Application of Aspergillus aculeatus to rice roots reduces Cd concentration in grain
Background and aims
‘Cd toxicity in rice’ events have resulted in vast public concern and uncertainty. Effective bioremediation could be accomplished via applying microbes that are capable of alleviating Cd content in rice grains.
Here, we investigated the effect of inoculating Aspergillus aculeatus on tolerance, uptake and transportation of Cd in rice cultivated in Cd contaminated growth medium.
A. aculeatus facilitated rice growth in Cd polluted growth medium and alleviated Cd toxic effects according to our observations on biomass, leaf and root length and grain yield. Cd accumulation analysis indicated that the plants which were inoculated with A. aculeatus exhibited minimum Cd level in all organs. Particularly in grain we observed a 40.5% reduction compared to the Cd only treated plants. Differences in Cd accumulation in rice inoculated with A. aculeatus might be attributed to the enhancement of cell wall-bound Cd, decreasing the Cd inorganic forms in roots, and inhibiting the expression of OsNRAMP5 and OsNRAMP1. A. aculeatus inoculation also led to minimum growth medium DTPA-Cd concentration, which possibly reduced the availability of the metals for plant uptake.
These results suggested that A. aculeatus might potentially be applicable to improve Cd tolerance and reduce Cd transportation in grains of rice.
KeywordsAspergillus aculeatus Cadmium Rice Tolerance Transport
Cadmium (Cd) is considered as one of the three major toxic heavy metals posing tremendous environmental threat (along with mercury and lead) (Jamers et al. 2013). Nriagu and Pacyna (1988) reported that global Cd production has risen by >1000-fold to 20,000 tons per year since the beginning of the twentieth century. Correspondingly, anthropogenic release of Cd into the environment now outstrips natural fluxes. As Cd is non-degradable, it is acutely toxic to all biological species (Begg et al. 2015). Under Cd stress, plant display inhibited plant growth, decreased carbon assimilation, generated oxidative stress, inhibited chlorophyll synthesis, reduced nutrient uptake and impaired photosynthesis (Das et al. 1997).
The highly substantial natural variation in Cd accumulation and tolerance is present among different plant species. As a staple crop for more than half of the global population, rice (Oryza sativa L.) tends to accumulate more Cd compared to other staple crops (Yao et al. 2015). Cd intake through the soil-crop-food chain is the most principal route for human exposure to its toxicity (Chang et al. 2002). Therefore, rice quality and its derivatives present a major health risk to the vast global rice consumers. Thus minimum rice grain Cd concentration is a highly desirable trait for food safety. China produces over 184 million tonnes of rice annually, which represents the highest global production (Borlaug 2007). However, the buildup of Cd in paddy irrigation water and soils has led to the elevation of Cd concentrations in Chinese rice grains (Bi et al. 2013). On May 16, 2013, Guangzhou Food and Drug Administration in China reported that 44.4% of sampled rice contained Cd concentrations above the national standard (the allowable maximum level for Cd in rice is 0.2 mg kg−1) (Bi et al. 2013). This ‘Cd toxicity in rice’ event has presented vast public concern and anxiety. In this sense, the need to ensure safety of China’s food production arising from polluted soil environment of the major grain producing areas is urgent.
Recently, some alleviating techniques have been applied in Cd polluted paddy soils to reduce the Cd contents of rice grains. One of the most reliable techniques is ‘soil dressing’, which is accomplished by removing the polluted soil layer and replacing it with clean soil (Ishikawa et al. 2005). However, this technique is subjected to certain restrictions in remediation of a large area with low Cd-contaminated soils because of limited clean replacement soil, and its enormous cost ($300,000–500,000 ha−1) (Ishikawa et al. 2005). A combination soil flooding and alkaline amendment application is widely employed in Cd-polluted paddy fields. However, the effectiveness of this technique depends on soil properties and weather conditions (Ke et al. 2015). In addition, the exploitation of rice cultivars which can absorb and translocate less Cd to the grain is a reliable technique for Cd content reduction in rice grain. One method is the modification of the genes related to Cd uptake or translocation. OsNramp5 is a major transporter responsible for Cd uptake and accumulation, knockout of this gene caused decreased Cd uptake and Cd accumulation in the grain (Sasaki et al. 2012). In addition to OsNramp5, OsIRT1, OsIRT2, OsNramp1 and OsLCD, have been proposed to contribute to Cd uptake and accumulation (Nakanishi et al. 2006; Shimo et al. 2011; Takahashi et al. 2011). These genes have served plant geneticists and breeders appropriate, however, the lengthy process of breeding new varieties has limited their application in breeding programs.
Recently, the application of natural soil microorganisms has attracted unprecedented attention because of their promising potential to provide a cost-effective approach to remediate heavy metal contaminated soil (Lloyd and Lovley 2001; Xie et al. 2014). Environmental Cd toxicity could be minimized by microorganisms via absorption, fixation, and covalently transformation of heavy metal ions (Xie et al. 2014). On the other hand, microorganisms (bacteria and fungi) that can produce indole acetic acid (IAA), siderophores and 1-aminocyclopropane-1-carboxylate (ACC) deaminase are capable of stimulating plant growth and helping plants acquire sufficient iron for optimal growth (AbouShanab et al. 2003; Burd et al. 2000). It has been rigorously confirmed that Cd contamination in soil is often associated with iron deficiency in a range of different botanical species because of their similar chemical properties (Ogo et al. 2014). Generally, the low iron content in plants grown under Cd polluted environment resulted in chlorosis, since iron deficiency inhibits chloroplast development and chlorophyll biosynthesis (Burd et al. 1998). The microorganisms can bind iron in the soil to form the microbial iron-siderophore complexes, which supply plants with iron. However, there is limited information on the potential of microorganisms to alleviate Cd toxicity in Cd polluted paddy soils.
Aspergillus aculeatus (A. aculeatus) is a fungus belonging to the genus Aspergillus that is isolated from Cd polluted soil. Our previous study showed that inoculation of A. aculeatus resulted in reduction of Cd translocation to plant shoots and promotion of bermudagrass growth under Cd stress (Xie et al. 2014). This observation suggested that A. aculeatus might be potentially applied to improve Cd tolerance and reduce Cd transportation to the plant’s shoots. Therefore, the objectives of this study were to: (1) investigate the effects of A. aculeatus on growth and grain yield of rice; (2) determine effects of Cd-resistant fungi A. aculeatus on Cd uptake and transportation within the roots of rice. It may therefore provide a new fungi-assisted phytoremediation for Cd-polluted paddy soils.
Materials and methods
Cd resistance of the strains
The tolerance of isolated fungus to Cd (minimum inhibitory concentration, MIC) was investigated. The fungus strains stored at −80 °C were activated with Martin medium, and then inoculated in liquid Martin medium (10.0 g glucose, 5.0 g peptone, 1.0 g KH2PO4 and 0.5 g MgSO4·7H2O per litre of medium, pH 6.0). The spores of the A. aculeatus were cultivated in liquid Martin medium. When the germination rate of spores was up to 80%, the spores were collected by centrifugation at 8000 rpm for 8 min, washed with sterile distilled water and resuspended. Under liquid cultivation, the MICs were determined by observing the fungal growth on Martin medium with Cd2+ at desired concentrations (0–200 mg L−1). One mL of above spore suspension (ca. 106 cfu mL) was inoculated into 250 mL Erlenmeyer flask containing 50 mL of liquid medium with the appropriate Cd concentration. Each treatment was replicated three times. The Erlenmeyer flasks were shaken at 180 rpm and 30 °C for 3 d. The fungal biomass was then determined. During solid cultivation, the mycelia of the A. aculeatus obtained from liquid cultures were spotted on plates. The plates were incubated at 30 °C for 3 d. Changes in colony diameter on the spotted position were then determined.
Effect of A. aculeatus on Cd tolerance and transport in rice
Preparation of growth matrix
This research was conducted at Wuhan Botanical Garden, Chinese Academy of Science in 2016. In April, the growth matrices were prepared for all the experiments. Sand and sawdust (sawdust/sand = 3/1 in volume, pH 6.5, organic matter 22.32 g kg−1, cation exchange capacity 2.65 cmol(+) kg−1, organic carbon 12.95 g kg−1, N 638 mg kg−1, P 572 mg kg−1, K 1.58%, Mg 392 mg kg−1, Fe 1.12%, Zn 96.4 mg kg−1, Cu 22.0 mg kg−1, total soil porosity 54.93%) were used as growth matrix in this study. The growth matrices were sieved with stainless steel mesh (1 mm) and sterilized at 127 °C for 1 h. Subsequently, the matrices were divided into three groups, namely control (CK), Cd treatment only (Cd) and Cd plus A. aculeatus (Cd + A. aculeatus). For Cd + A. aculeatus group, 10 mL of above -mentioned spore suspension (ca. 106 cfu mL) were inoculated into per kilogram growth matrix before the plants were planted.
Plant materials and growth conditions
The seeds of indica rice ‘9311’ (Oryza sativa L.) were sterilized with H2O2 (8%) for 20 min and washed with deionized water on April 16. To obtain seedlings, these seeds were germinated in filter paper soaked with distilled water at 30 °C under dark condition. After 3 days incubation, uniformly germinated seeds were selected and raised in plastic pots (20 cm in diameter and 35 cm deep) filled with 5.0 kg pre-prepared growth matrix. All the pots were sterilized using UV for 3 h before planted. The pots were set into the glasshouse at standard conditions (30/25 °C for day/night, 14 h light/10 h dark, 60% relative humidity and average photosynthetically active radiation of 720 mmol m−2 s−1). After 2 weeks, seedlings were thinned to three plants per pot. During the growth period, plants were irrigated three times per week and fertilized weekly with 800 mL of nutrient solution (pH 5.5) with the following composition: 0.7 mM K2SO4, 0.1 mM KCl, 0.1 mM KH2PO4, 2.0 mM Ca(NO3)2, 0.5 mM MgSO4, 10 μM H3BO3, 0.5 μM MnSO4, 0.2 μM CuSO4, 0.5 μM ZnSO4, 0.05 μM Na2MoO4, and 0.1 mM Fe-EDTA (Ishimaru et al. 2006).
Treatment and experimental design
The experiment was carried out in the glasshouse during the rice-growing season (from early May to mid-September). In May 8, 2016, the 3-week old seedlings grown in the above-mentioned growth matrix were treated. For Cd and Cd + A. aculeatus groups, seedlings were exposed to 20 mg Cd kg−1 growth matrix as CdSO4.8/3H2O (CdSO4.8/3H2O dissolved in ultrapure water was poured to the growth matrix directly). Controls were grown in the absence of Cd. The rice plants were grown under oxidative upland conditions with growth medium moisture at 60% of field capacity in order to reveal the intrinsic capacity for Cd uptake as previously described (Ishikawa et al. 2005). During the growth period, plants were fertilized weekly with 800 mL of above-mentioned nutrient solution. The experiments were arranged in a randomized complete block design with four replicates for each treatment and the pots were rotated randomly every three days. At maturity (September 10, 2016), the plant height, tiller number, ear length and number of grains per panicle were measured. And then, the treated roots were immersed in the solution that contained 10 mM Na2EDTA for 30 min and then thoroughly rinsed three times with distilled water to remove any nonspecifically bound Cd. Plants were harvested from of four replicates (pots) from each treatment for the measurement of grain yield and biomass. Stems, grains, leaves, glumes and roots were separated and oven-dried at 75 °C to constant weight for inductively coupled plasma mass spectroscopy (ICP-MS) analysis.
The experiment was carried out from May 8 to May 22, 2016. The 3-week old seedlings were treated. Treatments and growing conditions are the same as in experiment 1. The experiments were arranged in a randomized complete block design with eight replicates for each treatment and the pots were rotated randomly every three days. After plants were treated with Cd for 7 d (in May 15), half of the plants (four replicates for each treatment) were immersed in the solution that contained 10 mM Na2EDTA for 30 min and then thoroughly rinsed three times with distilled water to remove any nonspecifically bound Cd. The root and leaf length of plants were measured. And then, roots and shoots were separated and stored at −20 °C until analyzed. In May 22 (after plants were treated with Cd for 14 d), the rest of the plants were harvested and treated same as 7 d. The net flux of Cd2+ and microscopic imaging of Cd in roots were observed. The extraction of chemical form of Cd and separation of tissue fractionations in roots and leaves were analyzed. And the Cd concentrations in root, shoot and growth medium were measured by ICP-MS.
In May 8, 2016, the 3-week old seedlings grown in the above-mentioned growth matrix were treated. Treatments and growing conditions are the same as in experiment 1. The experiments were arranged in a randomized complete block design with four replicates for each treatment. The root samples, however, were harvested for gene expression analysis at 0, 3, 6, 12, 24, 48 and 72 h after Cd treatment began, immediately placed in liquid nitrogen, and stored at −70 °C until analyzed.
Measurement of Cd2+ flux
The net flux of Cd2+ in the treated roots was measured using scanning ion-selective electrode technique (SIET) (BIO-001A; Younger USA Science and Technology) (Li et al. 2012; Xu et al. 2012). The tips of ion-selective microelectrodes were backfilled with a commercially available ion selective cocktail. The microelectrodes were calibrated in 50 and 500 μM Cd2+ before the flux measurement. The concentration of the ions and gradients were measured by moving the ion-selective microelectrodes between two positions close to the root in a preset excursion (30 μm). The measuring solution contained 50 μM CdCl2, 0.5 mM KCl, 0.1 mM CaCl2, 0.1 mM NaCl, 0.1 mM MgCl2 and 0.3 mM 2-(N-morpholino) ethane sulfonic acid (MES). The steady-state fluxes of Cd2+ in the apical region (500 μm from the root apex) were recorded (3–5 min). And the fluxes of Cd2+ in the roots were measured along the root apex (0–600 μm from the tip) at intervals of 50–100 μm. The data were obtained with the ASET software. Three -dimensional ionic fluxes were calculated using MageFlux (http://xuyue.net/mageflux).
Microscopic imaging of Cd in roots
Cd probe Leadmium™ Green AM dye (Molecular Probes, Invitrogen, Calsbad, CA, USA) was performed to investigate the distribution of Cd in roots of rice which were pre-treated with 20 mg kg−1 Cd for 14 d (Lu et al. 2008). A stock solution of Leadmium™ Green AM was made by adding 50 μL of DMSO to one vial of the dye, and then diluted with 1:10 of 0.85% NaCl. Roots were immersed in this solution for 120 min in the dark. Samples were observed using a confocal laser scanning microscope (Olympus FV-1000, Tokyo, Japan) with excitation at 485 nm and emission at 520 nm, and serial confocal optical sections were taken. All images were taken at ×10 magnification. Images were pseudo-coloured with METAMORPH software (Universal Imaging, Downingtown, PA, USA).
Separation of tissue fractionations
Frozen roots and shoots were homogenized in pre-cold extraction buffer containing 250 mM sucrose, 1 mM dithioerythritol (DTT, C4H10O2S2, Sigma D 8255) and 50 mM Tris-HCl (pH 7.5) (Wu et al. 2005). The homogenate was strained through a nylon cloth, and the residue was washed three times with the extraction buffer. All of the filtrate was centrifuged at 300 g for 30 s. This precipitate combined with the first residue constituted the cell wall-containing fraction or Fraction I. Then, the supernatant of the first centrifugation step was centrifuged at 20000×g for 45 min and the pellet was the organelle fraction or Fraction II. The supernatant solution was the soluble fraction or Fraction III. All steps were performed at 4 °C. Fractions I, II and III were digested in a mixture of concentrated HNO3, HF and HCl (5:2:2, v/v/v). The concentration of Cd was determined using ICP-MS as described above.
Extraction of chemical form of Cd
Five chemical forms of Cd were extracted step by step using the method described by Wu et al. (2005). Different chemical forms of Cd were extracted in the order of the extraction solution listed below. Frozen roots and shoots were homogenized in extraction solution, diluted at ration of 1:100 (w/v). The homogenate was transferred into 50 mL centrifuge tubes and shaken for 22 h at 25 °C. After shaking, the tubes were centrifuged at 5000×g for 10 min and the supernatant was collected in a conical flask. The sediment was then re-extracted twice with the same 20 mL extraction solution and shaken for 2 h at 25 °C, then centrifuged at 5000×g for 10 min and the supernatants was collected. The three supernatants were evaporated and digested for the determination of corresponding Cd form using ICP-MS as described above. The order of the extraction solutions was as follow: (1) 80% ethanol, extracting inorganic Cd, which included nitrate/nitrite, chloride, and aminophenol Cd (FE); (2) deionized water, extracting water-soluble Cd of organic acid complexes and Cd(H2PO4)2 (FW); (3) 1 M NaCl, extracting Cd integrated with pectates andprotein (FNaCl); (4) 2% HAC, extracting insoluble CdHPO4 and Cd3(PO4)2 and other Cd-phosphate complexes (FHAC); (5) 0.6 M HCl, extracting oxalate acid bound Cd (FHCl).
The oven-dried tissues were ground into fine powder and then digested in a mixture of concentrated HNO3, HF and HCl (5:2:2, v/v/v). The mixture was digested in a Microwave Sample Preparation System (ETHOS ONE, Milestone) using the following procedure: 12 min at 130 °C, 25 min at 180 °C and finally, 25 min at 180 °C. The volume of each sample was adjusted to 50 mL using 1% HNO3 and, then filtrated with 0.45 μm filter membrane. The concentration of Cd was determined using ICP-MS. Each experiment was repeated at least six times.
Analysis of gene expression
Analysis of genes expression was performed on roots of different treated condition before and after exposure to Cd stress. Total RNA was extracted and purified according to the protocol of the Trizol reagent (Invitrogen, Carlsbad, CA) and then reverse-transcribed using an oligo (dT) primer. Expressions of the selected genes in roots were analyzed by real-time quantitative reverse transcriptase PCR (RT-PCR) using the fluorescent intercalating dye SYBRGreen with a detection system (Opticon 2, MJResearch, Waltham, USA). Total 18 genes, relating to Cd transportation and distribution, were used in this study. Primer sequences used to amplify the genes of interest are listed in Supplementary Table 1. Actin and HistoneH3 were used as internal controls, with primers 5′- GACTCTGGTGATGGTGTCAGC-3′ and 5′-GGCTGGAAGAG GACCTCAGG- 3′ for Actin and, 5′-AGTTTGGTCG CTCTCGATTTCG-3′ and 5′-TCAACAA GTTGACCACGTCACG-3′ for HistoneH3.
Analysis of DTPA-Cd in growth medium
Diethylenetriaminepentaacetic acid (DTPA) extraction of Cd was carried out by shaking 5.00 g of growth medium with 25 ml of 0.005 M DTPA solution, 0.1 M triethylamine (TEA) and 0.01 M CaCl2 in an end-over-end shaker at 25 °C for 2 h. The suspension was then centrifuged for 30 min at 6000×g. The supernatant was used for ICP-MS analysis.
The results were given as mean ± SD. For statistical analyses, we used ANOVA with the SPSS 20.0 software package (SPSS, Chicago, Illinois, USA). Differences between the treatments were test by the least significant difference (LSD) test at a 0.05 probability level.
Cd resistance of the strains
Effects of A. aculeatus on growth and yield of rice under Cd stress
Inoculations with Cd-resistant strain A. aculeatus subsequently improved the grain yield by 41.6% compared to Cd only regimes (Fig. 2d). Increase in grain yield under Cd + A. aculeatus treated was mainly attributed to the significant increase in the number of grains per panicle and grain weight (P < 0.05) (Fig. 2d). There were no significant differences in the tiller number and ear length between two treatments.
Effects of A. aculeatus on Cd transportation and distribution
Cd concentration and translocation factor in root and shoot were measured to determine the influence of Cd-resistant strain A. aculeatus in short period (7 d and 14 d). A. aculeatus decreased the Cd concentration in shoot and root at 7 and 14 d of treatment, compared to Cd only regime. After 14 d of Cd treatment, the plants subjected to Cd + A. aculeatus had 30.2% lower translocation factor compared to Cd only treated plant (Fig. 3b). The DTPA-Cd concentration in the rice rhizosphere medium was obtained to determine the impact of the strain. After 7 d and 14 d of Cd + A. aculeatus treatment, the DTPA-Cd concentration was reduced by 24.5% and 25.8% in rice rhizosphere medium, respectively, compared to the non- inoculated (Fig. 3b).
These results demonstrated that A. aculeatus reduced Cd accumulation. Therefore, we wondered whether A. aculeatus affected the root Cd fluxes. As a result, we used SIET to detect the effects of A. aculeatus on Cd uptake. Although difference was not significant, Cd + A. aculeatus treated roots had a slightly lower level of net Cd2+ flux at 14 d of treatment, compared to Cd only treated roots (Supplementary Fig. 1).
Cd sub-cellular distribution and chemical forms
The Cd concentrations bound to the different chemical forms in rice plant were shown in Supplementary Fig. 2. Similar to the sub-cellular distribution, the plant roots and shoots treated with Cd + A. aculeatus displayed lower Cd concentration of the five chemical forms than those of plant treated with Cd only (Supplementary Fig. 2b). Cd extracted by 80% ethanol (FE) and 1 M NaCl (FNaCl) was predominant in rice roots and shoots. Cd forms extracted by 80% ethanol (FE), deionized water (FW), 1 M NaCl (FNaCl), and 0.6 M HCl (FHCl) from root account for 29.0%, 10.1%, 56.2% and 2.9% of the total Cd amount for Cd only treated plants, and 20.3%, 12.3%, 54.4% and 11.1% of the total Cd amount for Cd + A. aculeatus treated plants, respectively (Supplementary Fig. 2a). No significant difference in the proportion of above five Cd chemical forms was observed in shoots.
Expression of Cd responsive genes
Soils contaminated by heavy metal may result in the development of selection pressure on soil fungi that eventually have increased levels of heavy metal resistance and adsorption capacity. As expected, the strain A. aculeatus tolerating up to 200 mg L−1 Cd in solid media was isolated from Cd polluted soil. This result implied that A. aculeatus we isolated was capable in Cd tolerance than other microorganisms reported in the literature. Although Cd2+ tolerant bacteria and yeasts have been isolated (Villegas et al. 2004; Huang et al. 2005; Wei et al. 2011), the filamentous fungi, such as A. aculeatus, were able to develop a significantly higher biomass to sequestrate more metals. Previous study showed that certain Aspergillus were remarkably tolerant to Cd than other filamentous fungi (Zafar et al. 2007). Our results are consistent with the findings. The fungus A. aculeatus showing high tolerance to Cd is very useful in Cd-polluted soil recovery systems.
Cd stress-induced plant growth inhibition and yield reduction has been rigorously described by many researchers (Cao et al. 2015). Cao et al. (2015) found that the grain yield of rice was markedly impaired at the external Cd levels above 5 mg kg−1. Our results showed that Cd exposure led to growth inhibition and yield reduction. However, inoculation with A. aculeatus could alleviate stresses caused by Cd, which was determined based on the biomass, leaf and root growth rate and grain yield parameters (Fig. 2). These observations demonstrated that A. aculeatus facilitated rice growth in Cd polluted growth medium and alleviated the Cd toxic effects.
Suppression of Cd transportation to shoot was a critical mechanism of plant Cd tolerance (Xie et al. 2014). Cd, with high soil–plant mobility, easily accumulates in plants’ tissues. High Cd concentration in plants was not only deteriorating crop quality and yield, but also posing threats on human health via/along the food chain (Masood et al. 2012). Chen et al. (2007) reported that Cd content in 47.2% of barley grain of 600 genotypes grown in non-contaminated soil exceeded the maximum permissible threshold (MPC: 0.1 mg kg−1) (Chen et al. 2007). Hence, focus should be shifted to strategies necessary for reducing Cd accumulation in the edible parts of crops. Recent studies indicated that restricted translocation from root to shoot might result in lower Cd concentration in grains than it in leaves or roots (Grant et al. 1998). The results of this study indicated that Cd immobilization in growth medium was highly enhanced by A. aculeatus. Rice plants inoculated with A. aculeatus had a lower Cd level in all organs (root, stem, leave, grain and shell), especially decreased by 40.5% in grain, compared to the Cd only treated plant. Correspondingly, inoculation with A. aculeatus had 30.2% lower translocation factor compared to those uninoculation. These results suggested that inoculation of A. aculeatus might actively reduce Cd translocation to plant shoots or diluted Cd via promoting plant growth. Consistent with this result, our previous study found that exogenous A. aculeatus can reduce Cd translocation in bermudagrass.
Consistent with Cd level in roots and translocation factor, the intensity of green fluorescence and root Cd influxes were lower in inoculation treatment with A. aculeatus. This indicated that inoculated with A. aculeatus in rice rhizosphere medium suppressed Cd uptake, which thereby decreased shoot Cd accumulation. These decreases in Cd uptake might translate into lower DTPA-Cd concentrations in rhizosphere medium of the inculated plants. Previously researchers have reported that Cd shared many physical similarities (charge and ionic radius) with Ca (Perfus-Barbeoch et al. 2002). In many plants, Cd may permeate through Ca channels from guard cells and root cells. These studies suggested that Cd accumulation and detoxification in some plants might be associated with metal transport through Ca channels or transporters. Suppressing Cd uptake across the inoculated root might be contributed by the inhibited H+-ATPase activities by Cd.
Many genes might be involved in regulation of Cd tolerance and transport in many plants. The natural resistance-associated macrophage proteins (Nramps) constituting a large family have been implicated in the uptake, translocation, intracellular transport, and detoxification of transition metals (Nevo and Nelson 2006). Only two Nramp genes (OsNramp1 and OsNramp5) in rice have been characterized at the molecular level and showed transport activity for Cd (Sasaki et al. 2012; Takahashi et al. 2011). OsNramp1 is suggested to be involved in cellular Cd uptake and Cd transport within the plant, and higher levels of OsNRAMP1 expression in the roots of rice were responsible for higher Cd uptake in the root cells (Takahashi et al. 2011). OsNramp5 is the major transporter for Cd uptake in rice, and knockout of OsNramp5 resulted in almost loss of function to take up Cd (Sasaki et al. 2012). Under Cd stress, the exogenous of A. aculeatus could inhibit the expression of OsNRAMP5 and OsNRAMP1 in rice roots, especially after 6 and 12 h of Cd treatment. Although the underlying physiological and molecular mechanisms for the regulation of gene expression by A. aculeatus is unknown, to some extent, our results might explain that A. aculeatus in rice rhizosphere growth medium suppressed Cd transport, which thereby decreased shoot Cd accumulation. More interestingly, inoculation with A. aculeatus significantly improved the transcription levels of OsIDEF1, compared to non-inoculated. Kobayashi et al. (2007) identified the rice transcription factor IDEF1, a key component regulating the response to tolerance of iron deficiency. Also, Fe and Cd have a similar chemical property (Ogo et al. 2014). Thus, Cd polluted soil is often associated with iron deficiency. Under conditions of Cd contamination, Fe deficiency may increase the severity of Cd phytotoxicity (Ogo et al. 2014). A. aculeatus may alleviate Fe deficiency, thus alleviate the toxicity symptom of Cd. Those results are consistent with the finding that plants inoculated with A. aculeatus grew better than non-inoculated under Cd stress.
Regionalization of vacuolar compart-mentation and cell wall deposition also play an important role in Cd detoxification and tolerance in plants (Zhang et al. 2014). Therefore, vacuoles and cell walls are considered to have great potential for Cd accumulation in plant roots. Zhang et al. (2014) observed the Cd distribution and chemical form in rice seedling and indicated that the first defense mechanism against Cd toxicity in rice might be cell wall deposition. In the present study, Cd mainly accumulated in cell walls, particularly in leaves, demonstrating that this structure was the primary subcellular fraction for Cd storage, which has been confirmed by other studies. However, significant increase in the proportion of Cd bound to cell walls in roots were observed in inoculation with A. aculeatus, compared to non-inoculated in the presence of Cd, indicating that the Cd bound to cell walls in root was highly enhanced by A. aculeatus. The different chemical forms related to the toxicity of this metal could indicate one of the most important detoxification mechanisms. The 80% ethanol-extractable Cd (inorganic forms) contributed most to Cd stress in plants, while NaCl-extractable Cd consisted of organic forms, might be responsible for the adaption of plants to Cd stress (Wang et al. 2008). Zhang et al. (2014) reported that the complexation of metals with organic ligands could reduce their toxicity by decreasing the free ion activity. Our result indicated that A. aculeatus was able to change in inorganic form Cd to other less harmful form Cd in root under Cd stress, In addition, the concentration of NaCl-extracted Cd was higher than that of other forms, which mirrored the accumulation of Cd in cell walls and soluble fractions in rice plants.
The authors sincerely thank Zhongqi Liu and Shenjun Zhang, Agro-environmental protection institute, Chinese academy of agricultural sciences for excellent technical assistance. This word was financially supported by the National Natural Science Foundation of China (Grant No. 31470363).
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