While defining Al responsive mechanisms is suggestive of the type of damage Al causes, it is crucial to define the genotoxic consequence of Al that triggers this response, whether real or perceived. Additionally, unanswered questions also persist regarding which factors participate in conjunction with ATR and ALT2 to respond to Al-dependent damage since clearly Al responsive stoppage of root growth is a multi-step process progressing through cell cycle arrest to terminal differentiation associated with endore duplication or DNA replication without cytokinesis. Therefore, not only is further identification of Al tolerance factors crucial to our understanding of Al response signaling, it is also of critical importance to determine how these factors function together to promote Aldependent cell cycle arrest causing terminal differentiation and subsequent plant growth inhibition. While the genotoxic consequences of Al in plants have yet to be elucidated, it is clear from our als3-1 suppressor mutagenesis approach that components of the DNA damage response machinery are mediating root growth inhibition in response to the likely negative impact of Al on DNA structure, integrity,flower display buckets or conformation. The identification of ATR and ALT2 as mediators of Al-dependent root growth inhibition presents new strategies and opportunities to engineer crop species capable of growing in Al toxic soils.
Based on the critical roles of ATR and ALT2 in mediating Al-dependent root growth inhibition and the current knowledge gained from the field of plant DNA damage, further studies need to be done to identify mediators and effectors within the ATR- and ALT2-mediated Al response pathway, especially those responsible for controlling cell cycle arrest, damage repair, and subsequent promotion of endocycling at the root apical meristem to stop root growth. Our understanding of the genomic consequences caused by Al is still in the beginning stages, and more work is needed. Continued testing of DNA damage response mutant responses to Al can give us the opportunity to elucidate further how genomic maintenance factors are involved in this biological problem. In addition to the value of gaining a better understanding the role of DNA damage response factors and cell cycle checkpoints in mediating Al-dependent DNA damage, Al toxicity represents a novel and biologically relevant model to study ATR dependent mechanisms in the DNA damage response in general.In previous studies, seeds of als3-1 were mutagenized with ethyl methanesulfonate and M2 seedlings were screened for roots capable of sustained growth in the presence of 0.75 mM AlCl3 in a soaked gel environment. Identified seedlings were rescued and allowed to set seeds, after which progeny were rescreened to identify bona fide als3-1 suppressors. In order to further study Al-dependent terminal differentiation of the Arabidopsis root, an als3-1 suppressor mutant was chosen from this screen that was capable of sustained root growth in comparison to als3-1 in the presence of a range of AlCl3 concentrations for further analysis. Subsequent work showed this mutant to be an allele of SUPPRESSOR OF GAMMA RESPONSE1 .
Since six mutant alleles have been previously published , this suppressor will be referred to as sog1-7.The sog1-7;als3-1 double mutant was studied further to determine if the als3- 1 suppression resulted from increased Al resistance or tolerance. Internalization of Al has been associated with deposition of the β-1,3-glucan, callose, in the plasmodesmata between cells in the root tip . Seedlings of Col-0 wild type, als3-1, and sog1-7;als3-1 were grown hydroponically for 6 days in 0 μM AlCl3 , and then exposed to either 0 or 25 μM AlCl3 . This is a concentration that causes inhibition of wild-type root growth in hydroponic growth conditions for 24 hours. After these treatments, the seedlings were stained with Aniline Blue to detect callose with the use of fluorescent microscopy. Roots of sog1- 7;als3-1 accumulated callose similarly to both Col-0 wild type and als3-1 which is consistent with plants being tolerant to internalized Al . This suggests that while callose deposition is correlated with Al toxicity and has been suggested to be integral to Al dependent stoppage of root growth, it may not primarily responsible for Al inducible growth inhibition . To determine whether sog1-7;als3-1 showed Al-responsive gene expression, it was tested whether sog1-7;als3-1 showed increases in transcripts known to be induced following Al exposure, as would be expected for enhanced Al tolerance rather than increased Al exclusion. For this experiment, seedlings of Col-0 wild type, als3-1, and sog1-7;als3-1 were grown hydroponically for 6 days, after which seedlings were exposed to 0 or 25 μM AlCl3 for 24 hours. Following this, roots were collected and total RNA was isolated for a Northern analysis with the Al-inducible probes ALS3 and ALMT1 . When grown in the presence of Al, Col-0 wild type, als3-1, and sog1-7;als3-1 all resulted in an increase in expression of both Al-responsive genes.
This further supports characterization of sog1-7 as an Al tolerant mutation, as sog1-7 suppresses the hypersensitivity of als3-1 following the internalization of Al . To quantify total Al that accumulated in the root tissue of Col-0 wild type, als3-1, and sog1-7;als3-1, dried and ashed root tissue was subjected to inductively coupled plasma-optical emission spectrometry . For this experiment, seedlings were grown hydroponically for 6 days in the absence of Al, after which roots were exposed to 0 or 50 μM AlCl3 for 24 hours. Root tips were subsequently harvested, washed with nutrient medium, dried, and then ashed in pure HNO3 in preparation for analysis. All Al-treated root samples showed significant and equivalent accumulation of Al, demonstrating that the sog1-7 mutation is not excluding Al internalization to suppress the als3-1 hypersensitive response . Considering the quantitative uptake of Al by root tissue along with callose deposition and Al-inducible gene expression, evidence supports that the sustained root growth for the sog1-7;als3-1 mutant is due to enhanced Al tolerance rather than Al exclusion from the root tip.A map-based cloning approach was used to identify the nature of the als3-1 suppressor mutation. For this, the als3-1 line carrying the suppressor mutant in the Col-0 background was crossed to an als3-1 line that had been introgressed into the La-0 background . Because of the recessive nature of the als3-1 suppressor mutation, F2 progeny from the cross were grown on gel plates soaked with 0.75 mM AlCl3 ,flower bucket and seedlings with roots that were capable of sustained growth were rescued. Following isolation of genomic DNA, PCR-based analyses were conducted and showed that the als3-1 suppressor mutation localized to the top arm of Arabidopsis chromosome 1 . Fine mapping resulted in a genetic window that allowed identification of candidate genes for sequence analysis. The als3-1 suppressor mutation was subsequently found to be in exon 4 of At1g25580, which was previously reported as the ATM-regulated transcription factor SOG1 that is responsible for initiation of endoreduplication following exposure to DNA damage agents . The als3-1 suppressor mutation represents an amino acid substitution in the predicted NAC domain of this NAM , ATAF1/ATAF2, CUC family transcription factor . To confirm the suppression of the als3-1 phenotype is caused by a single base change in At1g25580, functional complementation was subsequently performed using a full-length genomic SOG1 construct that was previously reported . Seedlings of Col-0 wild type, als3-1, sog1-7;als3-1, and sog1-7;als3-1 carrying a wild-type genomic version of SOG1 were grown in the presence of 0.75 mM AlCl3 in a soaked gel environment. After 7 days of growth, roots were assessed for terminal differentiation. Introduction of a wild-type genomic version of SOG1 into sog1-7;als3-1 fully restored Al hypersensitivity to sog1-7;als3-1, as demonstrated by the transgenic root being terminally differentiated in a manner indistinguishable from Al-treated als3-1 . While there have been six previously published alleles of sog1, only one allele has been maintained since publication, sog1-1 .
This sog1 mutant represents a single amino acid mutation from R to G at residue 155 of SOG1. In order to determine if this allele could also suppress the als3-1 phenotype, the double mutant, sog1-1;als3-1 was generated. Subsequently, seedlings of Col-0 wild type, als3-1, sog1-1;als3-1 and sog1-7;als3-1 were grown in the presence of 0.75 mM AlCl3 in a soaked gel environment. After 7 days of growth root tips were assessed for terminal differentiation. The sog1-1 allele was capable of suppressing the extreme Al response of als3-1 in a manner indistinguishable from sog1-7, since both sog1-1;als3-1 and sog1-7;als3-1 failed to exhibit the severe root growth inhibition seen for Al-treated als3-1 . To analyze sog1-7 roots growth without the als3-1 mutation in the genetic background, sog1-7 was backcrossed to Col-0 wild type, and homozygous sog1-7 F2 progeny were identified by PCR analysis. Col-0 wild-type and sog1-7 seedlings were then grown for 7 days in the absence or presence of increasing concentrations of AlCl3 in a soaked gel environment, after which root lengths were measured. In the absence of als3-1, the sog1-7 mutant roots showed greater growth than wildtype roots in the presence of a range of normally highly inhibitory levels of AlCl3 . This indicates that SOG1 has a prominent role in actively halting root growth following Al treatment. To determine if SOG1 expression is regulated by Al, real-time PCR analysis was performed. Col-0 wild-type seedlings were grown in a hydroponic environment for 6 days and subsequently treated with 0, 25, or 100 μM AlCl3 for 24 hours. Root tissue was collected, total RNA was isolated for cDNA synthesis and RT-PCR was performed using SOG1 specific PCR oligonucleotide primers . There was no indication that SOG1 is transcriptionally induced by Al. Similarly, seedlings of sog1-7 were grown in a 0 μM AlCl3 hydroponic environment for 7 days and subsequently analyzed with real-time PCR and the sog1- 7 mutation was not found to affect transcript stability since Col-0 wild type and sog1-7 showed comparable levels of SOG1 transcript . In previous studies, loss-of-function mutations in cell cycle checkpoint factors ATR and ALT2 resulted in increased root growth in the presence of Al . This tolerance was correlated with failure to arrest cell cycle progression in conjunction with forced quiescent center differentiation. In order to determine if this is also true for roots of a sog1 loss of-function mutant, sog1-7 was crossed to either a transgenic Arabidopsis line carrying a reporter for cell cycle progression, CYCB1;1:GUS , or a reporter for QC status, QC46:GUS . Seedlings of Col-0 wild type and sog1-7 carrying the CYCB1;1:GUS reporter were grown in the absence or presence of 0.75 mM AlCl3 in a soaked gel environment for 7 days, after which they were stained for GUS activity. Col-0 wild type carrying the CYCB1;1:GUS reporter results in a substantial increase in GUS activity following exposure to Al . This is consistent with a large number of root cells being incapable of exiting the G2 phase of mitosis and incapable of proceeding into actual cell division. Unlike previous reports for the atr-4 and alt2-1 loss-of-function mutations where GUS reporter levels were eliminated , sog1-7 carrying CYCB1;1:GUS had substantially reduced levels of the CYCB1;1:GUS reporter compared with Col-0 wild type . This may indicate that the role of SOG1 in Al-dependent inhibition of cell cycle progression at the G2 phase, while likely acting in conjunction with ATR and ALT2, may also function through other factors to prevent CYCB1;1 turnover. 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 sog1-7 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 sog1-7 roots had QC46:GUS accumulation in the absence of Al . Treatment with high levels of Al in Col-0 wild type resulted in the loss of the QC, but not in sog1-7 . This indicates that SOG1 plays an active role in differentiation of the QC following Al treatment and likely functions as a step in the transition to endore duplication in the root tip.In support of this model, it was found that Al treatment leads to terminal differentiation in conjunction with substantial increases in cell and nucleus size in als3-1 roots. For this analysis, Col-0 wild- type, als3-1, atr-4;als3-1, alt2-1;als3-1, and sog1-7;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 .