Studies in yeasts and animal cells suggest that RPA-coating of single stranded DNA act as a signal to activate ATR dependent downstream phosphorylation, primarily through an associated protein called ATRIP . SUV2 is required for repair of UV induced damage, as is its namesake, and suv2 mutants are also sensitive to HU, MMC and CDDP. In sum, ATR, ALT2, SOG1 and SUV2 are all required for DNA damage response as a result of replication fork stalls. This requirement of ATR, ALT2, SOG1 and SUV2 for a plant’s survival in the response to specific genotoxins as seen with HU, MMC and CDDP heightens the curiosity as to what the true nature of Al’s impact on DNA is. As the sensitivity of atr,alt2, sog1 and suv2 mutant roots to defined genotoxins demonstrates, loss of these factors should lead to sensitivity to Al rather than tolerance if Al directly causes DNA damage. It seems counter intuitive that a plant gains Al tolerance by reducing the function of factors necessary for DNA damage response. If atr, alt2, sog1 and suv2 mutant roots can maintain root growth even in the presence of Al, what actual damage is detected by ATR, ALT2, SOG1 and SUV2 in the presence of Al? Identification of four factors that have clear roles in DNA damage responses suggests that a primary effect of Al toxicity is directly related to compromised genomic integrity, with Al possibly serving as a genotoxic agent, whether real or perceived. It is curious that loss of any one cell cycle checkpoint results in increased tolerance to Al rather than sensitivity as is observed with known genotoxic agents like HU, MMC and CDDP. This may suggest that these checkpoints are either so sensitive that even the limited amount of genomic stress that might directly or indirectly occur with Al could activate these factors yet in reality be relatively inconsequential to growth, or that Al is inappropriately perceived as a genotoxic agent by ATR, ALT2, SOG1 and SUV2. Based on the current findings on SOG1 and SUV2 in conjunction with the previous reports on ATR and ALT2,plastic nursery plant pot a DNA damage response is the primary cause of Al dependent root growth inhibition in Arabidopsis.
What kind of Al dependent DNA damage these factors are detecting is still unknown; however, in concurrence with research from the fields of Al toxicity and DNA damage responses, there could be a multitude of sources of this damage, including but not limited to: Reactive Oxygen Species, phosphate deficiency leading to dNTP depletion, competition with Mg2+ causing ATP depletion and enzymatic dysfunction, and topological strain affecting replication fork stalls or reduced transcriptional capabilities.Chronic high levels of Al exposure have been shown to result in peroxidation of lipids within the membranes of cells . Lipid peroxidation is likely a downstream result of Al damage, perhaps caused by Reactive Oxygen Species known to be generated by Al . ROS can cause DNA damage, where damage to individual bases may also be implicated, and could be tested for in a comet assay. In peripheral blood lymphocytes treated with Al, a high incidence of oxidized bases, particularly purines and apurinic/apyramidinic sites, were attributed to Al-generated ROS . In the human genome such base lesions are indeed repaired by ATR-mediated nucleotide excision repair . In Arabidopsis, over expression of a variety of factors in the antioxidant pathway have resulted in increased Al tolerance in Arabidopsis such as glutathione S-transferase and peroxidase . However, a loss-of-function mutant of At4g10500, an uncharacterized member of the 2-oxoglutarate and Fe-dependent oxygenase super family, tested for possible scavenging of ROS in response to Al exposure did not show any phenotypic changes in response to Al . Additionally, if Al-generated ROS were indeed primarily responsible for root growth inhibition, theloss-of-function mutants for DNA damage response factors would result in heightened sensitivity rather than tolerance to Al. Unrepaired damage caused by ROS leads to oxidative damage of lipids, amino acids, and DNA which can lead to cell death. Although ROS is likely a detrimental symptom of Al exposure, as Al toxicity is a complex and widely destructive biological assault, it is unlikely that ATR, ALT2, SOG1, and SUV2 are detecting damage caused by ROS.Al toxicity and phosphate deficiency typically coexist due to acid soil conditions that promote Al bio-availability while simultaneously reduce Pi uptake by the roots , and ALS3 has been identified as a required factor in a Pi starvation response in a sucrose-dependent manner . While Pi deficiency has many varied symptoms as phosphorous is required for photophosphorylation, genetic metabolism, transportation of nutrients, and phospholipid composition of cell membranes , it has dire effects on DNA replication as massive quantities of Pi are needed in the form of dNTP’s that are polymerized in order to form DNA as well as in the ATP consumed in the polymerization reaction. dNTP depletion caused by Pi deficiency would cause replication fork stalls, and lead to replication catastrophe, much like HU treatment.
A Pi starvation growth assay where seedlings of Col-0 wild type, als3-1, and atr- 4;als3-1 could be tested for Pi starvation responses. If atr-4 is capable of suppressing the als3-1 phosphate sensitivity, this could resolve whether or not Pi deficiency plays a predominant role in the Al-dependent damage response. However, damage caused by Pi deficiency would logically result in heightened Al sensitivity rather than tolerance as is observed for the loss-of-function mutants for ATR, ALT2, SOG1 and SUV2. Alternatively, with sucrose being a required factor for Pi sensitivity in als3-1, an Al growth assay where seedlings of Col-0 wild type and als3-1 are grown in the absence of sucrose with increasing amounts of AlCl3 could assess the ALS3-dependent PI starvation response. If Al toxicity is actually the result of Pi starvation, als3-1 should grow similarly to Col-0 wild type in the absence of sucrose, rather than a hypersensitive response to Al. It seems highly improbable given that Al exposure forces terminal differentiation through means of endore duplication, requiring rounds of DNA replication, as possible mechanism to inhibit root growth caused by Pi deficiency. Al3+ ions complete with magnesium ions for binding sites on the plasma membrane and decrease the uptake of magnesium into the root. Increasing concentrations of available magnesium in soil or nutrient media or over expressing magnesium transport genes enhance Al resistance as increased magnesium released into the rhizosphere competes with Al. Magnesium is the predominant ionic regulator of metabolism, largely through its role as a cofactor for all phosphoryl transfers in the cell.It also acts as a second messenger for growth factors in regulation of protein synthesis and is required to maintain genomic stability. Besides its stabilizing effect on DNA and chromatin structure, magnesium is an essential cofactor in almost all enzymatic systems involved in DNA processing. Most obvious in DNA replication, its function is not only charge-related, but very specific with respect to the high fidelity of DNA synthesis . Furthermore, as an essential cofactor in nucleotide excision repair, base excision repair and mismatch repair, magnesium is required for the removal of DNA damage generated by environmental mutagens, endogenous processes, and DNA replication . More studies are warranted to study how Al interferes with the function of magnesium in plants under Al toxic conditions. Al growth responses in the presence of excess magnesium could be tested for atr-4;als3-1, alt2;als3-1, sog1-7;als3-1, and suv2;als3-1 in comparison with Col-0 wild type and als3-1 to determine if magnesium supplementation can alleviate the effects of Al toxicity.
This test would likely be indicative of Al resistance, showing magnesium outcompeting Al for entry into the cells of the root. If Al interferes with DNA replication machinery,seedling starter pot in vitro investigations such as PCR assays could be tested for amplicon lengths in the presence of Al to measure processivity, as such assays has been demonstrated in yeast . Such assays could be performed with commercially available DNA ploymerases, as Arabidopsis polymerase enzymes are not readily available.The effects of Al at the nuclear level are poorly understood. Al rapidly accumulates at high levels in root meristem nuclei and is especially concentrated around interphase chromatin as well as mitotic figures . Al does not appear to have a base composition preference and it is likely that this lack of base discrimination is due Al binding to the phosphate backbone of DNA . This could result from an electrostatic attraction of Al3+ to the negative charges of the phosphodiester bonds. Such an association could increase the rigidity of euchromatin and relax supercoiled heterochromatin destabilizing genome topology through an ever-fluxing torsional tug-of-war. DNA gyrases and topoisomerases regulate topological strains such as supercoiled and relaxed DNA, especially caused by replisome progression, and are required to prevent replication fork stalls caused by supercoiled DNA in front of unwinding . Loss-of-function mutants of topI or topII could be tested for growth responses to Al. Perhaps in reality, overexpression mutants of a topoisomerase would be capable of counteracting the strain Al exerts on DNA and should be generated and tested. However, despite the effectiveness of topoisomerases to alleviate replication induced topological strains, their functions would not rid the nucleus of Al3+ and would likely be unable to ameliorate the affect of Al on the whole genome. Such a topological strain caused by binding of Al to DNA or chromatin could condense DNA molecules and inhibit cell division by reducing its capacity to provide proper DNA separation as is necessary for DNA replication and mitotically relevant transcriptional events . Others have shown that Al causes DNA compaction, as well as compaction of chromatin, potentially through inhibition of unwinding of genomic DNA since Al3+ will raise the Tm of the double helix . Al has been shown to precipitate DNA out of solution in vitro, especially templates normally found in transcriptionally active euchromatin, extending the implication that Al compaction could lead to transcriptional repression. Importantly, many of these gene-silencing effects can be explained due to the extraordinary charge density of the Al ion, and perhaps this alone is reason enough to disrupt DNA processes that activate checkpoint responses. While gene silencing may indeed result from internalized Al, this is likely not a significant consequence of the effect of Al on DNA, as Al-inducible gene expression is a confirmed response to the internalization of Al . However, it is not unreasonable to predict that such conformational changes to the DNA could be perceived by ATR as being deleterious to replication fork progression. Such a topological strain on DNA caused by Al may cause a conformational change reminiscent of covalent crosslinkers, such as MMC and CDDP. Since Al3+ is expected to have high affinity for the negatively charged phosphodiester backbone of DNA and would presumably interact with this backbone differently than divalent cations and could cause an electrostatic interaction where Al acts as a non-covalent pseudo-crosslink. If Al acted as a pseudo-crosslinker that disrupts or restricts unwinding of DNA during processes such as DNA replication and RNA transcription, this may trigger ATR-dependent checkpoint activation, similar to a replication fork stall caused by a true DNA crosslink. As discussed, ATR, ALT2, SOG1 and SUV2 are all required to respond to DNA cross linking agents and all are linked to Al-dependent stoppage of root growth .Many uncertainties persist in regards to the nature of the unknown damage detected by ATR, ALT2, SOG1 and SUV2. It seems counter intuitive that a plant gains tolerance to an agent that causes DNA damage by reducing the function of a factor necessary for DNA-damage detection. Peculiarly, the genomic stability of Arabidopsis roots is secure enough to endore duplicate following Al exposure, indicating that unlike true DNA cross links, the effect of Al on the DNA is not severe enough to inhibit multiple rounds of DNA synthesis phases that would be required for the endore duplication cycle. Yet, visualization of micro-nuclei formation in cells of the root tip indicates that real breakage of chromosomes occurs following long term exposure to Al. Once more, it is perplexing that ATM is largely uninvolved with the Al response despite the consequent DNA breakage in the formation of micro-nuclei. Perhaps these factors force root growth inhibition in the presence of Al in order to prevent passage of minor damage to subsequent generations.