Mutation Research/Genetic Toxicology and Environmental Mutagenesis
Molecular instability induced by aluminum stress in Plantago species
Introduction
Soil contains an average of 7% total aluminum (Al), which represents the third most abundant element in the earth's crust [1], [2]. Al constitutes a phytotoxic barrier to crop production and its toxicity is one of the major factors that limit plant growth and productivity in acid soils [1], [3], [4]. Acid soils account for 30–40% of the Word's arable land and are spreading due to natural, agricultural and industrial processes [5]. Although Al naturally exists as an insoluble silicate and oxide at neutral or weakly acid pH, the problem becomes particularly severe when the soil pH drops below 5.0 because Al is solubilized into the most phytotoxic form Al3+.
Al rapidly affects a number of cellular processes in the root apex, which is considered to be the primary target of Al stress, causing an inhibition of elongation and lateral root formation as the first symptoms of Al toxicity in plants [6], [7]. This response indicates that Al quickly disrupts root cell elongation, which leads to the decrease in water and nutrient uptake, and eventually to limited growth of the whole plant due to cell division inhibition [8], [9].
Al also affects a wide range of physiological processes. At cellular level, the strong binding affinity of Al with oxygen donor ligands, such as proteins, nucleic acids and phospholipids, results in the inhibition of cell division, cell extension and transport, interfering with cytoskeleton structure and function, phosphorus metabolism and disruption of calcium homeostasis [10]. At the molecular level, Al stress causes major changes in the expression patterns of important oxidative stress responding genes, which may cause damage to cellular components if antioxidant defenses are overwhelmed [10].
To prevent or avoid Al toxicity, some plant species evolved and developed the ability to exclude Al from plant tissue (Al excluders), or to tolerate high internal Al concentrations (Al accumulators), allowing them to grow in acid soils with a high concentration of Al3+. The first mechanism involves the secretion through the root apices of Al-chelating substances, such as organic acid anions, that can form stable complexes with Al which are less phytotoxic than the ionic form, Al3+ (Al resistance). The other one involves an organic-mediated mechanism to detoxify Al internally through Al3+ accumulation at the simplest either by Al cheating in the cytosol to form harmless complexes on by sequestering it into organelles (Al tolerance) [11], [12].
Despite the considerable progress made in deciphering the physiological and genetic basis of Al toxicity and resistance/tolerance, the understanding of the molecular mechanisms underlying, these processes still remain limited.
The assessment of genotoxic effects of metals in plants, such as cadmium, copper, lead, Al and other metals is an important topic in environmental research and has been paid increasing attention to this field in the last years [13], [14], [15], [16]. Plants are ideal systems for genotoxicity assays and have been used as a bioindicator to evaluate the toxic effects of pollutants, once they could overcome environmental stress by developing efficient and specific physio-biochemical mechanisms, thus providing vital information from the standpoint of safeguarding biodiversity and ecological resources [17], [18].
Several studies have been using the comet assay to detect DNA degradation and micronucleus assay or chromosome aberration assay to measure the genotoxic effect of metals on different plants species, namely in lupin [19], tobacco [20] and onion [21]. However, these assays are limited by their relatively low sensitivity and low ability to provide information on the effects of toxic metals at the DNA level [22].
Advances in molecular biology have led to the development of selective and sensitive DNA analysis methods [14], making the study of the mechanisms involving metal-induced genotoxicity easier and more accessible [16]. Despite the existence of a large variety of DNA molecular markers that can be used to assess the genotoxicity, the analysis of Random Amplified Polymorphic DNA (RAPD) has been successfully used to evaluate the mutagenic effects of several metals, namely in rice [22], barley [14], common bean [23] and eggplant [15]. However, Inter Simple Sequence Repeat (ISSR) assay could be more advantageous since it is described as a more reproducible technique. This molecular marker becomes of great importance since the detection of genotoxic effects involves the comparison of profiles generated from control (unexposed) and treated (exposed) plants.
The current study was designed to detect the impact of Al contamination on two Plantago species, Plantago almogravensis Franco and Plantago lagopus L. in terms of DNA damage, using ISSR molecular markers. The genus Plantago belongs to the family Plantaginaceae and has approximately 483 species [24]. At the moment, there are no DNA sequences databases available and are scarce molecular studies in both species. P. almogravensis is endemic from Portugal where only one population is known and was described as the only Al-hyperaccumulating species of the Plantaginaceae family [25]. On the other hand, P. lagopus has a wide distribution and its usage as a traditional medicine is mainly due to the antioxidant activity of its extract [26]. The presence of an apparent B chromosome in P. lagopus (2n = 12) was reported by Dhar et al. [27]. Later on, in 2014, this supernumerary chromosome was described as genetically inert, with a heterochromatic structure, rich in repetitive DNA and transposable elements [28]. However, this species is not known to possess Al-hyperaccumulation activity despite showing tolerance to certain herbicides [29]. Both species can have a potential application for phytoremediation. However, this application of P. almogravensis can be limited due to the fact that this species is presently in risk of global extinction and protected under the European Habitats Directive and the Portuguese law. For this reason, currently, these plant species are being conserved through micropropagation [30]. The main goal of this study was to evaluate the genetic instability induced by Al stress in roots and leaves of both species using ISSR markers.
Section snippets
Plant material and Al treatment
At least 1000 seeds were randomly collected from 100 individuals of P. almogravensis and P. lagopus (50 of each species) in wild populations growing in Odemira (Portuguese Southwest coast) and in the center of Algarve, respectively. These were combined by species and a total of 40 seeds, 20 from each species, were randomly selected and germinated in vitro, and the shoots obtained multiplied in MS medium [31] containing 0.2 mg L−1 6-benzyladenine [30]. Plantlets were obtained by cultivating the
Results and discussion
Metal toxicity in plants is described as a cause of various damages in growth and development processes such as photosynthesis, lipid metabolism and nucleic acid biosynthesis. However, some plant species have developed the ability to tolerate metals in their organs. These plants have attracted much attention for phytoremediation purposes [9], [38], [39].
In the present study we report the application of ISSR markers analysis for the assessment of Al genotoxicity in P. almogravensis and P. lagopus
Conflict of interest
None.
Acknowledgements
This work was funded by a research grant from the Portuguese Foundation for Science & Technology (PTDC/AGR-AAM/102664/2008); FCT supported the doctoral fellowship of Neusa Martins (SFRH/BD/48379/2008) and the postdoctoral fellowship of Sandra Gonçalves (SFRH/BPD/84112/2012).
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