Seedlings were subsequently collected for RNA isolation, cDNA synthesis, and RTPCR analysis. Treatment with Al resulted in the same patterns of induction com- pared with γ-radiation for all genes tested for Col-0 wild type and als3-1 with the exception of SYN2 , which encodes a key sister chromatid cohesion protein that is inducible with γ-radiation but not Al . In contrast, there was no apparent induction of any of these SOG1-regulated genes in sog1-7;als3-1, alt2-1;als3-1, or atr-4;als3-1 . Thus, Al tolerance in each is correlated with failure to trigger the SOG1-dependent increase in expression of these DNA damage response genes following Al exposure. Loss of expression of this subset of genes was not observed for Al-treated atm-2;als3-1, which had clear induction of all SOG1-dependent Al-responsive genes to a level that was comparable to als3-1 . 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 and alt2 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 sog1-7 seedlings followed canon. Seedlings of Col-0 wild type and sog1-7 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 . Bleomycin acts by inducing DNA double strand breaks . Seedlings of Col-0 wild type and sog1-7 grown in increasing concentrations of either HU or Bleomycin, and both exhibited root growth inhibition . MMC is an interstrand cross-linking agent ,square black flower bucket and CDDP is an intrastrand cross-linking agent . Roots of Col-0 wild type and sog1-7 grown in increasing concentrations of MMC exhibited inhibition; however, roots of sog1-7 were more sensitive to MMC .
Similarly, seedlings of Col-0 wild type and sog1-7 grown in media with increasing concentrations of CDDP also exhibited root growth inhibition in which sog1-7 demonstrated greater sensitivity than roots . Loss-of-function mutations for both BRCA1and PARP2 , which are Al-inducible SOG1 targets, were tested for their capability to grow in the presence of AlCl3 in a soaked gel environment . Two independent brca1 loss-of-function mutants were modestly sensitive to a range of Al concentrations compared with Col-0 wild type, suggesting that BRCA1 plays a role in repair of Al-dependent DNA damage rather than transition of the root tip to endore duplication . PARP2, in conjunction with PARP1, is a key component of microhomology-mediated end joining, which is one type of nonhomologous end joining DNA repair mechanism that is related to base excision and single strand break repair . Loss of these two key components of microhomology-mediated end joining results in increased sensitivity to Al , consistent with Al acting as a DNA damage agent. Al hypersensitivity was even more pronounced for a parp1;parp2;ku80 triple loss-of function mutant, which represents a severe reduction in capability to carry out both classical NHEJ and alternative NHEJ . These results suggest that Al has substantive negative effects on DNA integrity that in part requires ATR and SOG1-dependent induction of BRCA1 and PARP2 to repair the damage.The suv2-3;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 suv2-3;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 moderate 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. Consistent with plants having enhanced tolerance to internalized Al, suv2-3;als3-1 roots accumulated callose similarly to both Col-0 wild type and als3-1 . It was also tested whether suv2-3;als3-1 showed Al-responsive increases in gene expression, 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 suv2-3;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 RNA gel blot analysis with the Al-inducible probe ALMT1 . Exposure of Col-0 wild type, als3-1, and suv2-3;als3-1 to Al resulted in increased expression of the Al-responsive gene indicating that suv2-3 suppresses the hypersensitivity of als3-1 following the internalization Al similarly to Col-0 wild type and als3-1 . Finally, total Al that accumulated in the root tissue of Col-0 wild type, als3-1, and suv2-3;als3-1 was measured using 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, including those of suv2-3;als3-1, showed significant accumulation of Al. In conjunction with callose deposition and Al-inducible gene expression, the quantitative intake of Al in root tissue indicates that the observed restoration of root growth for the suv2-3;als3-1 mutant was dependent on enhanced Al tolerance rather than Al exclusion from the root tip.
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 , 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 chromosome 5 of Arabidopsis . 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 At5g45610, which was previously reported to encode a homologue of the ATR-interacting protein, ATRIP . The als3-1 suppressor mutation represents a premature stop codon in the eighth exon of SUV2 at amino acid 359 . There have been two previous publications on the Arabidopsis ATRIP homologue in Arabidopsis using different phenotypes to describe the gene name: Sensitive to UV2and Hydroxyurea Sensitive2. One of these mutant alleles was obtained, suv2-1,square black flower bucket wholesale and was introgressed into the als3-1 background. This suv2 mutant represents a chromosomal rearrangement within SUV2 caused by ion-beam irradiation resulting in a recessive loss-of-function mutation. A double mutant was generated and a suv2- 1;als3-1 double mutant was tested in order to determine if it could also suppress the als3-1 phenotype. Seedlings of Col-0 wild type, als3-1, suv2-1;als3-1 and suv2-3;als3- 1 were grown for 7 days in the presence of 0.75 mM AlCl3 in a soaked gel environment, after which root tips were assessed for terminal differentiation. This showed that the suv2-1 allele can suppress the extreme Al response of als3-1 in a manner indistinguishable from suv2-3 since both suv2-1;als3-1 and suv2-3;als3-1 failed to exhibit the severe root growth inhibition seen for Al-treated als3-1 . With this allelic comparison of suv2-1 yielding similar capabilities as suv2-3 in suppressing the hypersensitivity of als3-1, crosses were made between suv2- 1;als3-1 and suv2-3;als3-1 to establish that these two mutant alleles within the same complementation group. Seedlings of Col-0 wild type, als3-1, suv2-1;als3-1, suv2- 3;als3-1 and the F1 progeny of the resulting crosses of als3-1 X suv2-1;als3-1, als3-1 X suv2-3;als3-1, and suv2-1;als3-1 X suv2-3;als3-1 were grown for 7 days in the presence of 0.75 mM AlCl3 in a soaked gel environment. The F1 seedlings of suv2-1;als3-1 X suv2-3;als3-1 suppressed the hypersensitivity of als3-1 in a manner comparable to both suv2-1;als3-1 and suv2-3;als3-1 . Despite the reported recessive nature of the suv2-1 mutation , als3-1 X suv2-1;als3-1 is marginally capable of suppressing the als3-1 hypersensitivity; and als3-1 X suv2-3;als3-1 also showed marginal suppression of als3-1 . Due to these results, suv2-3 is likely a semi-dominant loss-of-function mutation. To analyze suv2-3 roots growth without the als3-1 mutation in the genetic background, suv2-3 was back crossed to Col-0 wild type, and homozygous suv2-3 F2 progeny were identified by PCR analysis.
Col-0 wild-type and suv2-3 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 suv2-3 mutant roots showed greater growth than wild type roots in the presence of a range of normally highly inhibitory levels of AlCl3 . This indicates that SUV2 has a prominent role in actively halting root growth following Al treatment. To determine if Al regulates SUV2 expression, RT-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 SUV2 specific PCR oligonucleotide primers . There was no significant indication that SUV2 transcript is altered following Al treatment Al although there is an observable decreasing trend with increasing concentrations of Al. Seedlings of suv2-3 were also grown in a 0 μM AlCl3 hydroponic environment for 7 days and subsequently analyzed with RT-PCR and the suv2-3 mutation was not found to affect transcript stability since Col-0 wild type and suv2-3 showed comparable levels of SUV2 transcript . Al toxicity is most pronounced at the root tip , and as SUV2 transcript levels are marginally reduced with Al treatments, it was of interest to determine the tissue localization pattern for SUV2 in the absence and presence of Al. For this analysis, a transgenic Arabidopsis line carrying a SUV2:GUS fusion construct was generated and grown in the absence or presence of 1.50 mM AlCl3 in a soaked gel environment for 7 days, after which seedlings were stained for GUS activity. GUS activity was clearly observed throughout the meristematic region of the root tip in the absence of Al . Root tips treated with Al for 7 days also showed GUS persistence in the meristematic region of the root tip, but there is a discernable change to the morphology of the root tip as the zone of division is compacted leaving the zones of elongation and maturation in closer proximity to the meristematic region of the root tip . This reduction in the meristematic zone may account for the insignificant but observable reduction in SUV2 transcript levels following Al treatment. In order to determine the subcellular localization of SUV2, a transgenic Arabidopsis line carrying a SUV2:GFP fusion construct was generated to visually analyze seedlings via confocal microscopy. Transgenic seedlings of Col-0 wild type carrying native SUV2 promoter controlled SUV2:GFP fusion construct were grown in 0µM AlCl3 hydroponic growth media for 7 days. Root tips were subsequently examined using confocal microscopy and treated with the nuclear specific stain, Hoechst 33342. SUV2:GFP is found in the cytoplasm and within nuclei of root tip cells . In previous studies, loss-of-function mutations in cell cycle checkpoint factors ATR, ALT2 and SOG1 resulted in increased root growth in the presence of Al correlated with failure to arrest cell cycle progression in conjunction with forced quiescent center differentiation . In order to determine if this is also the case for roots of an suv2 loss-of-function mutant, suv2-3 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 suv2-3 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.