Physiological, Biochemical and Molecular Responses of Barley Seedlings to Aluminum Stress

Barley (Hordeum vulgare L.) is one of the most Aluminum (Al) sensitive cereal species. In this study, the physiological, biochemical, and molecular response of barley seedlings to Al treatment was examined to gain insight into Al response and tolerance mechanisms. The results showed that superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity were inhibited to different degrees following Al exposure. The MDA content also significantly increased with increasing Al concentrations. SRAP results indicated significant differences between Al treatments and controls in terms of SRAP profile, and the genomic template stability (GTS) decreased with increasing Al concentration and duration. These integrative results help to elucidate the underlying mechanisms that the barley response to Al toxicity.

In this study, we defined Al-induced physiological and biochemical changes, and DNA damage in barley seedlings under Al stress. These results help elucidate the mechanisms of Al tolerance in barley, and offer new perspectives for improving the barley tolerance in acidic soil.

Plant Materials, Growth Conditions, and Stress Treatments
The barley cultivar, Franklin, was assessed in this study. Seeds were surface sterilized in 0.2% NaClO for 20 min, rinsed with distilled (5 times) and germinated in moist sand. When the second leaf emerged, seedlings were selected for uniformity and transplanted into containers. The composition of the basic nutrient solution (mg L -1 ) was: (NH4)2SO4 48.2, MgSO4 154.88, K2SO4 15.9, KNO3 18.5, KH2PO4 24.8, Ca(NO3)2 86.17, Fe-citrate 7, MnCl2 4H2O 0.9, ZnSO4 7H2O 0.11, CuSO4 5H2O 0.04, HBO3 2.9, and H2MoO4 0.01. The pH of the solution was adjusted to 4.0. Growth inhibition tests were performed in barley plantlets exposed to 0, 50, 100, and 200 μmol L -1 Al for 5 or 10 days. Containers were incubated in a growth chamber at 26 o C ± 1 o C and a 14/10 h day-night photoperiod with a light intensity of 10000 lux. Each treatment was repeated on three occasions. After harvesting, each plant was separated into the leaf and roots, washed thoroughly with distilled water, frozen in liquid nitrogen and maintained at -80 o C for biochemical and molecular analysis.

Cellular Antioxidant Enzyme Activities and MDA Content Measurement
Barley leaves (0.3 g) were homogenized 50 mmol L -1 sodium phosphate buffer (pH 7.8). Homogenates were centrifuged (10000 × g) at 4 o C for 20 min and supernatants were collected for enzymatic analysis. SOD, POD, CAT and Malondialdehyde (MDA) content were measured based on previous studies [19].

DNA Isolation, SRAP-PCR, Cloning and Sequencing of SRAP Fragments
For both control group and Al treatments groups, roots were collected 5 d and 7 d post-germination and flash frozen in liquid nitrogen prior to storage at -80 o C. Total DNA was extracted from ~ 100 mg of fresh roots using a CTAB protocol [20]. SRAP amplification was performed using 30 primer combinations, including 9 forward and 8 reverse primers (Tab. 1). PCR amplification for SRAP was performed in a 20 μl volume containing 80 ng of template DNA, 50 μM forward primer, 50 μM reverse primer, 1.0 μl dNTPs (2.0 mmol/l each), 2.0 μl MgCl2 (20 mmol/l), 1U Taq DNA polymerase, and 1 × PCR buffer. The reaction conditions were as follows: pre-denaturation at 94 o C for 5 min, 5 cycles at 94 o C for 1 min, 35 o C for 50 sec, 72 o C for 1 min, followed by an additional 30 cycles at 94 o C for 1 min, 50 o C for 50 sec, 72 m o C for 1 min, and a 10 min final extension at 72 o C. Amplified products were analyzed by electrophoresis on 1.5% agarose gels.

Estimation of Genomic Template Stability (GTS)
Changes in the SRAP profiles were expressed as GTS which qualitatively measures evident changes. GTS was calculated using the formula: where a is the average number of changes in DNA profiles and n is the number of bands in control DNA profiles. Polymorphisms in the SRAP profiles included the disappearance and appearance of new PCR bands, in comparison to control SRAP profiles. Averages were calculated for each test group exposed to different Al treatments [19]. For the comparison of the sensitivity of each parameter (SOD, CAT, MDA, POD and GTS), changes were calculated as a percentage of their control value (100%).

Statistical Analysis
All statistical analyses were carried out using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). One-way analysis of variance, Duncan's new multiple range method and Student's t test were used to perform multiple comparison of physiological indices.

Influence of Al Stress on Antioxidant Enzyme Activity of Barley Seedlings
SOD can remove contaminants and toxic material produced by cells [21]. At Al concentrations of 50 and 100 μM, SOD activity increased. When the concentration of Al reached 200 μM, SOD activity began to decline ( Fig. 1(A)). When the Al concentration was lower than 100 μM, SOD effectively eliminates oxygen free radicals. This represents plant defense responses, as plants under abiotic stress resist ROS induced stress and subsequent plant damage. SOD activity increases to remove ROS and reduce membrane lipid peroxidation. When the concentration of Al increased to 200 μM, SOD activity gradually decreased, but was active in comparison to controls. These results suggest that enhanced membrane lipid peroxidation and permeability leads to the collapse of antioxidant defenses when Al stress increases [22]. The POD antioxidant system in plants coordinates with SOD to maintain low levels of free radicals, thereby preventing toxicity [21]. The influence of Al on the activity of POD in barley is shown in Fig.  1(B). POD activity increased as the concentration and time of Al exposure increased, indicating that POD deoxygenates excessive H 2O2 to H2O and O2 in plants, remove damaging ROS.
CAT is an enzyme scavenger that promotes H2O2 decomposition into O2 and H2O, removing hydrogen peroxide in plants, and decreasing H2O2 toxicity. As shown in Fig. 1(C), when the concentration of Al increased to 50 μM, the activity of CAT increased. However, upon further increases in Al concentrations, CAT activity was inhibited. These results suggest that as the concentration of Al rises, H2O2 accumulates in barley cells to levels in which CAT is no longer effective, eventually leading to apoptotic induction.

Influence of Al Stress and MDA Content of Barley Seedlings
Under abiotic stress, plant membranes undergo lipid peroxidation. MDA is a final product of membrane lipid peroxidation and an important indicator of membrane damage. MDA is used as a measure of lipid peroxidation and can be used as a marker of plant stress conditions [23]. Under Al stress, MDA levels significantly change in barley ( Fig. 1(D)). MDA increases in a time and concentration dependent manner in response to Al ( Fig. 1(D)) suggesting that Al toxicity enhances lipid peroxidation.

Effect of Al Stress on SRAP Profiles of Root Genomes
SRAP-PCR analysis was performed on each pool of genomic DNAs extracted from control and Altreated roots. Each pool contained DNA from five plantlets to avoid intra-population genetic polymorphisms. In total, 72 10-mer priming oligonucleotides were used to analyze PCR products of which nine provided specific and stable data (Tab. 2). In all cases, the SRAP patterns generated by Alexposed plantlets clearly differed to those obtained of control DNA. The results from 9 of the primers are shown in Tab. 2. The principal events observed following Al exposure were variations in the normal banding patterns in comparison to normal control seedlings. We observed 16, 21, 22, 17, 21, and 23 mutated loci amplified by only nine primers from 50 to 200 μmol L -1 after 5 and 10 days, respectively. Al-treated plantlets were determined through the comparison of Al-treated SRAP fingerprints with controls (Tab. 2). DNA polymorphism values (SRPA strip increase or disappearance) of Al exposure after 5 days (GTS5) were 57.9%, 47.7% and 42.1%, respectively. The GTS10 was 55.2%, 44.7% and 39.5%, respectively (Fig. 2). These results implied that the genomic stability of barley seedling root tip cells is   Asterisks indicate significant differences at P < 0.05

Discussion
The inhibition of Al stress is crucial to plant growth and development. However, the mechanisms through which Al ions lead to root cell damage remain controversial [24]. It has been proposed that Al ions inhibit enzyme activity and DNA replication, inhibit cell transduction, lead to cell wall damage, block transportation processes at the cell surface, and disturb Ca 2+ balance [25].
The normal metabolism of oxygen in chloroplasts and mitochondria generates ROS, which is accelerated by the heavy metal content in the soil, and other environmental stresses [19,26]. ROS accumulation leads to lipid peroxidation and increased MDA levels [19,23]. In this study, the activity of SOD, POD and CAT increased at low Al concentrations, but high Al concentrations for an extended period of time led to the inhibition of these enzymes. Plants that were protected produced a large number of metabolites to minimize heavy metal poisoning. However, excessive metabolic activity leads to toxicity. This study showed that the activity of SOD and CAT significantly increased when Al concentrations increased from 50 to 100 μM. However, when the Al concentration increased to 200 μM, SOD activity decreased. POD activity increased with increasing Al stress. These results suggest that in conditions of low Al, the major threat to the health of barley plants was H2O2, consistent with previous studies. However, the activity of SOD and CAT decreased at Al concentrations of 200 μM and 50 μM. This may have led to the collapse of protective enzyme function in barley leaves.
Under Al stress, single strand breaks in DNA occur, leading to base pair modifications and a loss of DNA repair. DNA damage leads to mutations in DNA, apoptosis induction, and a variety of toxic sideeffects if insufficiently repaired. To date, RAPD has been used to detect the genetic toxicological effects of pollutant biomarkers [27]. However, RAPD technology is limited by low reproducibility and stability. SRAP is a new form of DNA fingerprinting based on PCR technology [28]. SRAP enabled the detection of ORFs permitting gene amplification in the absence of sequence information. SRAP is widely accepted due to its rapid and simple operation, low cost, good reliability, high reproducibility and ease of sequencing. SRAP has been successfully applied to plant genetic maps [18], and for the assessment of crop genetic diversity [29]. SRAP can detect DNA damage and mutations in response to environmental pollutants, characterized by DNA fragmentation. We assessed genome polymorphisms at the DNA level using SRAP, in which a series of random primers were used for amplification, allowing detection of the entire genome. In this study, the barley root genome SRAP bands were amplified under Al stress conditions and clear differences in the response to Al treatment were observed (Tab. 2). This might be related to DNA crosslinking (reduction in hyperchromic effect) under Al stress. Thus the priming sites of some oligonucleotides have a high affinity for primers, so the primers cannot bind to primer binding sites. This is the reason for the appearance of new SRAP bands. The SRAP marker is amplified by open reading frames (ORFs) using specifically designed primers and is very polymorphic. Thus it better reflects the changes in genes in response to Al toxicity. GTS dramatically declined under Al stress conditions. The differential band patterns in response to Al stress were likely due to genome rearrangements, point mutations, double chain fractures, DNA insertions/deletions, DNA-protein crosslinking, methylation, and other forms of DNA damage. In comparison with the molecular level of plants affected by heavy metals as reported in previous studies [9,10,19], the polymorphic changes were therefore more significant and reliable, and DNA polymorphisms therefore act as biomarkers for the detection of Al pollution in plants.
In summary, the physiological and biochemical changes in barley seedlings under Al stress correlated with the SRAP profiles. This was the first study to employ SRAP for the assessment of DNA damage in barley roots under Al stress. SRAP could detect plant damage more rapidly than other physiological and biochemical indicators including germination rates, chlorophyll content, and enzyme activity. SRAP in combination with plant physiology thus provides a rapid and reliable method to analyze DNA damage in response to environmental stress. These methods represent an effective tool to detect genetic toxicity in response to Al pollution. This enhances our understanding of the plant defense mechanisms employed by barley in response to Al toxicity.