Consistent with prior results, it was found that Al treatment results in loss of the QC as measured by QC46 dependent GUS activity that is localized to the root stem cells . For this analysis, QC46:GUS transgenic seedlings in either the Col-0 wild type or suv2-3 backgrounds were grown for 7 days in the absence or presence of 1.50 mM AlCl3 in a soaked gel environment, after which seedlings were stained to visualize the QC. Both Col-0 wild-type and suv2-3 roots had QC46:GUS accumulation in the absence of Al . Treatment with high levels of Al resulted in loss of the QC in Col-0 wild type but not suv2-3 . This indicates that SUV2 plays an active role in differentiation of the QC following Al treatment likely as a step in the transition to endore duplication in the root tip. It was found that Al treatment leads to terminal differentiation in conjunction with substantial increases in cell and nucleus size in als3-1 roots but not in atr-4;als3-1, alt2-1;als3-1, and sog1-7;als3-1 roots . For this analysis, Col-0 wild- type, als3-1, and suv2-3;als3-1 plants were grown in the absence or presence of 0.75 mM AlCl3 in a soaked gel environment for 7 days, after which seedlings were fixed and stained with 4’,6-diamidino-2-phenylindole . Root tips were subsequently visualized using confocal microscopy at the same magnification for each. Al treatment results in substantial increases in both cell and nuclear size for als3-1 roots,plastic square flower bucket which is consistent with terminal differentiation in conjunction with endore duplication . In contrast, suv2-3;als3-1 roots did not show the dramatic Al-dependent increases in cell and nuclear size as seen for Al-treated als3-1 roots .

This shows that all als3-1 suppressor mutants, atr- 4, alt2-1, sog1-7 and suv2-3, block the Al hypersensitivity of als3-1 in conjunction with prevention of terminal differentiation and endore duplication. It was of interest to determine whether SUV2 is part of the transcriptional response pathway that leads to this root growth inhibition, as do the Al tolerant cell cycle checkpoints ATR, ALT2, and SOG1. The conditions that would allow for the best capture of these changes elicited by these cell cycle check points was determined to be 3 days after germination on 1.50 mM AlCl3 in a soaked gel environment when SOG1 is still present at the root tip and the QC has not yet differentiated . Because of this, seedling tissue was collected after 3 days of exposure to Al to assess whether Al causes upregulation of genes in a similar SOG1-dependent manner as seen for γ-radiation. For this experiment, Col-0 wild-type, als3-1, suv2-3, suv2-3;als3-1, suv2-1 and suv2-1;als3-1 seedlings were grown in the absence or presence of 1.50 mM AlCl3 in a soaked gel environment for 3 days. Subsequently, whole seedlings were harvested for isolation of total RNA, cDNA synthesis, and subsequent RT-PCR analysis. Several genes that have been found in a previous study to be highly induced by γ-radiation in a SOG1-dependent manner were used to perform a survey with regard to Al response . For this experimental approach, EF-1α was chosen as the normalization gene, which is expressed consistently in different genetic lines in the absence and presence of Al . Genes tested were those that were shown to be inducible following Al in an ATR-, ALT2-, and SOG1-dependent manner: a Zn finger of unknown function , a protein with an unknown role in DNA damage repair , a putative ubiquitin conjugating enzyme , a putative telomere maintenance protein , an ortholog of the human breast cancer susceptibility gene , and a key component of microhomology-mediated DNA repair .

Primers for these genes were generated and tested for parameters essential to accurate RT-PCR analysis including single amplicon replication verified via melts curves and amplification efficiencies .Consistent with the previous work on Al inducible DNA damage transcriptional response , treatment with Al resulted in a measurable increase in expression of this subset of genes in Col-0 wild type and in Al-treated als3-1 seedling . In contrast, increased in expression of these genes was not observed for suv2-3, suv2- 3;als3-1, suv2-1 and suv2-3;als3-1 roots in comparison to the respective controls . This result indicates that SUV2 functions as part of the Al inducible DNA damage transcriptional response that is similar to that observed following treatment with γ-radiation. Canonically, DNA damage checkpoints are responsible for the arrest of the cell cycle with concomitant induction of DNA repair mechanisms following DNA damage. Roots of atr, alt2, and sog1-7 loss-of-function seedlings have been observed to be sensitive to the DNA damage agents, hydroxyurea , Mitomycin C and cisplatin , thus it was of interest to determine whether suv2-3 seedlings followed canon. Seedlings of Col-0 wild type and suv2-3 were grown in a myriad of DNA damage agent containing medias for 7 days and their roots were subsequently measured . HU is a replication fork poison, as it reduces available pools of dNTPs by inhibiting ribonucleotide reductase . Seedlings of Col-0 wild type and suv2- 3 grown in 0 or 1 mM HU in nutrient media and both exhibited root growth inhibition with suv2-3 being significantly more sensitive . MMC is an interstrand cross-linking agent , and CDDP is an intrastrand cross-linking agent . Roots of Col-0 wild type and suv2-3 grown in nutrient media containing 0 or 2.5 µM MMC exhibited inhibition; however, roots of suv2-3 were again significantly more sensitive to MMC than Col-0 wild type .

Similarly, seedlings of Col-0 wild type and suv2-3 grown in 0 or 10 µM CDDP in nutrient media and yet again roots of suv2-3 demonstrated greater sensitivity than roots of Col-0 wild type .Since loss-of-function mutants for SUV2 and ATR are similar with regard to their roles responding to Al toxicity as well as other DNA damage agents , it might be expected that these two cell cycle checkpoint factors act together to trigger Al-dependent terminal differentiation of the root. In order to test whether there is a relationship between these two factors in Al-dependent stoppage of root growth, a suv2-3;atr-4 mutant was generated and tested for its capability to grow in the presence of Al. For this experiment, Col-0 wild type, suv2-3, atr-4, and suv2-3;atr-4 were grown for 7 days in the absence or presence of 1.50 mM AlCl3 in a soaked gel environment. Root lengths were measured and the suv2-3;atr-4 double mutant was comparable to suv2-3 and atr-4 for Al tolerance . This result suggests that SUV2 and ATR are part of the same pathway that halts root growth following exposure to Al. SUV2/HUS2 is a homologue of ATR interacting protein from vertebrates, Lcd1 from S.cerevisiae, and Rad26 from S. pombe. These all exhibit evolutionary conservation as DNA binding proteins that partner with ATR/Mec1/Rad3 and recruit their respective kinase partner to sites of DNA damage . Along with this conserved, functional relationship between homologues of ATR and its interacting partner, ATRIP, in a DNA damage response,plastic plant pot there is now also a relationship between ATR and SUV2 in Arabidopsis with regard to terminal differentiation of the root tip following Al exposure. It was of interest to determine whether SUV2 is a phosphorylation target of ATR as has been shown for SOG1 . For this analysis, the entire coding sequence of Arabidopsis ATR representing 2702 amino acids was produced as a GST fusion protein in a baculovirus protein expression system. In conjunction with this, the entire CDS of Arabidopsis SUV2, representing 646 amino acids, was produced as a Maltose Binding Protein fusion in an Escherichia coli BL21-DE3 pLysS protein expression system. Approximately 50 ng of GST-ATR was subsequently incubated with 1 mg of either MBP or MBP-SUV2 in the presence of [γ – 32P] ATP, after which samples were separated on an SDS-PAGE gel without ATR-GST. Phosphorylation results were visualized by autoradiography. While incubation of MBP with GST-ATR did not result in measurable phosphorylation of MBP, in contrast, MBP-SUV2 with GST-ATR resulted in a distinct radio labeled band that was the same size as that predicted for MBP-SUV2 . At least in vitro, SUV2 is a phosphorylation target of the Arabidopsis ATR kinase.

Al toxicity poses a significant agricultural problem in acid soil regions, especially in countries lacking industrialized methods to overcome the stress resulting from this metal ion, which reduces plant growth and moreover crop yields . Research investigating Al tolerance mechanisms in plants seeks to address this problem in hopes of overcoming the agricultural challenge in the development of Al tolerant crop plants. This has the potential to alleviate this reduced crop growth on Al toxic soils, thus holding the promise to improve the capability to feed an ever-expanding global population. The previously studied and recently introduced als3-1 suppressor mutations represent four Al tolerance genes, ATR, ALT2, SOG1 and SUV2, which all hold the potential to be introduced into crop species as their mutant alleles in order to augment the basal level of Al tolerance. Further research is needed to determine if these candidate genes are actually appropriate for conferring Al tolerance into crop species like barely or rice since even further research is needed to determine the effects of Al consumption in humans. Besides their capabilities to confer Al tolerance, these als3-1 suppressor mutations represent four genes involved the plant DNA damage response mechanism that responds to agents that result in the stalling of the replication fork. Identification of these four factors that have previously established roles in DNA damage responses suggests that a primary effect of Al toxicity is directly related to compromised genomic integrity. Whether the genotoxicity of Al is real or perceived, plants have evolved a DNA damage response to internalized Al. The previously reported Al tolerance DNA damage response genes are ATR and ALT2. The first factor reported was ATR, which encodes a PIKK family kinase that is a cell cycle checkpoint responsible for detection and responding to DNA damage by arresting the cell cycle and activating repair mechanisms . This gene along with its related PIKK factor ATM are highly conserved in all eukaryotes. While both these related gene products play partially overlapping roles in the DNA damage response, there is a clear division of function between ATR and ATM in response to Al . Loss-of function atr mutants, while tolerant to Al stress, are hypersensitive to the replication fork poison HU . In contrast, atm loss-of-function mutants do not present Al tolerance. The second mutation reportedly capable of suppressing the als3-1 sensitivity phenotype was found in ALT2, which encodes a protein homologous to Arabidopsis Cockayne Syndrome type A protein . AtCSA has been described as having a DDB1-binding WD-40 motif and is integral to the mechanism required for detection of UV-B-dependent DNA damage . Together, ATR and ALT2 have been shown to function in a non-additive manner to detect Al-dependent damage and actively halt root growth . Much like atr-4, the alt2-1 mutant, while tolerant to Al stress, is hypersensitive to DNA cross linking agents like CDDP and MMC . As described in Chapter 4, a third mutation was isolated using this unbiased mutagenesis screen for als3-1 suppressors. sog1-7 was identified as a single nucleotide change in SOG1, a gene that encodes a NAC domain family transcription factor responsible for initiation of a transcriptional response leading to endore duplication following exposure to Al. This previously described DNA damage response related transcription factor, which has been described as a direct effector of ATM, showed no evidence of acting in conjunction with its established kinase activator in the presence of Al. Instead, results show that SOG1 acts almost exclusively with ATR to halt root growth in the presence of Al. Therefore, both cell cycle arrest and differentiation of the QC are dependent on SOG1 following Al exposure and that these responses ultimately lead to endore duplication as a means for terminal differentiation of the root meristem following Al treatment. Thissue- and cell-specific localization of SOG1 was shown to be within the nucleus of actively dividing cells in the root tip. While it localizes throughout the meristematic regions of the root tip in the absence of Al, there is a progressive loss of SOG1expression when grown in an Al toxic environment, which is lost entirely once the root tip is terminally differentiated.