Inoculation with mycorrhizal fungi modifies proline metabolism and increases chromium tolerance in pepper plants (Capsicum annuum L.)

RESEARCH ARTICLE

Inoculation with mycorrhizal fungi modifies proline metabolism and increases chromium tolerance in pepper plants (Capsicum annuum L.)

Marcela Ruscitti^I; María Arango^I; Marta Ronco^{I, II}; José BeltranoI, II,^*

^IInstituto de Fisiología Vegetal, Facultad de Ciencias Agrarias y Forestales, CCT CONICET La Plata Universidad Nacional de La Plata, C.C. 327, 1900 La Plata, Argentina

^IICICBA, Comision de Investigaciones Científicas de la Provincia de Buenos Aires, Argentina

ABSTRACT

In general, heavy metals interfere with several physiological processes and reduce plant growth. Plants naturally establish symbiotic associations with soil microorganisms, such as mycorrhizal fungi. The aim of this research was to determine if inoculation with mycorrhizal fungi increases tolerance to Cr, evidenced by growth and biochemical parameters and the effect on roots membranes in Capsicum annum. Plants were either non-inoculated or inoculated with Glomus mosseae or Glomus intraradices, and grown in the presence of different concentration of Cr (K₂Cr₂O₄) in soil. Pepper plants grown without Cr behaved as mycotrophic species. At the highest concentration (200 μM K₂Cr₂O₄), Cr reduced root colonization by G. mosseae or G. intraradices (to 23 and 20% respectively). Moderate and high concentrations of Cr reduced all growth parameters. The interaction of inoculation and Cr increased leaf chlorophyll and proline content while reduced the leaf protein and root proline content. Carotenoid content was not affected by treatments. High Cr concentrations increased significantly electrolytes leakage in roots, either non-inoculated or inoculated plants. At the highest Cr concentration, inoculated plants had double the biomass of non-inoculated plants. Cr content in roots of inoculated plants was significantly higher than in non-inoculated plants. Chromium accumulation was low in leaves and showed no differences between treatments. Mycorrhization increased pepper plant tolerance to Cr in the soil, modifying proline metabolism to assure a more efficient response.

Key words: Electrolytes leakage, Glomus intraradices, Glomus mosseae, heavy metals.

INTRODUCTION

Heavy metals (HM) are one of the main sources of environmental pollution (Il'yasova and Schwartz, 2005) and are responsible for several environmental problems, associated with industrial and agricultural activities: decrease of microbial activity, soil fertility and crop yield (Yang et al., 2005). Interest in chromium originates from widespread use in various industries, such as metallurgical and chemical. Due to industrial process, large quantities of Cr compounds are discharged into the environment, resulting in significant adverse biological and ecological effects (Kabata-Pendias and Pendias, 2001).

Heavy metals interfere with several physiological processes reducing the plant growth, photosynthesis and consequently the biomass (Jamal et al., 2006). Decrease in total chlorophyll, chlorophyll a and b and carotenoids have been well documented for Cr stressed plants (Panda and Choudhury, 2005). In the soil, Cr exists in two different oxidation states: trivalent (Cr³⁺) and hexavalent (Cr⁶⁺). Both Cr³⁺ and Cr⁶⁺ differ in terms of mobility, solubility and toxicity. Hexavalent Cr⁶⁺ is more toxic and mobile than Cr³⁺, it forms chromate and dichromate, are highly soluble in water and there is no evidence of the potential role in plant metabolism (Panda and Patra, 1997). Chromium phytotoxicity can result in inhibition of seed germination, pigments degradation and induced oxidative stress in plants (Panda and Patra, 1997, 2000). Beside, these effects, Cr can alter membrane ultrastructure in plants (Choudhury and Panda, 2005). Toxic ions penetrate cells by the same mechanisms used by essential minerals (Patra et al., 2004). The pathways for Cr⁶⁺ transport underlie active mechanisms involving carriers of essential anions, such as sulphate, Fe and P (Cervantes et al., 2001; Barbosa et al., 2007) and moves mainly by plant xylem. Golovatyj et al. (1999) have demonstrated that Cr accumulation was great in roots and lower in vegetative and reproductive organs.

Arbuscular mycorrhizal (AM) fungi establish symbiotic associations with many plant species. This evolution strategy has been known for more than a century but just in recent decades it has become an useful tool in horticulture, agriculture and forestry, where there are evidences of its efficiency in the competitive and sustainable development of the production systems (Pawlowska and Charvat 2004, Pawlowska, 2005). Mycorrhizal fungi enhance plant tolerance to biotic and abiotic stresses (Auge, 2001; Beltrano et al., 2003). The influence of mycorrhizal fungi on plant nutrition is important for the absorption of elements with low mobility, such as P and heavy metals (Clark and Zeto, 2000). Participation of mycorrhizal fungi in heavy metals metabolism has been proposed as a mechanism to increase plant tolerance, in some cases heavy metals can be absorbed by fungi hyphae and transported to the plant (phytoextraction), in other cases fungi contribute to heavy metals iμMobilization in the soil (phytostabilization) (Gaur and Adholeya, 2004; Khan, 2005).

Mycorrhizal roots may act as a barrier against metal transport from roots to the aerial part of the plant. This effect is attributed to metal adsorption on the hyphal walls, since chitin has an important metal-binding capacity (Joner et al., 2000). Another possible tolerance mechanism is the dilution of heavy metals concentrations by growth increase of mycorrhizal plants. Liu et al. (2005) determined that biomass of tomato mycorrhizal plants increased by 30% compared to nonmycorrhizal with increasing concentration of arsenic in the soil.

The hypothesis of our work was that mycorrhiza fungi have a protective action against heavy metals stress, increasing pepper plant (Capsicum annuum L.) tolerance to Chromium. The aim of this investigation was to test this hypothesis by determining the effect of different Cr concentrations on pepper plant growth parameters (dry weigh, leaf area and partition of assimilated), biochemical parameters (Cr content in roots and leaves, chlorophyll, carotenoids, proline), and roots and leaf cell membranes stability in non-inoculated and inoculated plants with the mycorrhizal fungi Glomus mosseae or Glomus intraradices.

MATERIALS AND METHODS

Growth conditions: Seeds of pepper (C. annuum L. 'California Wonder 300') were sown in plastic pots previously filled with substrate composed of a mixture of soil (Argiudol vertic, pH 5.5, 12 mg/Kg^-1 total P, 3.5% organic matter, 2% total C and 0.24% total N) perlite and vermiculite (2:1:1) tindalized at 100ºC for 60 minutes, during 3 consecutive days. G. intraradices Schenck & Smith isolate GA1 and G. mosseae (Nicolson & Gerdemann) Gerdemann & Trappe (Banco de Glomeromycota In Vitro BGI. Buenos Aires, Argentina) were bulked-up through culture with Trifolium repens L. for four months in a semi-controlled grown chamber.

The inoculum (10% of the substrate weight), a mix of soil, spores (50 spores per g^-1 inoculum) mycelium and root fragments colonized by G. mosseae (μMos) or G. intraradices (Mintra), was added to the substrate at sowing time. The same amount of sterilized inoculum plus 10 ml mycorrhizal fungal-free filtrate from the inoculum suspension was added to non-inoculated pots in order to provide the same soil conditions.

When root colonization with G. intraradices was approximately 50% and with G.mosseae, 40%, inoculated and non-inoculated plants, pepper young plants were transplanted to 500 ml pots containing the same soil. Before transplanting different doses of Cr in the K₂Cr₂O₄ form was added to the substrate to rich concentrations of 0 μM K₂Cr₂O₄ (Cr0); 10 μM K₂Cr₂O₄ (Cr1); 100 μM K₂Cr₂O₄ (Cr2) and 200 μM K₂Cr₂O₄ (Cr3). The experiments were conducted at La Plata (34º SL, 54' WL) (Argentina). Pepper plants were grown in a greenhouse between October to December, under natural conditions.

The treatments were: (a) control (NI), the plants received no mycorrhizal inoculation with G. mosseae or G. intraradices: NICr0, without K₂Cr₂O₄; NICr1, 10 μM K₂Cr₂O₄ ; NICr₂, 100 μM K₂Cr2O4; and NICr3, 200 μM K₂Cr₂O₄; and (b) inoculated plants (M): μMosCr0 (G. mosseae) or MintraCr0 (G. intraradices), without K₂Cr2O4 ; μMosCr1 or MintraCr1, 10 μM K₂Cr₂O₄ ; μMosCr2 or MintraCr2, 100 μM K₂Cr₂O₄; and μMosCr₃ or MintraCr3, 200 μM K₂Cr₂O₄ .

Variables measured: Ten plants per treatment were harvested at the end of the experimental period (51 days after transplanting (DAT)).

Estimation of AM colonization: Fungal colonization was assessed according to Trouvelot et al. (1986) and expressed as rate of mycorrhization (Myc%) and relative arbuscules and vesicles abundance (A% and V% respectively). Roots were cleared with 10% KOH (p/v) and stained with trypan blue in lacto-phenol (Phillips and Hayman, 1970). The viability of hyphae was determined by measuring succinate dehydrogenase activity (SDH) (Schaffer and Peterson, 1993). Three replicates of 10 randomly chosen root fragments were mounted on slides and examined microscopically. Myc% was calculated as the proportion of infected roots over total root fragments and A% was calculated as the arbuscular abundance per colonized roots. V% was calculated as the abundance of vesicles per colonized roots.

Mycorrhizal dependency (MD) was calculated according to the following formula:

Growth parameter: Plant height, leaves number, DW of leaves, stems and roots were obtained by drying the material in oven at 80ºC until constant weight and leaf area (LA) per plant (Li 3000 leaf area meter, LICOR, Lincoln, NE, USA) was measured.

Seed germination test: Replicates of 25 seeds of C. annuum L. were sown on two layers of filter paper in 11-cm Petri dishes. About 5 ml of deionized water or Cr solution was added to each Petri dish as K₂Cr₂O₄. The concentrations used were: 10 μM, 100 μM and 200 μM, and a control without metal. Petri dishes were incubated in a grown chamber at 25 ºC and 12 h photoperiod. The seeds were considered germinated when radicle emerged 1μM. Percentage of germination was determined daily and after 15 days, the length of the root and the aerial part was measured. Each treatment was replicated four times.

Carotenoids, chlorophyll and leaf proteins content: Carotenoids and chlorophyll contents were determined in one leaf disc (1 cm diameter) per plant, and protein content was estimated in five leaf discs (1 cm diameter) per plant. The concentration of carotenoids and the content of chlorophyll were measured according Wellburn (1994), and proteins concentration, according to Bradford method (1976) using bovine albumin as standard. All absorption spectra were recorded in a Shimadzu UV-160 spectrophotometer (Kyoto, Japan). RESULTSwere expressed as mg carotenoids cm^-2, mg chlorophyll cm^-2 or mg protein cm^-2.

Proline content in roots and leaves: Proline content was determined from 1 g leaf or root fresh weight (FW), according to Bates et al. (1973). Extraction was made with an aqueous solution of 3% sulfosalicylic acid and the extract obtained reacted with ninhydrin acid and glacial acetic acid. Proline concentration was measured in a spectrophometer Shimadzu UV-160 (Kyoto, Japan) at 520 nm absorbance. Proline content was calculated per unit of FW according to: μMols proline g^-1 FW = [(mg proline / ml × ml toluene)/ 115.5 mg / μMols] / (g FW / 5)

Electrolyte leakage: This technique is based on the increase of cellular membrane permeability and concomitantly greater electrolyte diffusion out of cells when tissue is injured by a stress situation. The electrolyte leakage was measured as described by Lutts et al (1996) with a few modifications. After harvest, the uppermost fully expanded leaves of 10 plants per treatment were iμMediately cut into discs of 1 cm diameter. Leaf disc were washed briefly three times in deionized water to remove solutes released during cutting of the discs. Five discs of each leaf were placed in a vial filled with 10 ml deionized water and maintained at 25ºC for 4 h subsequently the electrical conductivity of the bathing solution was determined (ELi: initial). After the first measuring the vials were heated in boiling water for 60 min and the final electrical conductivity was obtained after equilibration at 25ºC (ELf: final). Electrolyte leakage was determined by measuring the electrical conductivity of the vial solution, using a conductimeter and data were expressed as mS cm^-1. Relative electrical conductivity (EL) was calculated as follows:

Determination of chromium in roots and leave: Chromium content was determined in 500 mg of root and leaf samples per treatment, according to method 3111B (APHAAWWA-WPCF, 1998). The samples were analyzed by atomic absorption spectrometry, direct air-acetylene flame with digestion pretreatment with nitric acid.

Statistical analysis: The experiment was a 3×4 factorial, in a completely randomized design with three mycorrhizal levels (NI, μMos, Mintra) and four levels of chromium (Cr0, Cr1, Cr2, Cr3). Data were analyzed by ANOVA, and comparisons among means were made using LSD (P<0.05). For the statistical analysis all inoculation percentage values were arcsine transformed to improve homogeneity. The number of replicates was: for growth data (n=10), and for mycorrhizal observations (n=3 replicate of 30 roots fragments).

RESULTS

Mycorrhyzation: None of the non-inoculated plants was colonized by G. mosseae or G. intraradices and there were very few colonization by native fungi. Without Cr, the level of plants colonization with G. mosseae was 44% and with G. intraradices was 58%. The percentage of arbuscles and vesicles was higher in plants inoculates with G. intraradices than in plants colonized with G. mosseae.

Chromium reduced inoculation with G. mosseae by 4%, 13% and 23% in Cr1, Cr2 and Cr3, respectively, compared to Cr0, while in plants inoculated with G. intraradices, reduction was 5%, 15% and 20% in Cr1, Cr2 and Cr3, respectively. Viability of hyphae, expressed by SDH activity, was higher in plants inoculated with G. intraradices than in inoculated with G. mosseae. The highest concentration of Cr reduced viability in 46% in MintraCr3 and 71% in μMosCr3 (Table 1).

Germination test: Percentage of germination and the length of the aerial part did not show significant differences with increasing Cr concentrations. Root length was significantly reduced with increasing Cr concentrations (Table 2).

Growth parameter: Moderate or high Cr concentrations reduced all plant growth parameters (roots, stems and leaves DW, and LA). Chromium reduced the height and leaf number in all treatments. In NI, all Cr treatments reduced these parameters compared to inoculated plants.

Height reduction was 16%, 14% and 22% in NICr3, μMosCr3 and MintraCr3, respectively, compared to Cr0 treatments (Figure 1A). Chromium concentration reduced the leaf number by 13%, 18% and 15% in NICr3, μMosCr3 and MintraCr3, respectively, compared to the control without chromium (Figure 1B). ANOVA showed a significant interaction (myco x chromium) for height though not for leaf number.

Chromium reduced LA in all treatments; the higher values were obtained in plants inoculated with G. intraradices in Cr0. Leaf area of plants inoculated with G. intraradices showed significantly higher compared to G. mosseae in all Cr treatments (Figure 1C). Myco x chromium interaction was not observed.

The total dry weigh (TDW) of plants inoculated with G. intraradices was higher than the inoculated with G. mosseae in all Cr treatments. NI plants showed the lowest values and decreased significantly with Cr treatments (Figure 1D). For TDW significant myco x chromium interaction was observed.

Figure 2

Aerial/TDW and aerial/roots DW ratios were not affected by mycorrhization although the ratios were affected by Cr concentrations. The leaf/TDW ratio was affected only by mycorrhization. Stem/TDW ratio was affected by mycorrhization and Cr concentration. The specific leaf area was not modified by mycorrhization although this was modified by Cr concentrations.

Mycorrhizal dependency without Cr was 18% and 33% for plants inoculated with G. mosseae and G. intraradices, respectively and 33% and 57% for inoculated with G. mosseae and G. intraradices, respectively at the higher Cr concentration (Table 1).

Proteins, chlorophyll and carotenoid contents of leaves: There were significant differences in foliar protein content at the different Cr concentrations. NI plants showed higher protein contents than inoculated ones. Plants inoculated with G. intraradices had significantly higher protein contents than those inoculated with G. mosseae. Protein content was significantly affected by mycorrhization and chromium and the myco x chromium interaction was significant (Table 3). Without chromium, chlorophyll concentration was not affected by inoculation with G. mosseae or G. intraradices. At the higher chromium concentrations, the leaves of μMos and Mintra plants retained greater chlorophyll levels than NI. Myco × chromium interaction was significant. Carotenoids content was not affected by the treatments (Table 3).

Proline content in leaves and roots: In NI plants, the proline content of leaves diminished 32% with an increase of chromium concentration (Cr0 versus Cr3). In μMos and Mintra plants, leaves proline content increased by 29% and 49%, respectively. In NICr3 plants, proline content of roots increased by 63% while inoculation with G. mossseae and G. intraradices induced a significant decreased in root proline concentrations (45% and 40%, respectively). Myco × chromium interaction was significant in leaves and roots (Table 3).

Determination of Electrolyte leakage: Leakage of root solutes increased due to high chromium concentrations but not due to mycorrhization (Figure 3B). Myco × chromium interaction was not significant. Treatments did not affect electrolyte leakage in leaves (Figure 3A).

Chromium determination in leaves and roots: At Cr0 and Cr1 no differences were observed in Chromium content in both aerial parts and roots of non-inoculated, μMos and Mintra plants. At the highest concentration, Cr content of NI, μMos and Mintra roots was significantly higher than aerial parts. Higher chromium content was at higher concentrations of chromium (Cr2 and Cr3) in μMos and Mintra roots than in non-inoculated roots. No differences were observed between G. mosseae and G. intraradices (Table 4).

DISCUSSION

Plant tolerance and/or resistance to heavy metals stress can be associated with one or more mechanisms, such as: (i) metal retention in roots preventing its translocation to the aerial part (Patra et al., 2004); (ii) metal iμMobilization in the cell wall (Cosio et al., 2005); (iii) homeostatic cellular mechanisms to regulate the concentration of metal ions inside the cell (Benavides et al., 2005); (iv) increase of tolerance to mineral deficiency or decrease of nutritional requirements; (v) increase in absorption of certain macronutrients or development of the capacity to absorb and use minerals in the presence of heavy metals (Meda et al., 2007). As a result of these tolerance and/or resistance mechanisms (alone or in combination) some plants can grow in environments contaminated with heavy metals where other species could not survive. Since seed germination is the first physiological process affected by Cr, the ability of a seed to germinate in a medium containing Cr would be indicative of its level of tolerance to this metal (Peralta et al., 2001). In our study, Cr did not affect seed germination of pepper or the growth of the aerial parts of the seedlings, though in agreement with findings by Nayari et al. (1997) and Panda et al. (2002) our RESULTSshowed that Cr toxicity significantly affected root growth. Meanwhile, Rout et al. (2000) observed that germination of Echinochloa colona was reduced to 25% with 200 μM Cr. Higher levels (500 ppm) of hexavalent Cr in soil reduced germination up to 48% in Phaseolus vulgaris (Parr and Taylor, 1982). Peralta et al. (2001) found that 40 ppm Cr⁶⁺ reduced by 23% the ability of seeds of lucerne (Medicago sativa cv. Malone) to germinate in contaminated soil.

Mycorrhizas can alleviate Cr toxicity and support greater plant growth in Cr rich soils (Davies Jr et al., 2001). Although little information is available on the influence of inoculation with mycorrhizal fungi as improvers of plant tolerance and phytoaccumulation of Cr (Davies Jr. et al., 2001), some authors have demonstrated that spores and pre-symbiotic hyphae of mycorrhizal fungi are sensitive to heavy metals and under certain conditions, metals inhibit spore germination and hyphal growth (Shalaby, 2003). This study demonstrate that inoculated pepper plants can tolerate the presence of Cr in the soil, and shows that in the treatments without chromium, mycorrhization was high, confirming that pepper is a mycotrophic species (Ronco et al., 2008). The increase in chromium concentration affected colonization and arbuscular and vesicular formation, in both Glomus species. Our data show that plants inoculated with G. intraradices showed the highest percentage of mycorrhization, although it was reduced with the increase of Cr in the soil. The sensitivity to Cr toxicity was revealed by reduction in arbuscular formation, followed by reduction in vesicular formation; hyphal formation was less affected. Also, high Cr levels affected hyphal viability, expressed by SDH activity, and it was higher in G. intraradices than in G. mosseae. Similar RESULTSwere obtained by Pawlowska and Charvat (2004) who demonstrated that G. intraradices was more tolerant in the presence of other HM. Similar effects of different HM had been reported by Rivera-Becerril et al. (2002) who observed that without Cd colonization of Pisum sativum was 45%, meanwhile in presence of HM was by 28%. Shalaby (2003) proposed that resistance to HM was likely due to phenotype plasticity rather to by genetic changes, since tolerance was lost after one generation in the absence of heavy-metals.

Chromium has not been recognized as an essential element for plant, however Shanker et al. (2005) reported that low concentrations of chromium can stimulate the plant growth, this fact was not observed in our study. In the absence of Cr, our RESULTSshowed that the higher growth was determined in plants inoculated with G. intraradices. The deleterious effect produced by the presence of Cr was lower in inoculated plants compared to non inoculated ones, and in those inoculated with G. intraradices compared to those inoculated with G. mosseae. These RESULTSare in agreement with Jamal et al. (2006), who demonstrated that Cr produced a significant reduction in the growth of Prosopis juliflora and with Bishnoi et al. (1993) who determined that the deleterious effect of Cr was more pronounced on the growth of roots than on the stems, this could be due to Cr accumulation in the roots, as was observed in our results. Cr reduced the height, leaves number, leaf area and dry weight in all treatments. The arbuscular mycorrhizae contribute in supporting partially Cr toxicity as demonstrated by higher growth of inoculated plants in the presence of Cr compared to the non-inoculated ones, as observed by Bagyaraj et al. (1988) in several crops and by Davies Jr. et al. (2001) in sunflower. Consequently, mycorrhizal dependency, that is the relationship between biomass of mycorrhizal plants compared to non-mycorrhizal, reaching higher values than 50% at the highest concentrations of Cr, regardless of the inoculum used, similar RESULTSare presented by Davies et al. (2002) in sunflower plants in similar Cr concentrations. The plant height and leaves number were the parameters less affected. Leaves, stem and root DW were the most affected by Cr and by inoculation in agreement with Anderson et al. (1972) who observed 11%, 22% and 41% reduction in oats plant cultivated in 2, 10 and 25 ppm of Cr in soil, respectively. The biomass reduction in pepper plants could be attributed to a competition mechanism between Cr and P, demonstrated by Davies et al. (2002) and previously reported by several authors. The roots of non-inoculated plants were the most affected organ by low and high concentrations of Cr. Our data showed that the effect of mycorrhization and the presence of Cr on the DW of leaves, stem and root and the interaction is highly significant (<0.001). Decrease root growth due to heavy metals is well-documented (Tang et al., 2001). The roots may act as a barrier against Cr uptake by plants, and this effect is increased by the presence of mycorrhizal fungi.

Our data show that the effect of Cr on the root growth and on the integrity of cell membranes, assessed by the electrolytes leakage, was significantly higher in NI compared to inoculated plants and in the roots compared to the leaves. Cr affects cell membranes, though the mechanism is not known, electrolytes leakage by root tissues is significantly increased by Cr. Electrolytes leakage by leaf tissues was not affected nor by chromium nor by mycorrhization. Davies et al. (2002) proposed that the first effects on the root membranes could be attributed to the high potential of reduction of Cr⁶⁺ which is retained in the vacuoles and cell walls of the root, while Cr reaching the leaves can be mainly at the Cr³⁺, although accumulation mechanisms of Cr are not well known, our RESULTSseem to confirm those presented by this author, since Cr content determined in the leaves did not promote significant damage in the membranes.

As found by James (2002), higher Cr content in roots than in leaves is observed, while the lower concentration is found in the stems. In this study, Cr content in leaves was low, in agreement with RESULTSby James (2002); and Sharma and Sharma (1993) for wheat and by Tripathi et al. (1999) for Albizia lebbeck; these last authors proposed that leaf growth might serve as suitable bio-indicators of heavy metal pollution and used in the selection of resistant species. According to Shanker et al. (2004) the reason of the high accumulation in roots could be caused because Cr is mobilized to the vacuoles of the root cells, thus producing lower toxicity to the aerial part. It is possible that when pass the endodermis via symplast Cr ⁶⁺ is reduce to Cr³⁺, which is retained in the root cortex cells and reduce Cr⁶⁺ concentration. Although higher plants do not contain enzymes reducing Cr⁶⁺, they have been widely reported in bacteria and fungi (Cervantes et al., 2001), and AM are likely to participate in this reduction. In addition to the reduction in growth and its effect on cell membrane stability, Cr⁶⁺ promotes inhibition of photosynthetic pigment synthesis (Vajpayee et al., 2000) and induce oxidative damages in biomolecules such as lipids and proteins (Vajpayee et al., 2001).

Our results show that carotenoids content was not modified by the presence of Cr, in discordance with Rai et al. (1992) who determined that Cr can induce degradation of carotenoids in plants, while chlorophyll content and proteins declined with increasing Cr concentration. Meanwhile, the moderating effect of AM reported in our study, as a possible expression of the protective action against stress by HM, was greater at the highest concentrations of Cr, avoiding a high degradation of both chlorophyll and proteins, in agreement with Abdul Razak (1985) who determined a decrease in the photosynthetic activity and in the chlorophyll synthesis by the accumulation of HM, and by Schützendübel and Polle (2002), that determined a significant reduction in proteins content and in enzyme activity by the interference produced of metal ions. The way in which mycorrhizal fungi modify these effects depends on several factors such as growth conditions, fungal species and metal concentration. The increase in mycorrhized plants tolerance to HM was observed in different species such as maize, barley and rye (Hildebrandt et al., 1999; Gaur and Adholeya, 2004).

Moreover, it was observed that many plants accumulate proline when were treated with toxic concentrations of heavy metals (Bassi and Sharma, 1993; Costa and Morel, 1994; Schat and Vooijs, 1997). The effect of HM on proline synthesis is contradictory. Some authors describe an increase in the intracellular concentration of proline in the presence of high concentrations of metals (Costa and Morel, 1994; Schat and Vooijs, 1997). Kavi Kishor et al. (1995) suggest that proline might protect plants from metal toxicity. Schat and Vooijs (1997) observed that metal-induced proline accumulation does not occur until damage has been caused; hence plants would not be protected against stress.

Rodriguez and Redman (2005) reported that proline protects fungal cells against abiotic stresses such as UV light, heat, salt, and hydrogen peroxide. Rodriguez et al. (2004) proposes that mutualistic fungi allow symbiotic plants to perceive stress more quickly than nonsymbiotic, resulting in the rapid activation of plant biochemical reactions that mitigate the impacts of stress. However, the mechanisms that conferred stress tolerance are poorly defined.

In our work, proline synthesis increased concomitant to Cr increase, in agreement with Bassi and Sharma (1993) and Costa and Morel (1994), however with different responses in leaves and roots and in none inoculated and inoculated plants.

In the roots of non-inoculated plants proline concentration increased with increasing Cr concentration, while it decreased in leaves. Though, in the roots of inoculated plants proline concentration decreased with increasing Cr concentration, while it increased in leaves, in agreement with data previously described for many species; this would respond to a modification in the partition of free Cr in plants.

This leaf values were higher and root values were lower in G. intraradices than in G. mosseae, due probability to a higher percentage of mycorrhized-roots. However, Porcel and Ruiz-Lozano (2004) demonstrated the accumulation of higher proline levels in soybean mycorrhizal roots and lower contents in mycorrhizal shoots compared to non-mycorrhizal plants under drought conditions. A more detailed analysis of both root and shoot samples during stress is necessary to be performed (Pinior et al., 2005).

Mycorrhizal fungi, in some cases increase absorption and accumulation of HM in roots and in other cases they favor HM translocation to the aerial part of the plant, this would explain the different behavior of mycorrhized and nonmycorrhized plants.

It is know that Cr is poorly mobile, so that in inoculated plants mycorrhizal fungi could be a protective barrier avoiding the translocation of metal towards the aerial part of the plant. Hence, the lower uptake of Cr in aerial part allows the proline metabolism to operate more efficiently, while in roots the high concentration of Cr can affect metabolism of this protecting amino acid. Although increase in HM concentration raised the cell proline content, inhibition of proline accumulation was evident beyond a certain threshold of the metal. Thus, in accordance with Mehta and Gaur (1999), high concentrations of heavy metals are inhibitory to proline biosynthesis, and the threshold sensibility change from organ and the mycorrhizal can modified the responses.

In conclusion, increase in Cr concentration modifies root colonization by mycorrhizal fungi, reduces height, leaf area, dry weight and other growth parameters, and modifies chlorophyll, protein and proline contents. Mycorrhization increases pepper plant tolerance to high Cr concentrations in the soil, modifying proline metabolism to make the response more efficient and confirming the hypothesis proposed.

Acknowledgments: The authors would like to thank O. Peluso and L. Wanhan (CONICET) for technical assistance, Cecilia Moreno (CIC BA) for translation and English revision, and the Universidad Nacional de La Plata and CIC BA for financial support.

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Received: 26 March 2010

Accepted: 22 February 2011

Publication Dates

• Publication in this collection
  16 Aug 2011
• Date of issue
  2011

History

• Received
  26 Mar 2010
• Accepted
  22 Feb 2011