Aluminum-Dependent Terminal Differentiation of the Arabidopsis Root Tip Is Mediated through an ATR-, ALT2-, and SOG1-Regulated Transcriptional Response

Abstract

By screening for suppressors of the aluminum (Al) hypersensitive Arabidopsis thaliana mutant als3-1, it was found that mutational loss of the Arabidopsis DNA damage response transcription factor SUPPRESSOR OF GAMMA RESPONSE1 (SOG1) confers increased Al tolerance similar to the loss-of-function mutants for the cell cycle checkpoint genes ATAXIA TELANGIECTASIA AND RAD3 RELATED (ATR) and ALUMINUM TOLERANT2 (ALT2). This suggests that Al-dependent terminal differentiation of the root tip is an active process resulting from activation of the DNA damage checkpoint by an ATR-regulated pathway, which functions at least in part through SOG1. Consistent with this, ATR can phosphorylate SOG1 in vitro. Analysis of SOG1's role in Al-dependent root growth inhibition shows that sog1-7 prevents Al-dependent quiescent center differentiation and endoreduplication in the primary root tip. Following Al exposure, SOG1 increases expression of several genes previously associated with DNA damage, including BRCA1 and PARP2, with gel-shift analysis showing that SOG1 can physically associate with the BRCA1 promoter in vitro. Al-responsive expression of these SOG1-regulated genes requires ATR and ALT2, but not ATAXIA TELANGIECTASIA MUTATED, thus demonstrating that in response to chronic Al exposure, ATR, ALT2, and SOG1 function together to halt root growth and promote terminal differentiation at least in part in a transcription-dependent manner.

Figure 1.

Growth Characterization of an als3-1 Suppressor Mutant in the Presence of Al. (A) and (B) Col-0 wild type, als3-1, and the als3-1 line carrying the suppressor mutant sog1-7 were grown in a soaked gel environment (pH 4.2) with either no or increasing concentrations of AlCl₃ (pH 4.2) for 7 d, after which root length was measured. Mean ± sd values were determined from 30 seedlings. (C) Seedlings of Col-0 wild type, als3-1, and sog1-7 als3-1 were grown for 6 d, after which they were exposed to either 0 or 25 μM AlCl₃ (pH 4.2) in hydroponics for 24 h and stained with Aniline blue for callose deposition. Seedlings were visualized using fluorescence microscopy. Bars = 50 μm. (D) Seedlings of Col-0 wild type, als3-1, and sog1-7 als3-1 were grown for 6 d, after which they were exposed to either 0 or 25 μM AlCl₃ (pH 4.2) in hydroponics for 24 h. Root tissue was harvested and total RNA was extracted for RNA gel blot analysis with Al-inducible genes. (E) Col-0 wild-type, als3-1, and sog1-7 als3-1 seedlings were grown for 6 d and then exposed for 24 h to either 0 or 50 μM AlCl₃ (pH 4.2) in hydroponics. Root tips were washed in nutrient medium without AlCl₃, harvested, dried, and ashed in nitric acid, and total Al content was measured using ICP-OES. Mean ± sd values were determined from five samples.

Figure 2.

Loss of SOG1 Results in Suppression of Al Hypersensitivity in als3-1. (A) Map-based cloning of the als3-1 suppressor (Col-0 background) crossed to als3-1 (Ws background) localized the second site mutation to the top arm of chromosome 1. Sequencing of candidates in the genetic window revealed a single nucleotide change in exon 4 of At1g25580, also known as SOG1. (B) Amino acid sequence of SOG1, showing the predicted NAC domain of the transcription factor (black box), the predicted nuclear localization signal (yellow box), and the effect of the nucleotide change in sog1-7 on primary structure (S206F). (C) Introduction of wild-type SOG1, including promoter, all exons and introns, and 5′ and 3′ untranslated regions, restores Al hypersensitivity to sog1-7 als3-1 grown for 7 d in the presence of 0.75 mM AlCl₃ (pH 4.2) in a soaked gel environment. (D) The sog1-1 allele suppresses the Al hypersensitivity phenotype of als3-1 as shown by growth of sog1-1;als3-1 for 7 d in the presence of 0.75 mM AlCl₃ (pH 4.2) in a soaked gel environment. (E) Growth of sog1-7 and Col-0 wild type for 7 d in the absence or presence of increasing concentrations of AlCl₃ in a soaked gel environment shows that sog1-7 roots are more Al tolerant than Col-0 wild type. Mean ± sd values were determined from 30 seedlings. Asterisk indicates significance at P ≤ 0.01 when comparing Al-treated lines using the Kruskal-Wallis one-way ANOVA test. (F) SOG1 transcript levels in Col-0 wild type, in the absence or presence of AlCl₃, and in sog1-7 were determined by real-time PCR using mRNA isolated from root tissue. Seedlings were grown for 6 d in a hydroponic environment, after which they were transferred to 0, 25, or 100 μM AlCl₃ (pH 4.2) for 24 h.

Figure 3.

Loss of SOG1 Function Prevents Terminal Differentiation and Blocks Al-Dependent Endoreduplication in als3-1. (A) Seedlings of Col-0 wild type and sog1-7, each of which carried the CDS of CyclinB1;1 including a predicted mitotic destruction box fused to the GUS reporter, were grown for 7 d in the absence or presence of 0.75 mM AlCl₃ (pH 4.2) in a soaked gel environment, after which seedlings were stained for 1 h for GUS activity and primary root tips were scored for blue color (1 = no color and 5 = intense blue color). Bars = 50 μm. (B) Seedlings of Col-0 wild type and sog1-7, both of which carried the GUS-based QC46 marker for the quiescent center, were grown for 7 d in the absence or presence of 1.50 mM AlCl₃ (pH 4.2) in a soaked gel environment, after which seedlings were stained for GUS activity for 24 h. Bars = 50 μM. (C) Seedlings of Col-0 wild type, als3-1, atr-4 als3-1, alt2-1 als3-1, and sog1-7 als3-1 were grown for 7 d in the absence or presence of 0.75 mM AlCl₃ (pH 4.2) in a soaked gel environment, after which samples were fixed in FAA and stained with DAPI. Root tips were visualized at 40× magnification via confocal microscopy for both cell and nucleus size. Bars = 25 μm.

Figure 4.

SOG1 Works in Conjunction with ATR to Promote Al-Dependent Stoppage of Root Growth. (A) A sog1-7 atr-4 double mutant was grown for 7 d in the absence or presence of 1.25 mM AlCl₃ (pH 4.2) in a soaked gel environment in order to determine whether the combination of mutations is additive for Al tolerance. Mean ± sd values were determined from 30 seedlings. Asterisk indicates significance at P ≤ 0.01 when comparing Al-treated lines using the Tukey HSD test. (B) SOG1 expression was found to be localized in part to the Arabidopsis root tip using a SOG1:GUS transgenic line grown for 7 d in either the absence or presence of 1.50 mM AlCl₃ (pH 4.2) in a soaked gel environment, after which seedlings were stained for GUS activity for 1 h. Al treatment resulted in loss of SOG1:GUS in Col-0 wild type but not in an atr loss-of-function mutant, indicating that Al-dependent changes in SOG1 levels are regulated by ATR likely as a part of ATR-dependent terminal differentiation. Bars = 50 μm. (C) Full-length Arabidopsis ATR protein was produced using a baculovirus protein expression system. Approximately 100 ng of recombinant ATR protein was incubated with either 1 μg of MBP or MBP-SOG1 protein in the presence of [γ-³²P]ATP, after which samples were separated by SDS-PAGE and analyzed by autoradiography. (D) Bacterially produced MBP-SOG1 protein was tested for its capability to physically interact with the promoter of one of SOG1’s predicted targets, BRCA1. Approximately 50 ng of MBP or MBP-SOG1 was incubated with radiolabeled BRCA1 promoter (−1 to −1500) using a standard EMSA approach. Analysis also included 50 ng of MBP-SOG1^R155G and MBP-SOG1^S206F, as well as MBP-SOG1 in the presence of increasing concentrations of unlabeled BRCA1 promoter. Following separation of samples using an agarose gel, results were examined using autoradiography.

Figure 5.

SOG1 Is Required for Al-Dependent Induction of DNA Damage Response Genes. (A) Seedlings of a SOG1:GUS transgenic line were grown in the absence or presence of 1.50 mM AlCl₃ (pH 4.2) in a soaked gel environment, after which roots were stained for 1 h for GUS activity on successive days. Bars = 50 μm. (B) Seedlings of a QC46:GUS transgenic line were grown in the absence or presence of 1.50 mM AlCl₃ (pH 4.2) in a soaked gel environment, after which roots were stained for 24 h for GUS activity on successive days. Bars = 50 μM. (C) Seedlings of Col-0 wild type, als3-1, sog1-7, and sog1-7 als3-1 were grown for 3 d in the presence of either 0 or 1.50 mM AlCl₃ (pH4.2), after which tissue was harvested for RNA isolation. Following cDNA synthesis, real-time PCR for previously described SOG1-regulated transcriptional targets was performed (Yoshiyama et al., 2009). Mean ± sd values were determined from three technical replicates.

Figure 6.

Response to Al in Arabidopsis Is an ATR-, ALT2-, and SOG1-Mediated Event Largely Independent of ATM. (A) Col-0 wild type, als3-1, atr-4 als3-1, and atm-2;als3-1 seedlings were grown for 7 d in the absence or presence of increasing amounts of AlCl₃ in a soaked gel environment (pH 4.2), following which root lengths were determined. Mean ± sd values were determined from 30 seedlings. Asterisk indicates significance at P ≤ 0.01 when comparing Al-treated lines using the Tukey HSD test. (B) Photos show representative control and Al-treated seedlings from each line. (C) Seedlings of 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 (pH 4.2) in a soaked gel environment for 3 d, after which tissue was collected for RNA isolation. Following cDNA synthesis, real-time PCR was performed to examine expression patterns for a group of previously documented SOG1-regulated genes (Yoshiyama et al., 2009). Mean ± sd values were determined from three technical replicates.

Figure 7.

Roots of brca1 and parp2 Loss-of-Function Mutants Are Hypersensitive to Al. (A) Two different T-DNA loss-of-function alleles of BRCA1 were grown with Col-0 wild type in the absence or presence of increasing concentrations of AlCl₃ in a soaked gel environment (pH 4.2) for 7 d, after which root lengths were measured. Mean ± sd values were determined from 30 seedlings. (B) Seedlings of Col-0 wild type and loss-of-function mutants representing key components of either C-NHEJ (ku80) or B-NHEJ (parp1 parp2) were grown for 7 d in the absence or presence of increasing concentrations of AlCl₃ in a soaked gel environment (pH 4.2), after which root lengths were measured. Mean ± sd values were determined from 30 seedlings.

Figure 8.

Model for Al-Dependent Stoppage of Root Growth. Based on our results from identification of als3-1 suppressors that increase Al tolerance, it is expected that Al acts as a genotoxic agent that in an unknown manner activates an ATR- and ALT2-dependent cell cycle checkpoint pathway to stop cell division following chronic Al exposure. Loss of ALS3 function is predicted to result in increased Al accumulation in root tip cells that likely would lead to greater impacts on DNA and consequently cause hyperactivation of the ATR- and ALT2-dependent response pathway (red arrows within the nucleus). At present, it is not clear what role the WD-40 protein ALT2 plays in this active process, although it could be argued that ALT2 functions analogously to other WD-40 proteins involved in the mammalian DNA damage detection pathways of GGNER and TCNER. Regardless, both ATR and ALT2, but not ATM, function to halt cell division, trigger QC differentiation, and promote endocycling at least in part through SOG1. This includes promotion of a transcriptional response to Al that is composed of a suite of genes that are related to two distinctly different functions. One set, as demonstrated by analyses of loss-of-function mutants for BRCA1 and PARP2, encodes products that are responsible for repair of the apparently limited DNA damage that occurs following treatment with Al. The other set, while still cryptic with regard to its members, represents factors that are responsible for transitioning from actively dividing root cells to terminal differentiation and endoreduplication following Al treatment. It could be argued that increased Al tolerance occurs following mutational loss of these cell cycle checkpoints because the negative consequences of cell cycle arrest far outweigh the actual genotoxic consequences of Al.