) in the leaves of Thlaspi caerulescens and Thlaspi japonicum, respectively.

It is well known that free radical induced peroxidation of membrane lipids is a reflection of stress-induced damage at the cellular level (Jain et al. 2001) and the level of MDA, produced during peroxidation of membrane lipids, is generally accepted as an indicator of severe oxidative stress (Katsuhara et al. 2005). Our results indicated that the oxidative stress caused by the Mn treatment varied between the maize varieties. Among them, var. Kneja 434 showed the lowest level of MDA in the leaves, suggesting that the Mn leaf content did not contribute to the lipid peroxidation level. In agreement with the above result, the increased carotenoid concentrations in the Mn-sensitive varieties (Fig. 4) could be considered to be a response to Mn-induced oxidative stress. Carotenoids are well known for their anti-oxidant activity within the chloroplasts, scavenging singlet oxygen and lipid peroxyradicals as well as preventing lipid peroxidation (Burton and Ingold 1984; Winston 1990). The fact that high light intensity increases the damaging effects of excess Mn on the photosynthetic apparatus (Clair and Lynch 2004) supports the view that Mn toxicity primarily affects chloroplast functions.

Although carotenoids were increased in var. Kneja 605 (Fig. 4), the photosynthetic apparatus was not efficiently protected against the oxidative stress induced by excess Mn in the sensitive varieties. As a result, chlorophyll levels decreased. In Mn-tolerant var. Kneja 434, chlorophyll a and b contents were maintained, despite the accumulation of high leaf Mn concentrations. Changes in chlorophyll levels are a good indicator of varietal differences in Mn tolerance in maize (Fig. 4), and support data reported previously for other crop species (Ohki 1985; Nable et al. 1988; Kitao et al. 1997). The photosynthetic activity and chlorophyll content of Mn-stressed plants have been used as suitable criteria for screening Mn tolerance in Triticum aestivum (Macfie and Taylor 1992; Moroni et al. 1991), Phaseolus vulgaris (González and Lynch 1997) and in deciduous broad-leaved tree seedlings (Kitao et al. 1998).

Electrolyte leakage as a consequence of the loss of membrane integrity can also be used as a marker to measure cell membrane functional status under stress conditions (Almeselmani et al. 2006). Our results showed that the leakage of electrolytes was more strongly expressed in the the Mn-sensitive var. Kneja 605, var. Kneja 509 and var. Kneja 537 compared with var. Kneja 434 (Fig. 5a). The formation of MDA can be used as a general indicator of the extent of lipid peroxidation resulting from oxidative stress (Skórzyn′ska-Polit and Krupa 2006). In the present study, the MDA content increased significantly in the leaves of var. Kneja 605, var. Kneja 509 and var. Kneja 537, suggesting that Mn leads indirectly to the production of superoxide radicals, resulting in increased lipid peroxidative products. Thus, both the MDA content and EL represent further evidence of the high ability of var. Kneja 434 to tolerate high Mn concentrations. This result implies that Mn tolerance, which is under genetic control (Meharg 1994) in maize, must be based on mechanisms of detoxification without decreasing Mn accumulation.

Conclusion

In conclusion, the results of the present study showed significant variations in the growth responses of four maize varieties to excess Mn. The varieties Kneja 605, Kneja 537 and Kneja 509 showed a stronger inhibition in biomass, photosynthesis, transpiration and loss of membrane integrity observed at the lower Mn leaf concentrations compared with var Kneja 434. The higher Mn tolerance of var. Kneja 434 may be accounted for by a stronger internal capacity for protection against the phytotoxicity of Mn and a higher potential for Mn detoxification. Maize variety Kneja 434 is a good candidate for improving maize productivity on acid soils under non-tropical conditions.

Acknowledgments

This work was supported by Grant No. B/1524/2006 of the National Science Fund, Ministry of Education and Science, Bulgaria, and the Spanish Government MEC ref. BFU2007-60332.