ALS1 encodes a half type ABC transporter that was localized to the vacuolar membrane of root tip cells, suggesting that it may be important for compartmentalization of internalized Al . Map based cloning revealed that the als1-1 mutation is a single amino acid substitution in this previously uncharacterized half type ABC transporter. It contains two distinct domains: the N-terminal domain contains at least four predicted transmembrane regions and appears to have a structure typical of the membrane spanning regions of ABC transporters and the C-terminal domain has all the required motifs associated with functional ATPases of ABC transporters . ALS1 also has significant homology to two distinct ABC transporters with different functions. ALS1 is homologous to mammalian TAP-type transporters associated with the endoplasmic reticulum to move short polypeptides for antigen processing. ALS1 is also homologous to yeast mitochondrial-localized subfamily B ABC transporter, Atm1p, and vacuolar localized Ycf1p. Atm1p is an essential component in the mitochondrial export of Fe/S clusters to the cytoplasm, indicating that it is essential for iron homeostasis in yeast. Ycf1p is required for movement of cadmium-glutathione into the yeast vacuole and loss of Ycf1p results in cadmium hypersensitivity. It is possible that ALS1 functions similarly to Atm1p and Ycf1p, thus transporting a metal complex in Arabidopsis . It was determined that ALS1 is primarily limited to vascular tissue in the roots, leaves, stems and flowers,bucket flower with high activity throughout the distal portion of the root tip. High GUS activity was also detected in the hydathodes and the area surrounding the water pore. ALS1 was found to be exclusively localized to the vacuolar membrane using ALS1 fused to GFP .
It was proposed that ALS1 is potentially transporting a metal complex from the cytosol to the vacuole of the cell, similar to two yeast homologs Atm1p and Ycf1p. This would allow sequestration of Al into the vacuole and remove interaction of the Al cation within the cytosol. Due to this activity, it is possible that ALS3 loads and unloads Al from the phloem for movement of Al to less sensitive cells, while ALS1 transports intracellular Al from the cytosol to the vacuole for sequestration. Mutation of either of these two factors can lead to inappropriate accumulation of Al in sensitive tissues within the plant . ALS7/SLOWWALKER encodes a transcription factor that among other things is required for regulation of expression of genes whose products participated in the production of polyamines such as spermine . Since als7-1 has Al hypersensitive roots, it is expected that reduced production of polyamines lowers the protective effect of these multi-charged cations because of reduced capacity to compete with Al3+ for binding to anionic sites within the root tip. Anionic targets of Al3+ are expected to include negative charges in the plant cell wall as well as symplastic targets such as genomic DNA, which directly binds to polyamines such as spermine and spermidine . Aluminum resistantmutations have been identified by using EMS mutagenized Arabidopsis seedlings that had enhanced root growth on a phytotoxic level of AlCl3. From this screen, five lines were explored further in depth. Four of these lines were mapped to a similar location and all had increased organic acid release of malate and/or citrate. The fifth line did not have enhanced organic acidrelease, suggesting the resistance is conferred in a manner other than organic acid release, which has already been described previously. Unfortunately, fine scale mapping could not be accomplished due to the difficultly of identifying a population that had a clear Al resistant phenotype .
Since als3-1 has such a profound phenotype that is specific to Al stress, it presented a unique opportunity to use this mutation to help identify factors that confer increased Al tolerance and/or resistance using a suppressor mutagenesis approach. EMS chemical mutagenesis on als3-1 was performed and plants that masked als3-1 hypersensitivity on low levels of Al were isolated. The als3-1 suppressor lines represent mutations in factors that confer increased Al resistance and/or tolerance. By using this method, 12 strong suppressor mutants were identified, and 3 were initially chosen for further analysis. All three gave similar phenotypes and all mapped to the same region. Therefore only one of those lines was studied further, alt1-1. When the alt1- 1;als3-1 suppressor mutant was grown in the presence of increasing concentrations of AlCl3, its growth not only reversed AlCl3 hypersensitivity of als3-1 but it also had increased root growth compared to Col-0 wt in higher levels of AlCl3. It was found that alt1-1 is a mutation that does not exhibit any enhancement in Al exclusion, due to callose accumulation and Al content of alt1-1 being similar to wild type . Through map-based clonging, alt1-1was discovered to be a loss of function mutation in ATAXIA TELANGEICTASIA MUTATED AND RAD3-RELATED. In higher eukaryotes, ATR encodes a cell cyclecheckpoint that senses DNA damage as part of a signal transduction pathway capable of halting the cell cycle in order to repair damaged foci . In Arabidopsis, ATR is known to signal repair of persistence of single-stranded DNA, single-stranded DNA breaks, replication fork stalls, and cross links . Alt1-1 confers a single amino acid substitution of G1098E in the highly conserved yet uncharacterized UME domain, which is speculated to function in protein-protein interactions. A second alt allele, alt1-2 was also the results of a point mutation in ATR a L2553F substitution in the conserved phosphatidylinositol 3- and 4-kinase domain of ATR . Both of these dominant-negative mutant alleles were originally found because of their capability to suppress the hypersensitivity phenotype of als3-1 .
Further work has shown that a T- DNA insertion allele, atr-2, is also capable of fully suppressing als3- 1, thus supporting the argument that alt1-1and alt1-2 are mutations that reduce the function of ATR even though they are dominant . This is particularly evident when one considers the response of atr-2 and atr- 4 to other DNA-damage agents, which only adds to the conundrum of why Al activates this ATR-dependent pathway. While these mutants are Al tolerant, as demonstrated by their capacity to maintain root growth due to failure to arrest cell cycle progression and force QC differentiation, atr-2 and atr-4 roots exposed to different DNA-damage agents such as the replication fork poison hydroxyurea and the DNA cross linkers cisplatin and Mitomycin C exhibit extreme sensitivity . From these results, ATR isabsolutely necessary to repair stalled replication forks as well as DNA crosslinks yet loss of this repair factor confers Al tolerance. It is difficult to reconcile increased Al tolerance with loss of such a key DNA-damage response factor, although it could be argued that Al may bind to DNA in a manner similar to covalent crosslinkers but without the extreme detrimental effects of these cross linkers. This argument is based on the likelihood that Al would interact with DNA electrostatically in a reversible manner, with binding likely holding DNA in an unfavorable conformation that subsequently is perceived as a replication fork stall by ATR. Interestingly, even though treatment with Al has been shown to result in DSBs and micronuclei , a loss-of-function atm mutant was incapable of suppressing the Al hypersensitivity phenotype of als3-1suggesting that DSBs are not important to activating the DNA-damage checkpoint following Al treatment. The second als3-1 suppressor mutant identified is alt2-1, which is a loss-of function mutation in the cell cycle checkpoint TANMEI/ALT2. ALT2 encodes a WD- 40 motif containing protein homologous to an integral component of the mechanism required for response to DNA damage, AtCSA,cut flower bucket which is part of the DNA- damage binding machinery . WD-40 proteins are commonly found in many different biochemical pathways, serving as scaffolds for protein complexes. In some cases, such proteins participate in SCF ubiquitin ligase complexes in order to tag target proteins for degradation. Such a role for ALT2 in response to Al is consistent with the observation that CULLIN4, which is a key component of SCF ubiquitin ligases, interacts directly with DWD motif containing proteins . Such a function is distinctly different from what was previously described for human CSA, which functions in TCNER to monitor for conformational changes in DNA as assessed by blockage of transcription . Cooperation of a WD-40 motif containing protein with CULLIN4 is characteristic of Global Genome Nucleotide Excision repair and is independent of RNA polymerase II . At this point, it is unclear in which if either NER response pathway ALT2 may participate although the potential linkage of ALT2 to either is consistent with Al acting as a genotoxin. Like atr-2 and atr-4, alt2-1 falls into the same conundrum since it fails to halt the cell cycle and force differentiation of QC in the presence of a normally inhibitory concentration of Al yet it is highly sensitive to DNA cross linkers .
A double mutant representing the loss-of-function of both ATR and ALT2 showed no additive Al tolerance compared with the single mutants, suggesting that ATR and ALT2 act together to detect Al-dependent damage and actively halt root growth. Interestingly, alt2-1 does not exhibit hypersensitivity to the replication fork poison HU while it does show extreme sensitivity to CDDP and MMC, which is consistent with it being a key regulator of cell cycle progression following exposure to DNA-damage agents . Therefore, at this point the genotoxic nature of Al has yet to be defined. However, it is clear from the unbiased als3-1 suppressor screens, in which DNA damage response factors were identified as Al tolerance mutations, an ATR- and ALT2-dependent cell cycle checkpoint pathway is key to stoppage of root growth and promotion of terminal differentiation following Al treatment. Based on the results with ATR and ALT2, one could argue that Al-dependent DNA damage is a critical determinant of root growth inhibition, yet the effects of Al on the nucleus are just beginning to be elucidated. Al rapidly accumulates to high levels in root meristem nuclei and is especially concentrated around interphase chromatin as well as mitotic figures . It is hypothesized that Al binds to the phosphate backbone of DNA , which would be expected to result from an electrostatic attraction of Al 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. Binding of Al to DNA or chromatin could condense DNA molecules and inhibit cell division by reducing its capacity to provide a viable template for replication, mitotically relevant transcriptional events, and even proper DNA separation . It is not unreasonable to predict that such conformational changes could be perceived by ATR as being deleterious to replication fork progression, thus activating this cell cycle checkpoint. It should be noted that chromosomal aberrations resulting in DNA breakage and intra-strand cross linking are a reported consequence of chronic exposure to Al that lead to micro-nuclei formation although it is not clear how relevant these are to stoppage of root growth since ATM does not appear to have a role in this process . Clearly, further studies are necessary to define if and how Al interacts with genomic DNA, especially since it is the Al3+ species that is predicted to bind to the negatively charged DNA backbone, yet intracellular pH is not expected to favor the formation of this species. In the end, utilizing Al toxicity as a real world model to study DNA damage in plant systems presents a novel system to study DNA damage response without the use of rare chemotherapy drugs or types of radiation not found in earthly environments. Ultimately, 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. This certainly begs the question—if atr and alt2 mutant roots can maintain root growth even in the presence of Al, what actual Al-dependent damage is detected by these cell cycle checkpoints? It cannot be ruled out that inappropriate activation of the cell cycle checkpoint machinery and the repair mechanisms that they regulate may actually be the cause of the damage such as DSBs and micro-nuclei. Consequently, one explanation could be that failure to activate this pathway prevents the program-dependent accumulation of the damage and results in roots that can grow in normally inhibitory levels of Al.