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, which is consistent with terminal differentiation in conjunction with endore duplication . In contrast, atr-4;als3-1, alt2-1;als3-1, and sog1- 7;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 three suppressor mutants block the Al hypersensitivity of als3-1 in conjunction with prevention of terminal differentiation and endore duplication.Since loss-of-function mutants for SOG1 and ATR are phenotypically similar with regard to Al tolerance , 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 sog1-7;atr-4 mutant was generated and tested for its capability to grow in the presence of Al. For this experiment, Col-0 wild type, sog1-7, atr-4, and sog1-7;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 sog1-7;atr-4 double mutant was comparable to sog1-7 and atr-4 for Al tolerance . This result suggests that SOG1 and ATR are part of the same pathway that halts root growth following exposure to Al.
Al toxicity is most pronounced at the root tip ,square flower bucket and since SOG1 works in conjunction with other factors to shut down root grown in the presence of Al, it was of interest to determine the tissue localization pattern for SOG1. For this analysis, a previously reported transgenic Arabidopsis line carrying a SOG1:GUS fusion construct was grown in the absence or presence of 1.50 mM AlCl3 in a soaked gel environment. After 7 days of growth, GUS activity in seedlings was assessed. GUS activity was clearly observed throughout the root tip, which is consistent with the role of SOG1 in promoting terminal differentiation of the root tip following Al exposure . In contrast, root tips treated with Al for 7 days showed no GUS activity. This indicates that SOG1 does not persist after a root has terminally differentiated following Al treatment. Since it was determined that ATR and SOG1 act within the same pathway, SOG1:GUS was introgressed into the atr-4 mutant. Loss of SOG1 expression in the presence of inhibitory levels of Al is apparently ATR-dependent since SOG1:GUS is maintained in root tips of the loss-of-function atr-4 mutant even following Al treatment . Even though effects of Al toxicity severely compromise root growth, the terminal differentiation seen in Al-treated roots is not associated with tissue death as shown by Evan’s blue staining in previous studies .In order to determine the subcellular localization of SOG1, examination of a previously published SOG1:GFP translational fusion allowed for visual localization via confocal microscopy. Transgenic seedlings of Col- 0 wild type carrying native SOG1 promoter controlled SOG1:GFP fusion construct were grown in 0µM AlCl3 hydroponic growth media for 6 days, after which they were exposed to either 0 or 100µM AlCl3 for 48 hours.
Root tips were subsequently examined using confocal microscopy and treated with the nuclear specific stain, Hoechst 33342. In the absence of Al, SOG1:GFP is found at the perimeter of nuclei of root tip cells . Exposure of roots to a highly inhibitory concentration of Al in a hydroponic system for 48 hours results in either a redistribution of SOG1:GFP to the interior of the nucleus or a morphological change to the nuclear architecture . While it is unclear what is occurring to affect the visualization of nuclear aggregation of SOG1 following Al treatment, analysis of SOG1:GFP localization in the atr-4 loss-of-function mutant shows that in both the absence and presence of 100µM AlCl3 in hydroponics, SOG1:GFP is maintained in a comparable fashion to SOG1:GFP in Col-0 wild type without exposure to Al . Despite the uncertainty of the nature of the change in SOG1:GFP visualization in response to Al, this result does indicate that this change is dependent on ATR.ATR is a member of the phosphoinositide 3-kinase-like kinase family and major regulator in the DNA damage response in mammalian systems . Since there is an apparent functional relationship between ATR and SOG1 with regard to terminal differentiation of the root tip following Al exposure, it was of interest to determine whether SOG1 is a phosphorylation target of ATR. 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 SOG1, representing 449 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-SOG1 in the presence of [γ- 32P] ATP, after which samples were separated on an SDS-PAGE gel.
While incubation of MBP with GST-ATR did not result in measurable phosphorylation of MBP, MBP-SOG1 incubated with GST-ATR resulted in a distinct radiolabeled band that was the same size as that predicted for MBP-SOG1 . At least in vitro, SOG1 is a phosphorylation target of the Arabidopsis ATR kinase. While Al treatment has been associated with upregulation of a large group of genes in multiple plant model systems , it has been difficult to identify which members of these Al-inducible groups are of primary relevance to Al toxicity and response. Therefore, demonstration that SOG1 is responsible at least in part for stoppage of root growth following chronic exposure to Al is expected to allow for determination of which Al-inducible genes are central to Al-dependent terminal differentiation. With this in mind, it was of interest to determine whether Al results in similar SOG1-dependent changes in gene expression as seen with γ-radiation . Several genes have been found to be substantially upregulated following exposure to γ-radiation, including many that are involved in response to and repair of damaged DNA. Examples include BRCA1, PARP2, XRI1, RAD50, and RAD51, along with a number of genes whose functions in relation to DNA damage response have yet to be elucidated . In order to determine whether Al causes changes in expression of these SOG1-regulated genes, it was first necessary to determine the conditions that would allow for the best capture of these changes. This was particularly problematic since SOG1 does not persist after Al-dependent terminal differentiation , making it necessary to determine at which point damage had accumulated to a high enough level to promote entrance into endore duplication but not late enough to where the transition had already been initiated. For this work, SOG1:GUS expression in the root tip was followed over a time course of Al exposure. Col-0 wild- type transgenic plants expressing SOG1:GUS were grown in the presence of 1.50 mM AlCl3 in a soaked gel environment, and samples were taken on successive days for visualization of GUS activity. GUS activity persisted in the root tip throughout the course of the experiment for untreated samples,black flower bucket whereas growth of roots in the presence of Al resulted in a progressive loss of GUS activity starting 3 days after planting . It was also necessary to assess the status of the root QC on a daily basis through the use of the QC46:GUS reporter line. Consistent with the loss of SOG1:GUS activity, the root QC disappeared by day 5 of growth in the presence of 1.50 mM AlCl3 in a soaked gel environment with a substantial decrease in GUS activity occurring between days 3 and 4 . When considered in conjunction with the results from the SOG1:GUS time course, the transition from an actively growing root tip to one that has transitioned to endore duplication occurs between days 3 and 4 of chronic Al exposure, indicating that SOG1-dependent increases in gene expression in response to Al would be most likely observed within this 3 to 4 day window. 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, sog1-7, and sog1-7;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. For this experimental approach, a normalization gene needed to be selected that acts as a house keeping gene, which is expressed consistently in different genetic lines in the absence and presence of Al. Generally, genes encoding cytoskeletal elements are often used; however, as discussed previously, Al disrupts micro-tubule and actin dynamics so this was not an option . Based on a previous study, At5g60390 was selected due to its consistent expression under other heavy metal toxicities such as nickel and copper . To ensure that EF-1 α would be an ideal normalization gene candidate, seedlings of Col-0 wild-type, als3-1, sog1-7, and sog1-7;als3-1 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. All genetic lines under all test conditions showed equivalent expression . 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 . Genes tested included those encoding 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 . Treatment with Al resulted in a measurable increase in expression of this subset of genes in Col-0 wild type compared with no Al . In Al-treated als3-1 seedlings, the expression of these genes also increased significantly, even more so in comparison to Col-0 wild type , consistent with the extreme Al hypersensitivity seen for this mutant. In contrast, an increase in expression of these genes was not observed for sog1-7 and sog1-7;als3-1 roots in comparison to the respective controls . This result indicates that Al triggers a SOG1-dependent transcriptional program that is similar to that observed following treatment with γ-radiation. SOG1 has been demonstrated previously to function downstream of ATM in response to γ-radiation . ATR and ATM are both members of the PIKK family of kinases, and now that ATR has been established to work in conjunction with SOG1 to inhibit root growth in the presence of Al, it was of interest to determine whether Al-responsive stoppage of root growth also required ATM. An atm-2;als3-1 double mutant was generated . The capability of the atm-2 loss-of-function mutation to suppress the Al hypersensitivity of als3-1 was compared with that of atr-4. Seedlings of Col-0 wild type, als3-1, atr-4;als3-1, and atm-2;als3-1 were grown for 7 days in the absence or presence of 0.75 mM AlCl3 in a soaked gel environment . Exposure to Al resulted in severe Al hypersensitivity in als3-1 roots compared with Col-0 wild type, whereas atr-4;als3-1 mutant roots were indistinguishable from Col-0 wild type in the presence of Al . In contrast, Al-treated roots of atm-2;als3-1 were only marginally longer than those of als3-1, with both displaying the same terminal differentiation phenotype following Al exposure . Because there is a clear discrepancy regarding the roles of ATR and ATM in mediating stoppage of root growth following exposure to Al, it was determined whether loss-of-function mutations for each had an impact on SOG1-dependent expression of genes following Al exposure. For this analysis, Col-0 wild type, als3-1, sog1-7;als3-1, alt2-1;als3-1, atr-4;als3-1, and atm-2;als3-1 were grown in the absence or presence of 1.50 mM AlCl3 in a soaked gel environment for 3 days.