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. 2013 Apr 24;162(2):831–841. doi: 10.1104/pp.113.216796

The ATM-Dependent DNA Damage Response Acts as an Upstream Trigger for Compensation in the fas1 Mutation during Arabidopsis Leaf Development1,[W]

Tetsuya Hisanaga 1,2,3,4, Ali Ferjani 1,2,3,4, Gorou Horiguchi 1,2,3,4, Naoko Ishikawa 1,2,3,4, Ushio Fujikura 1,2,3,4,2, Minoru Kubo 1,2,3,4,3, Taku Demura 1,2,3,4,4,5, Hiroo Fukuda 1,2,3,4, Takashi Ishida 1,2,3,4,4, Keiko Sugimoto 1,2,3,4, Hirokazu Tsukaya 1,2,3,4,*
PMCID: PMC3668073  PMID: 23616603

The ATM-dependent DNA damage response acts as an upstream trigger for compensation, an enhanced cell expansion caused by defective cell proliferation, during Arabidopsis leaf development.

Abstract

During leaf development, a decrease in cell number often triggers an increase in cell size. This phenomenon, called compensation, suggests that some system coordinates cell proliferation and cell expansion, but how this is mediated at the molecular level is still unclear. The fugu2 mutants in Arabidopsis (Arabidopsis thaliana) exhibit typical compensation phenotypes. Here, we report that the FUGU2 gene encodes FASCIATA1 (FAS1), the p150 subunit of Chromatin Assembly Factor1. To uncover how the fas1 mutation induces compensation, we performed microarray analyses and found that many genes involved in the DNA damage response are up-regulated in fas1. Our genetic analysis further showed that activation of the DNA damage response and the accompanying decrease of cell number in fas1 depend on ATAXIA TELANGIECTASIA MUTATED (ATM) but not on ATM AND RAD3 RELATED. Kinematic analysis suggested that the delay in the cell cycle leads to a decrease in cell number in fas1 and that loss of ATM partially restores this phenotype. Consistently, both cell size phenotypes and high ploidy phenotypes of fas1 are also suppressed by atm, supporting that the ATM-dependent DNA damage response leads to these phenotypes. Altogether, these data suggest that the ATM-dependent DNA damage response acts as an upstream trigger in fas1 to delay the cell cycle and promote entry into the endocycle, resulting in compensated cell expansion.

Organ size is determined by cell number and cell size, both of which can be modulated by various physiological and environmental changes. To reach proper organ size under various growth conditions, cell proliferation and postmitotic cell expansion must be highly coordinated during organogenesis. Despite the importance of these processes in the control of organ size, the underlying mechanisms are not well understood. Plant leaves usually reach constant size under a given growth condition, making them an excellent model system to study organ size control. All cells in a young leaf primordium actively proliferate at the beginning, but as leaf development proceeds, cell proliferation starts to be restricted toward the junction between the leaf blade and the leaf petiole (Donnelly et al., 1999; Nath et al., 2003; White, 2006; Ichihashi et al., 2010, 2011; Kazama et al., 2010), generating a proximal-distal gradient of cell proliferation activity in the leaf blade. Subsequently, cells that terminate proliferation in the distal region start postmitotic expansion, whereas cells in the proximal region still continue to proliferate (Beemster et al., 2005). The leaf eventually reaches its appropriate final size when all cells stop proliferation and expansion.

During leaf development, a defect in cell proliferation often triggers enhanced cell expansion. When wheat (Triticum aestivum) seedlings are irradiated with γ-rays, they develop leaves that have fewer but larger cells than nonirradiated seedlings (Haber, 1962). This phenomenon, called compensation, has been reported in various mutants and transgenic plants of Arabidopsis (Arabidopsis thaliana; for review, see Tsukaya, 2002; Beemster et al., 2003; Horiguchi and Tsukaya, 2011). For example, a loss-of-function mutation in a transcriptional coactivator, ANGUSTIFOLIA3 (AN3), and overexpression of a cyclin-dependent kinase inhibitor, KIP-RELATED PROTEIN2 (KRP2), both cause a compensation phenotype (De Veylder et al., 2001; Horiguchi et al., 2005; Ferjani et al., 2007). Kinematic analysis of various compensation mutants revealed that the size of dividing cells in most of these mutants is similar to that of wild-type plants, strongly suggesting that compensated cell expansion is not a result of uncoupling between cell proliferation and cell expansion (Ferjani et al., 2007). It is thought, instead, that some regulatory system operates postmitotically to coordinate cell proliferation and cell expansion. Such a system should be an advantage for plants to support their highly plastic development, but the molecular mechanisms underlying this control are largely unknown.

Several recent reports have begun to provide insights into the mechanism of compensation. A study on Arabidopsis oligocellula mutants suggested that compensated cell expansion is induced by the extent of reduction in cell proliferation in a threshold-dependent manner (Fujikura et al., 2009). Moreover, several different modes of compensation, in terms of duration and rate of cell expansion, are observed in various fugu mutants, implying that compensation might be mediated through multiple mechanisms (Ferjani et al., 2007). Chimera analysis also revealed that excess cell expansion induced by the an3 mutation is non cell autonomous, whereas the expansion induced by KRP2 overexpression is cell autonomous (Kawade et al., 2010). Genetic analysis using extra small sisters and the compensation-exhibiting an3 mutants showed that compensated cell expansion is governed by the hyperactivation of cell expansion pathways required for normal cell expansion (Fujikura et al., 2007). Among many multicellular organisms, a positive correlation exists between cell size and nuclear ploidy level (Nagl, 1976; Melaragno et al., 1993). Nuclear ploidy is increased by a process called the endocycle or endoreduplication cycle, in which nuclear DNA is replicated without mitosis. Several Arabidopsis mutants have defects in endocycle progression, and in many cases, these defects are accompanied by altered cell size, suggesting that ploidy regulation is important for the control of cell size (Sugimoto-Shirasu et al., 2005; Breuer et al., 2007; Kurepa et al., 2009; Sonoda et al., 2009). Some, but not all, mutants exhibiting compensation also show higher ploidy phenotypes, suggesting that an increase in ploidy may play some roles in compensated cell expansion (Ferjani et al., 2007).

Chromatin Assembly Factor1 (CAF-1) is a histone chaperon consisting of three subunits (p150, p60, and p48 in humans), and it functions in nucleosome assembly, recruiting histones H3 and H4 onto a newly synthesized DNA chain (Smith and Stillman, 1989, 1991; Shibahara and Stillman, 1999; Tagami et al., 2004). CAF-1 is well conserved among eukaryotes, but the consequence of CAF-1 disruption is not identical among different organisms. For example, cultured human cells with defective CAF-1 do not proceed into the cell cycle and instead undergo apoptosis (Hoek and Stillman, 2003; Ye et al., 2003; Nabatiyan and Krude, 2004). The CAF-1 mutation in Drosophila melanogaster similarly leads to complete arrest of the cell cycle and subsequent lethality (Song et al., 2007). Therefore, CAF-1 activity appears to be essential for the survival of animals and insects. In contrast, CAF-1 mutation only delays cell cycle progression in yeast (Saccharomyces cerevisiae), and these defects are accompanied by heterochromatin silencing (Kaufman et al., 1997; Enomoto and Berman, 1998). In Arabidopsis, FASCIATA1 (FAS1) and FAS2 encode the large and middle subunits of CAF-1, respectively. The fas1 and fas2 mutants were originally isolated as mutants exhibiting stem fasciation (Leyser and Furner, 1992), but they also exhibit other developmental defects, including abnormal phyllotaxy, abnormal structure of shoot and root apical meristem, and serrated leaves (Kaya et al., 2001). It is hypothesized that these phenotypes are caused by ectopic expression of key meristem regulators such as WUSCHEL (WUS) and SCARECROW (SCR) due to the compromised chromatin assembly (Kaya et al., 2001). The fas1 and fas2 mutants also exhibit various abnormalities within the nucleus, some of which might result from the open chromatin conformation in these mutants. These include increased DNA double-strand breaks, increased frequencies of transfer DNA insertion and homologous recombination, and loss of telomere and 45s ribosomal DNA repeat sequences (Endo et al., 2006; Kirik et al., 2006; Ono et al., 2006; Schönrock et al., 2006; Mozgová et al., 2010). At the cellular level, fas1 and fas2 leaves appear to display typical compensation phenotypes, since they have fewer but larger cells (Exner et al., 2006; Ramirez-Parra and Gutierrez, 2007). Given that DNA-damaging treatment partially phenocopies these phenotypes, it is speculated that the DNA damage response activates the cell cycle checkpoint, promoting exit from the mitotic cycle into the endocycle (Ramirez-Parra and Gutierrez, 2007). In support of this idea, Adachi et al. (2011) recently showed, using Arabidopsis roots and culture cells, that cells arrested by DNA damage switch into the endocycle and differentiate.

Various endogenous and exogenous stresses cause damage to genomic DNA, and eukaryotic organisms have mechanisms to respond to these damages. Two related kinases, ATAXIA TELANGIECTASIA MUTATED (ATM) and ATM AND RAD3 RELATED (ATR), are essential for the DNA damage response in mammals. ATM is activated by double-strand breaks, whereas ATR is activated by single-strand breaks or stalled replication forks (Harper and Elledge, 2007). These kinases are known to activate several downstream regulators to elicit cellular responses such as cell cycle arrest and DNA repair. Both ATM and ATR are widely conserved among eukaryotes (Garcia et al., 2003; Culligan et al., 2004; Harper and Elledge, 2007). For example, gene expression analysis of γ-irradiated Arabidopsis plants revealed that hundreds of DNA damage response genes, including POLY (ADP-RIBOSE) POLYMERASE1 (PARP1; Doucet-Chabeaud et al., 2001), PARP2 (Babiychuk et al., 1998), RAD51 (Doutriaux et al., 1998), and BREAST CANCER SUSCEPTIBILITY1 (BRCA1; Lafarge and Montané, 2003), are expressed in an ATM-dependent manner (Culligan et al., 2006).

We have previously reported that the fugu2 mutants display typical compensation phenotypes (Ferjani et al., 2007). In this study, we show that FUGU2 encodes FAS1 and that the two fugu2 alleles have mutations in the FAS1 locus. Our microarray analyses showed that genes up-regulated in the leaf primordia of fas1 largely overlap with a group of genes that respond to the genotoxic stress. Subsequent genetic analyses with fas1 atm and fas1 atr double mutants further revealed that ATM-mediated DNA damage response triggers cell cycle delay, entry into the endocycle, and compensated cell expansion in fas1. These results suggest that the ATM-dependent DNA damage response is one of the upstream triggers for compensated cell expansion in Arabidopsis.

RESULTS

FUGU2 Encodes FAS1, the Large Subunit of CAF-1

The three alleles of fugu2, fugu2-1, fugu2-2, and fugu2-3, were originally isolated from a screen of mutants with altered leaf size (Horiguchi et al., 2006). To gain molecular insights into compensation, we performed map-based cloning of the FUGU2 gene using the fugu2-1 allele. Sequencing of genomic DNA revealed that fugu2-1 has a transposition in the 10th exon of FAS1, the large subunit of CAF-1 (Kaya et al., 2001), and that fugu2-3 has a 3.5-kb deletion from the promoter region to the sixth intron (Fig. 1A). Although we failed to identify the precise molecular lesion in fugu2-2, these data suggest that FUGU2 encodes FAS1. We also examined the mRNA levels of FAS1 in fugu2 mutants using semiquantitative reverse transcription (RT)-PCR and found that the level of FAS1 transcripts is partially reduced in fugu2-1 and fugu2-2 and undetectable in fugu2-3 (Fig. 1B). These results confirm that the FUGU2 locus corresponds to FAS1; thus, we renamed fugu2-1, fugu2-2, and fugu2-3 as fas1-5, fas1-6, and fas1-7, respectively.

Figure 1.

Figure 1.

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Cloning of the FUGU2 gene. A, Mutation points of each fas1 allele are indicated. The exon, intron, untranslated region, and intergenic region are indicated by black boxes, white boxes, gray boxes, and solid line, respectively. Arrows indicate the primers used for RT-PCR. B, Semiquantitative RT-PCR analysis of FAS1 expression in the wild type (WT) and each fas1 allelic mutant. Complementary DNA fragments amplified with the RT1-RT2 primer set are indicated in the top row, and those of the RT3-RT4 primer set are given in the middle row. The bottom row shows TUB4 expression as an internal control.

To further substantiate that the fas1 mutation causes compensation, we reexamined the leaf phenotype of fas1-4 (SAIL_662_D10) previously reported by Exner et al. (2006). The fas1-4 mutant has a transfer DNA insertion in the sixth intron of FAS1; accordingly, our RT-PCR analysis failed to detect the first half of the FAS1 transcript (Fig. 1B). As shown in Figure 2, A and B, fas1-4 has narrower and more serrated leaves than the wild type. An examination of subepidermal palisade cells in the first leaf also confirmed that fas1-4 has fewer cells (40%) than the wild type but that the size of individual cells is on average 150% larger compared with the wild type (Fig. 2, C and D). As a consequence, fas1-4 leaves are smaller than wild-type leaves but only by 70% (Fig. 2D), clearly indicating that fas1-4 exhibits a typical compensation phenotype. Although the level of FAS1 transcripts varies between fas1-5, fas1-6, and fas1-7, the degree of compensation is comparable in these alleles (data not shown). Thus, we used the fas1-4 and fas1-5 alleles for further analyses.

Figure 2.

Figure 2.

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Leaf phenotypes of fugu2/fas1 mutants. A, Whole rosettes of wild-type (WT; left) and fas1-4 (right) plants at 25 d after sowing. Bar = 10 mm. B, First leaves of wild-type (left) and fas1-4 (right) plants at 25 d after sowing. Bar = 10 mm. C, Palisade cells in the first leaf of wild-type (left) and fas1-4 (right) plants. Typical cells are marked in red. Bars = 100 μm. D, Leaf area, subepidermal palisade cell number per leaf, and projected cell area (left to right, respectively). First leaves of plants grown on rock wool at 25 d after sowing were used for the analysis. Values are means ± sd; n = 8.

The fas1 Mutation Up-Regulates the Expression of DNA Damage Response Genes in an ATM-Dependent Manner

During the development of first leaves, cells in the leaf primordium stop dividing at 10 d after sowing (Ferjani et al., 2007). Therefore, the molecular response that causes compensated cell expansion is expected to be up-regulated around this developmental stage. To explore the basis that induces compensation, we performed microarray analysis using total RNA extracted from the first pair of wild-type and fas1-5 leaves at 10 d after sowing. Our data from two independent experiments suggested that 67 genes are up-regulated and 118 genes are down-regulated in fas1-5 leaf primordia by more than 3-fold. We then examined these expression changes using semiquantitative RT-PCR and found that 46 genes are up-regulated in the leaf primordia of fas1-4 and fas1-5 (Supplemental Fig. S1) while 39 genes are down-regulated (data not shown).

To obtain an overview of these expression profiles, we compared these results with publicly available microarray data sets using the Genevestigator V3 tool (https://www.genevestigator.com/gv/index.jsp). We found that genes up-regulated in fas1 largely overlap with those that respond to known DNA damage treatments, such as UV-B irradiation or bleomycin and mitomycin C treatment (Supplemental Fig. S2A). We also compared our data with microarray data obtained from γ-irradiated plants (Culligan et al., 2006) and found that many of the genes up-regulated in fas1 overlap with those that respond to γ-radiation (Supplemental Fig. S2B). To validate this trend, we tested whether the expression of several known DNA repair genes, including PARP1, PARP2, RAD51, and BRCA1, is up-regulated in fas1-4 leaf primordia. As expected, our real-time RT-PCR analysis detected significant up-regulation of these DNA repair genes in fas1-4 (Fig. 3), further supporting that the DNA damage response is activated in developing leaves of fas1.

Figure 3.

Figure 3.

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Expression of DNA damage response genes in fas1-4 atm-2 and fas1-4 atr-2. RT-PCR analysis is shown for DNA repair genes (RAD51, BRCA1, PARP1, and PARP2) in the wild type (WT), atm-2, atr-2, fas1-4, fas1-4 atm-2, and fas1-4 atr-2. Total RNA prepared from the first leaf pair at 10 d after sowing was reverse transcribed and amplified by RT-PCR. All values were normalized against the expression level of the ACTIN2 gene and are expressed relative to the wild-type level. We used three biological replicates, and error bars indicate sd.

Two closely related kinases, ATM and ATR, are the central players of the DNA damage response in Arabidopsis (Garcia et al., 2003; Culligan et al., 2004). To test whether they participate in the DNA damage response in fas1 mutants, we generated the fas1-4 atm-2 and fas1-4 atr-2 double mutants. As shown in Figure 3, the atm-2 mutation almost completely abolishes the up-regulation of PARP1, PARP2, RAD51, and BRCA1 genes in fas1-4, while the atr-2 mutation has very little effect (Fig. 3). These results suggest that the fas1 mutation activates the ATM-dependent DNA damage response, leading to the up-regulation of DNA repair genes.

The atm Mutation Partially Suppresses the Compensation Phenotype in fas1

To explore the link between the ATM-dependent DNA damage response and the compensation of fas1 leaves, we examined whether the atm mutation interferes with the compensation phenotype in fas1. As predicted, atm-2 single mutants do not have any obvious defects in leaf morphology (Fig. 4, A and B), and their leaf cell number and size are approximately the same as those in the wild type (Fig. 4, C and D). When this mutation is introduced into fas1-4, it partially restores the growth defects of fas1-4 leaves (Fig. 4, A, B, and D). Quantitative analysis of cell number and cell size in the fas1-4 atm-2 double mutants revealed that the atm-2 mutation partially rescues the decreased cell number phenotype and that this recovery is associated with suppression of the compensated cell expansion phenotype in fas1-4 leaves (Fig. 4, C and D).

Figure 4.

Figure 4.

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Leaf size, cell number, and cell size of fas1-4 atm-2 and fas1-4 atr-2. A, Whole rosette of the wild type (WT), atm-2, atr-2, fas1-4, fas1-4 atm-2, and fas1-4 atr-2 at 21 d after sowing. Bar = 10 mm. B, First leaf of the wild type, atm-2, atr-2, fas1-4, fas1-4 atm-2, and fas1-4 atr-2 at 21 d after sowing. Bar = 10 mm. C, Palisade cells in first leaf of the wild type, atm-2, atr-2, fas1-4, fas1-4 atm-2, and fas1-4 atr-2. Typical cells are marked in red. Bar = 100 μm. D, Bar graphs indicating leaf area, cell number per leaf, and projected cell area of the subepidermal palisade layer (left to right, respectively). First leaves of plants grown on phytagel plates were harvested at 21 d after sowing and used for the analysis. Values are means ± sd; n = 8.

Our gene expression data suggest that the ATR-dependent pathway does not play major roles in the activation of DNA damage response genes in fas1 (Fig. 3). Consistently, the atr-2 mutation does not restore the leaf growth defects in fas1-4; instead, it retards the growth further (Fig. 4, A, B, and D). At the cellular level, we found that atr-2 does not rescue the cell number phenotype in fas1-4 but independently suppresses the compensated cell expansion (Fig. 4, C and D). These results suggest that the ATM-mediated DNA damage response participates in triggering compensation in fas1, while the ATR-mediated pathway contributes to compensated cell expansion through some other mechanisms.

The atm Mutation Partially Restores the Cell Cycle Delay in fas1

The mature fas1-4 leaves have less than 40% of palisade cells compared with the wild type (Figs. 2D and 4D). To test whether this is caused by delays in cell cycle progression or the premature termination of cell production during leaf development, we performed the kinematic analysis on the first leaf of wild-type and fas1-4 plants harvested at 5 to 12 d after sowing. As shown in Figure 5A, wild-type leaves show a steady increase in cell number from day 5 and stop producing new cells by day 12. Cell number in fas1-4 leaves is strongly reduced at day 5, indicating that cell cycle progression is already perturbed during early primordium development (Fig. 5A). Although fas1-4 cells continue to produce new cells up to day 12, the rate of cell production between days 5 and 8 is slightly reduced in fas1-4 leaves (Fig. 5A). We calculated the slope of the graph in Figure 5A to estimate the rate of cell production and found that the slope drops from 1.5 in the wild type to 1.3 in fas1, implying that the fas1 mutation has prolonged effects on cell cycle progression. These results suggest that the decreased cell number phenotype of fas1-4 primarily results from delayed cell cycle progression rather than from the premature termination of cell production.

Figure 5.

Figure 5.

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ATM contributes to the cell cycle delay in fas1-4. A, Kinematic analysis of leaf cell number of the wild type (WT), atm-2, atr-2, fas1-4, fas1-4 atm-2, and fas1-4 atr-2. The average cell number per leaf was calculated as described by De Veylder et al. (2001). Values are means ± sd; n = 8. *P < 0.001 (Student’s t test, between the wild type and fas1-4), **P < 0.01 (Student’s t test, between fas1-4 and fas1-4 atm-2). B, Flow cytometry analysis of the wild type, atm-2, atr-2, fas1-4, fas1-4 atm-2, and fas1-4 atr-2. First leaves of plants at 8 d after sowing were used. Values are means ± sd; n = 3. C, RT-PCR analysis of S-phase (CYCA3;1 and histone H4) and G2/M-phase (CYCB1;1, CYCB1;2, and KN) genes in the wild type, atm-2, atr-2, fas1-4, fas1-4 atm-2, and fas1-4 atr-2. Total RNA prepared from the first leaf pair at 10 d after sowing was reverse transcribed and amplified by RT-PCR. All values were normalized against the expression level of the ACTIN2 gene and are expressed relative to the wild-type level. We used three biological replicates, and error bars indicate sd.

To further investigate how cell production is perturbed in fas1-4, we performed flow cytometry analysis using the first pair of leaves from 8-d-old plants. Both wild-type and fas1-4 leaf cells contain only 2C and 4C nuclei at this stage, but the proportion of 4C to 2C nuclei is much higher in fas1-4 compared with the wild type (Fig. 5B). Interestingly, similar phenotypes are also described for mutants arrested at the G2/M phase of the mitotic cell cycle, such as e2f target gene1 (Takahashi et al., 2008), implying that the duration of G2/M phase is also prolonged in fas1-4. Therefore, we analyzed the expression of the S-phase genes CYCLINA3;1 (CYCA3;1) and histone H4, the G2/M-phase genes CYCB1;1 and CYCB1;2, and the M-phase gene KNOLLE (KN; Breuer et al., 2007; Takahashi et al., 2008). Our real-time RT-PCR analysis revealed that the expression of CYCA3;1 and histone H4 is comparable between the wild type and fas1-4, whereas the expression of CYCB1;1, CYCB1;2, and KN is elevated in fas1-4 (Fig. 5C). These results support the view that the fas1 mutation delays cell cycle progression at the G2/M phase.

To explore whether the ATM-dependent DNA damage response leads to the cell cycle delay in fas1, we examined whether the atm mutation interferes with the cell cycle phenotypes in fas1-4. The duration and rate of cell production are similar between the wild type and atm-2 (Fig. 5A). Compared with fas1-4, fas1-4 atm-2 leaves contain more cells at day 5 and the rate of cell production, as estimated by the slope of the graph in Figure 5A, is restored to 1.4 in fas1-4 atm-2 leaves, suggesting that the ATM-dependent DNA damage response contributes to the perturbation of cell production in fas1-4. Consistently, our flow cytometry analysis and RT-PCR analysis of cell cycle genes suggested that the atm mutation also partially recovers the cell cycle delay at the G2/M phase (Fig. 5, B and C). We should note that the expression of CYCB1;1 is strongly induced by DNA damage (Culligan et al., 2006); thus, the activated DNA damage response may also account for the accumulation of CYCB1;1 transcripts in fas1.

The atr-2 mutation does not restore the cell number defects in fully mature leaves from 21-d-old fas1-4 seedlings (Fig. 4D). Similarly, the impact of the atr-2 mutation on the cell production phenotype in fas1-4 is minor in 5- to 12-d-old seedlings, although we occasionally see a limited degree of recovery in fas1-4 atr-2 (Fig. 5A). Our flow cytometry analysis suggested that the atr-2 mutation may partially recover the G2/M progression defects in fas1-4 (Fig. 5B), but these results are not consistent with our RT-PCR data showing that atr-2 does not modify the expression of S-phase, G2/M-phase, and M-phase genes in fas1-4 (Fig. 5C). Together, these results suggest that the ATM-dependent DNA damage response pathway primarily contributes to the cell cycle delay in fas1.

The atm Mutation Partially Represses the High-Ploidy Phenotype in fas1

In Arabidopsis leaves, cell size often correlates with the nuclear ploidy level (Melaragno et al., 1993). Consistently, enhanced cell expansion in fas1 is associated with its higher ploidy phenotypes (Exner et al., 2006; Ferjani et al., 2007; Ramirez-Parra and Gutierrez, 2007). Given that the DNA-damaging chemical, zeocin, phenocopies these phenotypes, it is thought that the DNA damage promotes the endocycle and accompanied cell expansion. To examine whether the high-ploidy phenotypes of fas1 are induced through the ATM-dependent DNA damage response, we performed flow cytometry analysis using fully mature leaves and calculated the endoreduplication index (Sterken et al., 2012). The nuclear ploidy levels of cells in first leaves of 21-d-old wild-type plants range from 2C to 32C, indicating that many cells in wild-type leaves have undergone several rounds of endocycles (Fig. 6A). Compared with this, cells in first leaves of 21-d-old fas1-4 plants display higher ploidy phenotypes, with their endoreduplication index significantly higher than in the wild type (Fig. 6). The ploidy distribution in the atm-2 single mutant is indistinguishable from the wild type, but when this mutation is introduced into the fas1-4 background, it rescues the high-ploidy phenotype in fas1-4 (Fig. 6). These results strongly suggest that the ATM-dependent DNA damage response participates in the induction of high-ploidy phenotypes in fas1-4.

Figure 6.

Figure 6.

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Ploidy level distribution of fas1-4 atm-2 and fas1-4 atr-2. A, Ploidy level distribution of wild-type (WT), atm-2, atr-2, fas1-4, fas1-4 atm-2, and fas1-4 atr-2 nuclei from the first leaf of plants at 21 d after sowing. The most representative data are shown. B, Endoreduplication index of the wild type, atm-2, atr-2, fas1-4, fas1-4 atm-2, and fas1-4 atr-2. Values are means ± sd; n = 3. * P < 0.01 (Student’s t test).

Unexpectedly, our flow cytometry also revealed that the atr-2 mutation alone gives reproducible defects in endocycle progression (Fig. 6), suggesting that the ATR-mediated DNA damage response pathway is required for DNA repair during the endocycle. The fas1-4 atr-2 double mutants display ploidy distribution intermediate between fas1-4 and atr-2; consequently, their endoreduplication index is similar to that of the wild type (Fig. 6).

The atm and atr Mutations Partially Restore the Meristem Defects in fas1 Roots

The fas1 mutants display severe defects in the structures of the shoot and root meristem (Leyser and Furner, 1992; Kaya et al., 2001). Given that these defects are associated with the ectopic expression of meristem regulators such as WUS and SCR, misexpression of these genes is thought to cause the fas1 phenotypes (Kaya et al., 2001). Since the ATM-mediated DNA damage response pathway triggers cell cycle defects in fas1 leaves, we asked whether the same pathway also contributes to meristem defects in fas1. As described by Kaya et al. (2001), the typical arrangement of initial cells and columella cells found in the wild type is lost in fas1-4 mutants (Fig. 7, A and B). In contrast, fas1-4 atm-2 roots have a similar arrangement of initial and columella cells to the wild type (Fig. 7, A and B), indicating that the atm mutation suppresses these aspects of the fas1-4 phenotypes. In addition, we found that the size of the root meristem in fas1-4 is shorter than in the wild-type and that the atm-2 mutation suppresses this phenotype (Fig. 7, A and C). Interestingly, we also noticed that the atr-2 mutation restores both of these meristem defects in fas1-4 roots (Fig. 7). These data suggest that both ATM- and ATR-dependent DNA damage response pathways contribute to the defects in the root meristem of fas1.

Figure 7.

Figure 7.

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Root meristem phenotypes of fas1-4 atm-2 and fas1-4 atr-2. A, Root meristems of the wild type (WT), atm-2, atr-2, fas1-4, fas1-4 atm-2, and fas1-4 atr-2 at 9 d after sowing. Bar = 100 μm. Arrowheads mark the positions of meristems. B, Initial cells and columella cells of the wild type, atm-2, atr-2, fas1-4, fas1-4 atm-2, and fas1-4 atr-2 at 9 d after sowing. Bar = 100 μm. C, The number of cortex cells in the root meristem was counted in 9-d-old wild-type, atm-2, atr-2, fas1-4, fas1-4 atm-2, and fas1-4 atr-2 roots. Values are means ± sd; n = 10. *P < 0.01, **P < 0.05 (Student’s t test).

DISCUSSION

In this study, we demonstrate that the fas1 mutants display compensation phenotypes through activation of the ATM-dependent DNA damage response pathway. Our data suggest that the ATM-dependent DNA damage response leads to delay in the mitotic cell cycle and promotion of the endocycle, finally resulting in the induction of compensated cell expansion in fas1 (Fig. 8).

Figure 8.

Figure 8.

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A scheme that explains how compensation is induced in fas1 leaves.

ATM-Dependent Up-Regulation of DNA Damage Response Genes in fas1 Leaves

Our microarray analysis and subsequent RT-PCR analysis revealed that the expression of DNA damage response genes is up-regulated in developing leaves of fas1 (Fig. 3; Supplemental Fig. S2). A previous study reported that the expression of genes involved in the DNA damage response is up-regulated in fas1 (Schönrock et al., 2006), but it was not clear in which organs the DNA damage response takes place, because total RNA extracted from whole seedlings was used for the analysis. In this study, we used total RNA extracted from leaf primordia. Our data largely agree with the data of Schönrock et al. (2006) and suggest that leaf primordium is at least one of the organs in which the DNA damage response is activated by the loss of FAS1 function.

The DNA damage response largely consists of DNA repair and cell cycle arrest, and γ-irradiation induces the up-regulation of hundreds of genes involved in these processes (Culligan et al., 2006). With γ-irradiation, ATM is responsible for the up-regulation and ATR has almost no roles in the process (Culligan et al., 2006). Our expression analysis using fas1 atm and fas1 atr demonstrated that only the absence of ATM suppresses the up-regulation of several DNA repair genes in fas1 leaves, suggesting that up-regulated expression of DNA repair genes in fas1 leaves depends only on ATM. Therefore, the transcriptional response of several DNA repair genes appears to be regulated similarly in γ-irradiation and loss of FAS1 function. A previous study reported that fas1 has an increased amount of endogenous double-strand breaks (Endo et al., 2006). Thus, it is likely that the accumulated double-strand breaks by the fas1 mutation activate the ATM-mediated DNA damage response.

The ATM-Dependent DNA Damage Response Leads to Compensation in fas1

Our genetic analysis revealed that the atm mutation partially suppresses the decrease in cell number in fas1 (Fig. 4D). Furthermore, kinematic analysis suggested that the cell number phenotype of fas1 is a result of the cell cycle delay and that the atm mutation also partly restores this phenotype (Fig. 5A). Our flow cytometry analysis and gene expression analysis also showed that young fas1 leaves contain more cells at the G2/M phase compared with the wild type and that this phenotype is also partly dependent on ATM (Fig. 5, B and C). Together, these data suggest that the ATM-dependent DNA damage response triggers the G2/M arrest in fas1, leading to a decrease in cell number. Our flow cytometry analysis on fully mature leaves indicated that the fas1 mutants display higher ploidy phenotypes in an ATM-dependent manner (Fig. 6), suggesting that activation of the ATM-mediated DNA damage response pathway also leads to the promotion of endocycles. Since the atm mutation suppresses both ploidy and compensated cell expansion phenotypes in fas1, our data support the potential role of the endocycle in compensated cell expansion. Based on our current knowledge of the function of ATM, it is unlikely that ATM directly regulates the endocycle. Instead, we speculate that ATM is required for the sequential process (i.e. DNA damage response, leading to endoreduplication). Interestingly, a recent study by Adachi et al. (2011) showed that Arabidopsis culture cells or root cells arrested by DNA damage transit into the endocycle and undergo cell expansion prematurely.

It is intriguing that, in contrast to the partial recovery of cell number, compensated cell expansion in fas1 is completely suppressed by the atm mutation (Fig. 4, C and D). Fujikura et al. (2009) proposed a threshold theory in which compensation is triggered only when the down-regulated cell proliferation activity is below a certain threshold. If this theory is applied to fas1, the observed phenomenon can be interpreted as follows. The level of decrease in cell proliferation activity in fas1 caused by the ATM-dependent cell cycle arrest is enough to trigger compensated cell expansion. However, when ATM is disrupted, cell proliferation activity is partially restored, and this recovery is sufficient to prevent exceeding the threshold, thereby leading to a complete suppression of compensation.

We should also note that the atm mutation does not fully restore the growth defects of fas1 leaves and that the cell number of fas1 atm leaves is still less than that of wild-type leaves (Fig. 4). These data suggest that additional mechanisms also contribute to cell cycle arrest and subsequent compensation in fas1. Given that fas1 mutants display various pleiotropic phenotypes (Kirik et al., 2006; Ono et al., 2006; Schönrock et al., 2006; Mozgová et al., 2010), some of these defects may have downstream consequences in cell cycle progression.

The ATR-Dependent DNA Damage Response Pathway Is Required for Endocycle Progression

Compared with fas1, fas1 atr seems to have a slightly increased number of cells in developing young leaves (Fig. 5A), but they have similar numbers of cells in fully mature leaves (Fig. 4D), suggesting that ATR has only a minor effect on the cell number phenotype in fas1. Furthermore, the induced expression of G2/M-phase genes in fas1 is not suppressed by atr, supporting that ATR is not primarily involved in cell cycle delay at the G2/M phase. Why our flow cytometry data are not consistent with these views and appear to show that atr partly cancels the accumulation of 4C nuclei in young fas1 leaf cells is not clear (Fig. 5B). One possibility that might explain this discrepancy is that some proportion of 4C nuclei we detected by flow cytometry might actually have entered into the endocycle, especially in fas1, and that with the role of ATR in the endocycle, as discussed below, the atr mutation may block this progression into the endocycle, resulting in the apparent reduction of 4C nuclei in fas1 atr. Since the transition into the endocycle is controlled both transcriptionally and posttranslationally (Komaki and Sugimoto, 2012), fas1 cells might be able to enter the endocycle while keeping the transcript level of G2/M genes relatively high (Fig. 5C).

In contrast to the minor effect on the cell number phenotype, compensated cell expansion in fas1 is completely suppressed by atr, suggesting that ATR has more direct impacts on compensated cell expansion in fas1. Our flow cytometry analysis of fully mature leaves revealed that atr mutants have decreased ploidy compared with the wild type (Fig. 6). It is known that the molecular function of ATR is to sense DNA replication folk stress; therefore, ATR seems to be required to deal with replication stresses associated with the successive progression of endocycles.

The DNA Damage Response Pathway Contributes to the Meristem Defects in fas1 Roots

The fas1 mutant was isolated as a mutant that has stem fasciation phenotypes (Leyser and Furner, 1992), and a previous study suggested that correct chromatin assembly by the CAF-1 complex is important to regulate the expression of genes required for meristem maintenance (Kaya et al., 2001). In contrast, our observations in this study revealed that the meristem defects in fas1 roots are partially suppressed by atm and atr, suggesting that the DNA damage response pathway contributes to the meristem defects in fas1 (Fig. 7). Although we did not investigate the DNA damage response in roots further, the fas1 mutation may induce the damage response similar to shoots, leading to G2/M arrest and/or premature onset of the endocycle in the root meristem. Adachi et al. (2011) reported that both ATM- and ATR-dependent pathways participate in the DNA damage response and the induction of endocycles in Arabidopsis roots. Our data are consistent with this and further support that both ATM and ATR pathways contribute to the DNA damage response in the root meristem. It will be interesting to test whether the DNA damage response also participates in fasciation or other shoot meristem defects in fas1. We did not address these questions because the fas1-4 allele we used in this study does not display strong shoot phenotypes under our growth conditions.

In conclusion, our study showed that one mechanism to induce compensation is mediated through the ATM-dependent DNA damage response pathway. Whether this type of compensatory mechanism also operates under normal growth conditions will be an interesting question for future studies.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

The Arabidopsis (Arabidopsis thaliana) wild-type accession used in this study was Columbia. As described previously, allelic fugu2 mutants were in the Columbia background (Horiguchi et al., 2006; Ferjani et al., 2007). The fas1-4 mutants (SAIL_662_D10) were obtained from the Arabidopsis Biological Resource Center. The atm-2 and atr-2 mutants in the Columbia background were kind gifts from Kevin Culligan and Anne Britt. For histological analyses, plants were grown either on rock wool or on plates containing Murashige and Skoog salts, pH 5.8, 1% (w/v) Suc, and 0.5% (w/v) phytagel at 22°C under a 16-h-light/8-h-dark photoperiod.

Microarray Analysis

For the microarray analysis, plants were harvested at 10 d after sowing, and total RNA was extracted from the first pair of leaf primordia using the RNeasy Plant Mini Kit (Qiagen). Microarray analysis was performed for two independent biological materials by using the ATH1 expression array (Affymetrix). Array data were processed and analyzed with Microarray Suites 5.0 software (Affymetrix).

Quantitative Real-Time RT-PCR Analysis

Plants were harvested at 10 d after sowing, and total RNA was extracted from leaf primordia using the RNeasy Plant Mini Kit (Qiagen). One microgram of total RNA was reverse transcribed using the Prime Script RT Reagent Kit with gDNA Eraser (TaKaRa). Each complementary DNA sample was diluted 1:9 in water, and 1 μL of this dilution was used as a PCR template. Quantitative real-time RT-PCR was performed using the THUNDERBIRD qPCR Mix (Toyobo) on an Mx3000P QPCR System (Agilent Technologies). The gene-specific primer sets used in this study are listed in Supplemental Table S1. Expression levels were normalized with respect to those of ACTIN2 and averaged over at least three technical and three biological replicates.

Microscopic Analysis

To measure leaf area, cell number, and cell size, leaves were fixed with formalin/acetic acid/alcohol and cleared with chloral solution (200 g of chloral hydrate, 20 g of glycerol, and 50 mL of distilled water) as described previously (Tsuge et al., 1996). Whole leaves were observed using a stereoscopic microscope (MZ16a; Leica Microsystems), and individual leaf cells were visualized using a microscope equipped with Nomarski differential interference contrast (DMRX E; Leica Microsystems). To observe the root meristem structure, roots were stained with 10 μg mL−1 propidium iodide and visualized using the Leica TCS-SP5 confocal laser microscope.

Ploidy Measurements

Ploidy levels were quantified by flow cytometry (PA-I; Partec) as described previously (Sugimoto-Shirasu et al., 2005). At least 7,000 nuclei isolated from the first pair of leaves were used for each ploidy measurement. Endoreduplication index (EI) was calculated as EI = (0 × %2C) + (1 × %4C) + (2 × %8C) + (3 × %16C) + (4 × %32C) (Sterken et al., 2012) and averaged over at least three technical replicates.

Supplemental Data

The following materials are available in the online version of this article.

Acknowledgments

We thank Kevin Culligan and Anne Britt (University of California, Davis) for providing atm and atr mutants.

Glossary

RT

reverse transcription

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