Since the negative short-term effects of Al on growth are apparently limited given that inhibition is alleviated without checkpoint function, conceivably these checkpoints have evolved to detect the minor strain on an individual’s genomic stability and serve to prevent transmission of Al-dependent genetic deffects to subsequent generations that ultimately would compromise the viability of the population. These checkpoints sacrifice the individual by halting the cell cycle in the root and forcing endore duplication to prevent an Al-dependent generational penalty with regard to heritable genomic integrity. Cells of the shoot meristem generate the floral reproductive organs of the plant, and thus the heritable genetic material. While the root is the most affected organ of the plant in the Al toxic response, Al is reallocated and sequestered to other regions of the plant body as Al-oxalate complexes can transport Al through the xylem from the roots to the shoots . In addition to a root hypersensitive phenotype, shoots of als3-1 grown in Al containing media display reduced cotyledon and leaf expansion, as well as a second shoot apex . Thus shoots are indeed affected by Al toxicity which could pose a threat to plant reproductive processes; therefore, it is plausible that Al-dependent root growth inhibition could serve as a means to preserve genomic integrity of the species. In all likelihood, it is plausible that Al induces an inappropriate activation of the DNA damage response by detecting Al-dependent pseudo-cross links. This could be the result of topological strain on the DNA and/or functional interference with Mg-dependent replication machinery,round nursery pots activating unnecessary repair of the DNA that may actually be the true detrimental effect of Al-dependent root growth inhibition.
Repair processes like nucleotide excision repair and nonhomologus end joining may be at work as shown by hypersensitivity to Al for loss-of function mutants in ALT2 and PARP1, PARP2 and KU80 respectively. The repair mechanisms could then result in subsequent damage inflicted on DNA, much of which may be related to double strand breaks that are regularly observed following Al treatment. The specific repair pathway activated in response to Al treatment is unknown, as well as whether or not this unknown process inflicts damage on the DNA during the repair. If indeed Al is acting to cause pseudo-cross links, it stands to reason that this effect would also inhibit proper DNA repair processes and could further cause damage to the DNA. As such, failure to activate this response pathway prevents the program-inflicted damage and results in roots that can grow normally in the presence of inhibitory levels of Al.While it is speculative at this time as to what the true nature of the effect of Al is on DNA integrity, an ATR-dependent DNA damage response pathway is clearly activated in the presence of internalized Al. Whatever the direct effect of Al is, based on the response factors, it can be speculated how these factors assemble and respond to Al stress based on their functional homology to related proteins: SUV2 likely aggregates at sections of persistent single stranded DNA coated by the heterotrimer, Replication Protein A, known to aggregate to single stranded DNA . SUV2 may act as a homodimer as it has been shown to bind to itself through its N-terminal coiled-coil domain as demonstrated by a yeast-2- hybrid . The proline-rich repeats at the N-terminus of SUV2 suggest a stiff elbow-hinge that could “wag” as a mechanism of recruiting other response factors to the locus.
It is possible that this homodimerization is controlled by phosphorylation of SUV2 since the dimerization domain has two SQ phosphorylation motifs associated with it. SUV2 would act to recruit ATR to this region of DNA in a manner similar to their homologues in yeast and mammals . The interaction of SUV2 and ATR with persistent single stranded DNA should induce autophosphorylation of ATR, and phosphorylation of SUV2 by ATR. It is possible that ATR will also be phophorylating other substrates, such as RPA2 and H2AX, to orchestrate additional responses. Once ATR has activated these sensors, likely in conjunction with other unidentified proteins, they would then induce the signal transduction pathway by activation of SOG1, again, likely through phosphorylation by ATR. Conserved serine-glutamine motifs are preferential ATM and ATR phosphorylation targets in mammals . SOG1 has five SQ motifs in the C-terminal transcriptional activation domain and SUV2 has two SQ motifs near the N-terminus, one of which is within the dimerization domain. Following activation of SOG1, expression of a group of genes is promoted, specifically genes tested from an established set of SOG1-mediated genes involved in a DNA damage response. This subset of Al-induced SOG1 mediated genes includes genes known to repair DNA e.g. BRCA1, RAD51, RAD17, GMI1, and PARP2 and halt the cell cycle e.g. CYCB1;1 as well as more transcription factors e.g. TRFL10,TRFL3, ANAC103 and WRKY25. Perhaps it is these transcription factors, as well as unidentified genes promoted as part of this response that are responsible in some unknown manner for a mechanism that forces a programmatic change in the root tip and QC, thus triggering this tissue to switch to endore duplication and causing terminal differentiation and permanent stoppage of growth of the primary root. While little is known about the placement of ALT2 within this signal transduction pathway, it likely functions as a scaffold protein, perhaps as part of a ubiquitin ligase signaling mechanism, acting analogously to other WD-40 proteins. WD40 repeat proteins are a class of proteins that are generally involved in mediating interactions between other proteins, associating with a variety of protein complexes, including E3 ubiquitin ligases . In eukaryotes, proteins are targeted for degradation via the ubiquitination-proteasome system, but ubiquitination also plays an important role in post-translational modification of proteins in the activation of signaling pathways.
As part of a crucial step in the DNA damage response pathway in mammals, following phosphorylation by ATM or ATR, one of the core histones of the nucleosome, γ-H2AX, is mono-ubiquitinated. This mono-ubiquitination is required for the recruitment of subsequent repair factors like BRCA1 and 53BP1 to both double and single stranded DNA breaks . In plants, CULLINs , which are part of a family of scaffolding proteins, form the largest family of E3 ligase complexes. Arabidopsis proteins containing WD40 domains, including ALT2, are proposed to be capable of interacting with the DDB1-CUL4-ROC1 complex . The recruitment structures and mechanisms are not well understood for CULLIN based ubiquitination signaling; however,plastic flower pots CUL4 has been shown to form a complex with DW40 proteins in response to UV damage that is ATR dependent . This establishes a potential link between ATR and ALT2 in a DNA damage response where resulting cross links would cause a replication fork stall, as Al likely causes.A model for stoppage of root growth following chronic exposure to Al can be developed in accordance with current evidence. In this model, Al impacts DNA in a currently unknown way, likely from a pseudo-cross linking effect resulting in a replication fork stall. Based on the genetic factors responsible for activating the Al dependent DNA damage response, it is a reasonable prediction that such an interaction would hold DNA in a conformation that inhibits replication fork progression. Regardless of the physical consequences of Al on DNA structure or integrity that have yet to be determined, the predicted genotoxic effects of Al are clearly sufficient to activate an ATR-, ALT2-, SOG1- and SUV2-dependent cell cycle checkpoint mechanism as demonstrated by the increase in Al tolerance seen for each loss-of-function mutant. This mechanism functions to promote transcription of a group of genes related to halting the cell cycle and to repair the perceived damaged DNA. Furthermore, it is likely that additional genes are included in this transcriptional response that are related in some unknown manner to a mechanism that forces a programmatic change in cells of the root tip and especially the QC. These genes would trigger cells to differentiate, losing their meristematic identity by switching from a normal cell cycle progression to endore duplication. Ultimately, it is this terminal differentiation that permanently stops growth of the primary root as the primary cause of Al toxicity. While significant work remains to be done, especially in determining the genotoxic consequences of Al that activate this DNA damage response pathway and developing a transcriptional profile of SOG1 targets that lead to inhibited root growth following Al treatment, it is clear that terminal differentiation of the root tip following chronic exposure to Al is an active event mediated by the DNA damage checkpoint factors ATR, ALT2, SOG1 and SUV2. 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 further elucidate how genomic maintenance factors are involved in this biological problem. In addition to the value of gaining a better understanding of 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 for studying ATR dependent mechanisms in the DNA damage response in general.For all growth experiments, seedlings were surface sterilized, vernalized, and etiolated before planting. Seeds were immersed in 70% ethanol and then washed 4 times with sterile water. Seeds were then immersed in 50% bleach for 5 minutes, after which seeds were washed 4 times with sterile water. The AlCl3 soaked gel environment was sterilely prepared by pouring a lower gel layer consisting of 80 mL of nutrient medium plus 0.125% gellan gum in Nunc Lab-Tek Extra-Depth Polystyrene Dishes 100 x 25 mm . Nutrient medium consisted of 2 mM KNO3, 0.2 mM KH2PO4, 2 mM MgSO4, 0.25 mM 2SO4, 1 mM Ca2, 1 mM CaSO4, 1 μM MnSO4, 5 μM H3BO3, 0.05 μM CuSO4, 0.2 μM ZnSO4, 0.1 μM CaCl2, 0.02 μM Na2MoO4, 0.001 μM CoSO4, and 1% sucrose. Al was introduced by overlaying the solidified lower layer with 20 mL of “soak solution” containing the proper concentration of AlCl3. Trail soak solution was made consisting of the nutrient solution medium described above, while only brought to 90% of the intended volume. 50 mL trail solutions were made consisting of 45 mL the slightly concentrated nutrient medium, X mL 25mM AlCl3, Y μL 0.1 N KOH and Z mL diH2O . The trail soak solution was made to determine the amount of 0.1 N KOH to use to adjust the pH of the nutrient soak containing AlCl3. The amount of base to add was determined empirically by adjusting the pH on an aliquot of the soak solution containing AlCl3. The amount of base determined from this trial soak solution was added to the actual soak solution prior to adding AlCl3. The sterilized soak solution was allowed to equilibrate with the lower layer for 2 days and was then poured off. This method was used for all concentrations of AlCl3 for plants grown in a gel soaked environment. In hydroponics experiments, Al-screening media was sterilely prepared as above without gellan gum and AlCl3. Seeds were sowed on 250-μm mesh, polypropylene screen in Parter Medical Products Quad Perti Dish 100 X 15 mm . After 6 days of growth unless otherwise specified, screens were transferred to new Al screening media supplemented with either 0 μM, 25 μM AlCl3 or 50 μM AlCl3. For treatment with hydroxyurea , mitomycin C , bleomycin , or cisplatin were added to plant nutrient media plus sucrose . Seeds were sowed and allowed to grow for seven days, after which roots were measured. For experiments on plant nutrient media plus sucrose , the medium consisted of 5 mM KNO3, 2.5 mM KH2PO4, 2 mM MgSO4, 2 mM Ca2, 50 μM FeEDTA, 1 μM MnSO4, 100 nM CaCl2, 100 nM CoSO4, 5 nM H3BO3, 50 nM CuSO4, 20 nM NaMoO4, 0.8 M Sucrose, 0.8% agar. Plants were grown in 24-hour light at 20°Cin I-36LLVL biological incubator . After one week, plants were repotted in Sunshine Special Blend potting soil with controlled release fertilizer, 15-9-12 + minors . Plants were grown in 24- hour continuous light at 22°C in a plant growth room with Sylvania Gro-Lite fluorescent bulbs until maturity.