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Tebichi null mutants display variable developmental defects
Previous work reported the phenotype of teb mutants, with stunted growth and deformed leaves (Inagaki et al., 2006). Five alleles of the mutant were initially described, three of which: teb1, teb2 and teb5 gave rise to the same phenotype and appeared to be full loss of function mutants (Inagaki et al., 2006) (Figure 1A). However, rice mutants did not show developmental defects (Nishizawa-Yokoi et al., 2020), and other groups reported much milder phenotypical defects for teb2 and teb5 mutants (van Kregten et al., 2016). To clarify this, we carefully re-examined the phenotype of these mutants. In our growth conditions, most of the teb2 and teb5 mutants appeared indistinguishable from the wild-type after 1 month of growth (Figure 1B). We classified teb mutants’ phenotypes in two categories: wild type like (WTL) plants appeared identical to the wild-type (Col-0) and plants with severe (S) developmental defects showed the previously described tebichi phenotype (Figure 1B). We first checked that both WTL and S plants were homozygous for the teb mutation, using primers flanking the T-DNA insertions in teb2 and teb5 mutants (Figure 1A, C). We also checked by qPCR that both teb alleles we used did not allow the expression of the full length POLQ mRNA. To this end we used three primer pairs: one (#1) at the 5’ end of the TEB gene, upstream both insertions, one flanking the T-DNA insertion site of the teb5 mutant (#2), and one (#3) in the 3’ moiety of the gene (Figure 1A). The first primer pair allowed detection of wild-type levels of mRNA in both mutants, indicating that the 5’ extremity of the gene is normally expressed (Figure 1D). However, the teb2 mutant accumulated no detectable transcripts produced downstream of the insertion. Expression of the 3’ moiety of the gene was drastically reduced in teb5 and no mRNA spanning the insertion site could be detected (Figure 1D). Thus, neither teb2 nor teb5 accumulate full length TEB mRNA, and are likely knock-out mutants, consistent with previous reports (Inagaki et al., 2006). We next quantified the distribution of teb mutants between the two phenotypic categories. In our growth conditions ~ 85-90% of teb mutants were in the WTL category and only 10% to 15% in the S category corresponding to the previously described phenotype (Figure 1E). Importantly, the S phenotype was never observed amongst wild-type plants, and is therefore characteristic of teb mutants.
Since teb mutants were shown to display a constitutive upregulation of DNA damage responsive genes (Inagaki et al., 2009), we asked whether the severity of the phenotype may correlate with the levels of expression for DDR genes. We thus determined the expression level of BRCA1 that is involved in the DNA repair (Lafarge and Montané, 2003) and SMR7 (Yi et al., 2014) which is an inhibitor of cell cycle progression, in rosette leaves of teb plants. Plants from the two phenotypic classes displayed upregulation both genes as previously reported (Inagaki et al., 2009), but no significant differences were observed between teb plants with different phenotype (Figure S1).
We next asked whether the observed variability in the teb mutant phenotype could also be observed earlier during development. Indeed, we observed that 15-day-old teb mutants displayed a higher proportion of plants with arrested root growth than the wild-type (Figure S2). Likewise, at 7 days after germination, plantlets displayed more variable sizes than the wild-type, with a higher proportion of small plantlets with shorter roots and smaller cotyledons (Figure S3A, B). To determine whether this phenotypic variability related to increased DNA damage accumulation, we performed immuno-labelling of phosphorylated-H2AX variant on root tips of wild-type plants and small and big plantlets of teb mutants, that forms foci at the site of DSBs (Charbonnel et al., 2010). As shown on Figure 2, we could observe a significant increase in-H2AX labelling in teb mutants: the percentage of root tip nuclei showing-H2AX foci was around 1% in the wild-type, and around 10 % in both teb mutant alleles. However, the percentage of labelled nuclei was not significantly different between big and small plantlets. Consistently, DDR genes activation did not differ significantly between small and big teb mutants (Figure S3C, D). Furthermore, plants with arrested root growth did not show severe teb mutant plants at later stages: we selected 20 of those plantlets and transferred them to the green house, but none of them developed a severe phenotype after 3 weeks. Collectively, these results indicate that loss of Pol results in an increase in DNA damage accumulation in proliferating cells, but that the appearance of the teb severe phenotype is stochastic and does not correlate with significantly higher levels of DNA damage or DDR activation.
One possible explanation for the stochastic appearance of the severe phenotype in teb mutants could be the accumulation of mutations as a consequence of defects in DNA repair. Under such a scenario, developmental defects would be expected to be transmitted to the next generation, or to aggravate in the next generation. To test this, the progeny of WTL and S plants was sown, and we evaluated the distribution of plants between the two classes in the next generation. However, the distribution of plants between the two classes was the same in the subsequent generation (Figure S4), suggesting that developmental defects are not due to heritable mutations.
Pol is involved in replicative stress tolerance
Pol θ has been proposed to play a key role in replicating cells (Inagaki et al., 20 09), we therefore asked whether replicative stress could increase the proportion of plants showing developmental defects in teb mutants. Wild-type and teb mutants were germinated on MS supplemented with Hydroxyurea (HU, 0.75 mM). At 10 days after germination, the survival rate of teb mutants was lower than that of wild-type plants (Figure S5), indicating that teb mutants are hypersensitive to replicative stress. After 10 days, surviving plants were transferred to soil, and the proportion of plants with a WTL or S phenotype was assessed after 3 weeks. Wild-type (Col-0) plants subjected to this treatment displayed a growth reduction but did not show other developmental defects such as deformed leaves (Figure S6). By contrast, as shown on Figure 1E, the proportion of plants with severe developmental defects was significantly increased in both teb2 and teb5 mutants. The proportion of S plants increased from less than 15% to almost 30%, indicating that replicative stress may be the cause for developmental defects observed in teb mutants.
To further explore the role Pol in response to replicative stress, we took advantage of the hypomorphic mutant pol2a-4. This mutant (also called abo4-1) is partially deficient for the replicative DNA polymerase Pol (Yin et al., 2009), and we have shown that it displays constitutive replicative stress (Pedroza-Garcia et al., 2017). The teb2 and teb5 mutations were therefore introduced in the pol2a-4 background by crossing, generating the pol2a teb2 and pol2a teb5 double mutants.
Six weeks old plants of all mutant combinations are shown in Figure 3A. Interestingly, pol2a teb double mutants displayed severe developmental defects that were fully homogeneous between individuals. To further characterize the developmental defects of pol2a teb double mutants, we quantified root length: we observed that teb and pol2a roots were shorter compared to wild type plants, as previously reported (Inagaki et al., 2006; Pedroza-Garcia et al., 2017). In addition, root length of pol2a teb double mutants was significantly reduced compared to single mutants (Figure 3B and 3C). Because teb mutants display disorganized meristem and spontaneous cell death in root tips (Inagaki et al., 2006), we evaluated whether these defects were exacerbated in pol2a teb double mutants. Root tips of eight-day-old plants from mutant combinations were observed by confocal microscopy after propidium iodide staining. We observed disorganized meristem and cell death in the teb mutants, confirming the result of the previous study (Inagaki et al., 2006). Furthermore, meristems were severely compromised in pol2a teb double mutants (Figure 4A-F) with disorganized patterning, extensive cell death and differentiation of root hair close to the tip of the root. Finally, meristem length was measured in these mutants, showing that pol2a and teb mutants have smaller root meristem size compared to the wild type Col-0, and more drastic reduction of meristem size was observed in the pol2a teb double mutants (Figure 4G). Futhermore, pol2a teb double mutants accumulated significantly higher levels of-H2AX foci than teb single mutants, whereas pol2a mutants did not accumulate more DSBs than the wild-type, as previously reported ((Pedroza-Garcia et al., 2017), Figure 2D). Together, these results indicate that cell proliferation is more severely compromised in pol2a teb double mutants than in parental lines, likely due to increased accumulation of DNA breaks, consistent with the notion that Pol plays a key role in the repair of replication-associated DNA damage.
Our results indicate that loss of Pol impairs the repair of replication-associated DNA damage, which could lead to the activation of the DDR response. To test this hypothesis, we next checked the expression of DNA damage responsive genes in all mutant combinations by qRT-PCR (Figure 5). We selected genes representative of different responses triggered by DDR activation such as DNA repair genes (RAD51 and BRCA1) and cell cycle regulation (SMR5/7, WEE1 and CYCB1; 1). Expression of all tested genes was induced in the teb single mutants and in pol2a compared to wild-type Col-0, consistent with previous reports (Inagaki Total RNA was extracted from twelve-day-old plantlets. Expression of selected genes was assessed by real-time qPCR and normalized to actin. We monitored the expressions of genes involved in cell-cycle arrest (SMR5, SMR7 and WEE1), DNA repair (RAD51 and BRCA2) and both (CYCB1;1). Values are Fold change compared to the wild-type Col-0. Graphs represent average of 3 technical replicates +/- standard deviation and are representative of 3 independent biological replicates. Different letters above bars denote statistically relevant differences (ANOVA followed by Tukey test, performed on raw data before normalization, p<0.01).
Abiotic stresses aggravate the severity of teb mutants’ phenotype.
Taken together, our results indicate that a key cellular function of Pol is to allow repair of replication-associated DNA damage. This led us to postulate that the discrepancies between our observations and previous reports regarding the severity of teb mutants’ phenotype could stem from different intensities of basal replicative stress between laboratories, due to different growth conditions. Under such a scenario, abiotic stresses would be expected to impact the severity of teb mutants’ phenotypes. To test this hypothesis, we subjected teb2 and teb5 mutants to various abiotic stress conditions: high light intensity (HL, 350 µmol x m-2 x s-1), salt treatment (50 or 100 mM of NaCl) and heat (growth at 32°C). Except for the HL treatment, plants were grown under a low light intensity (LL, 160µmol x m-2 x s-1). After 3 weeks, we counted the plants in each phenotype category (n>50). These treatments obviously modified the phenotype of wild-type plants but did not induce the appearance of the conspicuous teb-like phenotype in Col-0 plants (Figure S7). It is worth noting that the HL condition could not be considered as a stress condition for wild-type plants as they grew faster and reached a larger size than under LL conditions (Figure S7). The proportion of S plants increased under HL and high salt stress (100 mM) for both teb2 and teb5 mutants (Figure 6A, B). By contrast, a lower concentration of salt (50 mM) had no impact on the distribution of teb mutants between the different phenotypic classes. Likewise, growth at 32°C did not significantly affect the proportion of teb mutants with a severe phenotype. We tried increasing the temperature to 37°C, but the proportion of plantlets that did not survive in these growth conditions was over 50% in both wild-type and mutants, which prevented further analysis. To determine whether abiotic stress conditions affected the level of DDR activation in teb mutants, we monitored the expression of DDR marker genes. Results obtained on mature plants were too variable to draw robust conclusions, so experiments were performed on in vitro grown plantlets (Figure S8). The two DNA-repair genes (XRI-1 and BRCA2) and the cell cycle inhibitor SMR7 were induced by salt treatment but not by high-light in wild-type plants: the expression level of these three genes was induced about 1.7-fold by salt treatment in the wild-type. By contrast expression of SMR5 did not change between growth conditions.
Table of contents :
I- Introduction
I-1 Review: The plant DNA Damage Response: signalling pathways leading to growth inhibition and putative role in response to stress conditions
Overview
Abstract
INTRODUCTION
Main players in DDR signalling
Role of the plant DDR in abiotic stress responses
Role of the plant DDR in biotic stress response
Concluding remarks
I-2- DNA replicative stress
I-2-1 Overview
I-2-2 What is replicative stress?
I-2-3 Trans lesion synthesis (TLS)
I-2-4 Repriming synthesis
I-2-5 ATR dependent responses
I-2-6 Other processes regulated by ATR
3- Thesis objectives
II- Results
II-1-Exploring new players that contribute to plants’ replicative stress response
II-1-1 Identification of LUMINIDEPENDENS as a new player in the plant replicative stress response.
II-1-2 Distinctive and emerging roles of E2Fs transcription factors during plant replicative stress response
II-2- Role of DNA polymerase θ in the repair of replication-associated DNA damage
II-2-1 OVERVIEW
II-2-2 Article: The plant DNA polymerase theta is essential for the repair of replicationassociated DNA damage
II-3 Understanding the role of the novel interaction between CDT1 and Polymerase epsilon
II-3-1 OVERVIEW
II-3-2 INTRODUCTION
II-3-2 STRATEGIES
II-3-3 Conclusing remarks and future perspective
III- Material and methods:
III-1 Plant growth and genotyping
Plant material and growth conditions
CTAB DNA extraction
dCAPS genotyping
Root growth assay
III-2 Protein-protein interactions
Yeast to hybrid
Co-immunoprecipitation (Co-IP)
III-3 Imaging and flow cytometry
GUS staining
Flow cytometry
EdU labelling
Confocal microscopy imaging
RNA extraction and quantitative RT-PCR
Statistical analysis
RNA-seq analysis
IV References
