Sieda (Shade Book 3)

INTRODUCTION

B Inefficient splicing of intron 9 leads to reduced expression of functional SAV6 in the mutants. Failure to remove intron 9 would reveal a stop codon, leading to a truncated protein with 30 extra amino acids. Two independent transgenic lines SAV6 pro: More than three independent lines were analysed and expression patterns common in all three lines were shown. To verify the mapping result, we performed a complementation test.

The hypocotyl and root lengths of SAV6 pro: As shown in Figure 2C , D and Supplementary Figure S1C, the short root and hypocotyl phenotypes of sav6 were completely rescued by the transgene. Expression of SAV6 was stronger in the progenitor cells of the stomatal lineage, such as the primary and satellite meristemoids, than in fully developed stomata cells Supplementary Figure S2D.

Trichomes are highly modified single cells that undergo several endoreplication cycles during their morphogenetic development 30— The expression pattern of SAV6 therefore suggests that SAV6 may function in both dividing cells and some of the cells that undergo endoreplication. We compared the protein sequences of these Arabidopsis proteins with FEN1 from different organisms. Oryza sativa , rice; Dre: Danio rerio , zebra fish; Hsa: Homo sapiens , human; Mmu, Mus musculus , mouse; Dme: Drosophila melanogaster , fly; Sc: Saccharomyces cerevisiae , yeast.

Values below branches indicate bootstrap values. Alternatively, SAV6 may require other plant proteins for its full function. We further characterized the nuclease activity profile of SAV6 using five standard substrates, based on a series of publications describing the hFEN1 substrates 10 , These substrates include the following: The short hypocotyls and roots of sav6 mutants may result from a reduced cell number or reduced cell length, or both.

We compared the hypocotyl epidermal cell profile of the Col-0 wild type and sav6. Wild type hypocotyls should consist of around 20 epidermal cells, and elongation of hypocotyls results mostly from cell expansion reviewed by Our results showed that the epidermal cell number in sav6 was reduced compared to that in the wild type, and this phenotype was rescued by the wild type SAV6 gene Figure 5A. We then measured the cell lengths of the hypocotyl epidermal cells, but found no significant differences in cell length between the Col-0 and sav6 seedlings grown in Wc, simulated shade or darkness Figure 5B.

These results indicated that the short hypocotyls of sav6 resulted from reduced cell number rather than reduced cell length, and the sav6 mutation may affect hypocotyl cell division during embryogenesis. Mutation of SAV6 affects both cell division and elongation. C Root apical meristem RAM size is reduced in sav6. The white arrowhead indicates the quiescent centre QC cells. D Cells in the maturation zone of sav6 roots are shorter than those in the wild type.

E QC maintenance is compromised in sav6 mutants.

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Error bars represent the SEM. Integrated cell proliferation and cell expansion controls the root length. Cells in the elongation zone of the roots stop cell division and initiate differentiation. They undergo endoreplication and start to elongate. We measured the lengths of cells entering the maturation zone, where root hairs emerge. Therefore, SAV6 is required for both cell division and elongation in roots. During root development, QC cells function as stem cell organizing centres by creating a microenvironment that maintains the stem cell fate of its surrounding cells.

In sav6 mutants, however, signals from QC25 were dramatically reduced or completely absent, and those from QC46 were absent. Similar results were obtained when a WOX5 pro: The above results indicate that the maintenance of QC cells is impaired in sav6. Deletion of yeast Fen1 rad27 results in a high level of sensitivity to DNA damaging agents such as UV irradiation and methyl methane sulfonate, a strong mutator phenotype, and conditional lethality We tested the response of sav6 to UV-C.

The seedlings were treated with various doses of UV-C light and then allowed to recover for 5 days in light.

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As shown in Figure 6A , with increasing doses of UV-C irradiation, wild type seedlings became chlorotic and this response was much more dramatic in the sav6 mutants. UV-C treatment also inhibited root elongation Figure 6B , and this effect was again enhanced in sav6. A Seedling responses of sav6 to various doses of UV-C. B Root response of sav6 to UV-C.

We tested the sensitivity of sav6 to various genotoxic reagents. HU is a ribonucleotide reductase inhibitor that blocks DNA replication. As shown in Figure 7A , the roots of sav6 were hypersensitive to CPT-induced growth inhibition, and this effect was mostly rescued by expression of wild type SAV6. For Zeocin treatment, we used PI staining to detect dead cells in roots. Zeocin treatment induced cell death in wild type seedlings and this effect was enhanced in sav6 mutants. A The roots of sav6 were hypersensitive to CPT-induced growth inhibition.

L1 is one of the SAV6 pro: B sav6 is hypersensitive to Zeocin. Dead cells were detected by propidium iodide PI staining. Because dead cells were detected in sav6 mutants even without Zeocin treatment, we speculated that the responses to DNA damage were constitutively activated in sav6 mutants. The expression levels of three DNA damage response genes: As shown in Figure 7C , the expression levels of all three genes were elevated in the roots of sav6 mutants, which was also partially suppressed by the SAV6 transgene Supplementary Figure S5C , confirming that responses to DNA lesions are induced in sav6 roots.

It was previously reported that mild treatment with DSB-inducing reagents such as X-rays and radiomimetic drugs can induce cell death in stem cells and their early descendants, whereas, Aphidicolin, which specifically inhibits nuclear DNA replication, did not induce cell death 7. Because HU can also induce the expression of the above three marker genes, we identified a marker gene, At4g , which is specifically induced by DSB-inducing gamma rays and bleomycin 5.

As shown in Supplementary Figure S5D and S5E, both short term 1 and 3 h and long term 12 and 24 h Zeocin treatment elevated the expression of this gene. UV-C treatment also slightly increased its expression, but HU or Aphidicolin did not change its expression. In sav6 , we also observed a significant increase in At4g expression, which was completely rescued by introduction of the genomic SAV6 transgene Supplementary Figure S5F , supporting our hypothesis that the observed phenotypes and elevated expression of the marker genes in sav6 may result from DNA damage stress, likely to be DSBs, but not from DNA replication stress.

Both replication stress and response to DNA damage will induce cell cycle arrest. C Knocking down the expression of SMR7 in sav6 increases the susceptibility of the transgenic line 35S: Dead cells were detected by PI staining. The experiment was repeated three times in total and similar patterns were observed each time.

However, the development of the QC cells was not affected. It is widely believed that in response to DNA damage stress, the cell cycle is arrested to prevent damaged DNA from passing into the next generation. To evaluate DNA damage-induced cell death in sav6 and 35S: As shown in Figure 8C , there are more dead cells in 35S: QC cells rarely divide. They demonstrated that when these seedlings were transferred to a bleomycin-free medium later, the number of cells expressing QC marker gene, WOX5 , increased, which may be associated with the activation of PSK5 6.

We therefore examined the expression of PSK5 in sav6 seedlings and discovered that its expression was significantly elevated in sav6 roots Figure 9A. The altered expression of all three genes was fully or partially rescued by introduction of the SAV6 gene Supplementary Figure S7. We therefore propose that the constitutively activated DNA stress response pathways in sav6 may induce QC cell division through increased PSK5 expression, which subsequently leads to the loss of QC identity in those cells. B Long exposure to Zeocin abolished the expression of QC marker genes.

To test this hypothesis, we first examined if constitutively elevated PSK5 expression would affect QC development. Indeed, as shown in Supplementary Figure S8A, wild type seedlings grown on BL for 7 days exhibited elevated PSK5 expression in roots, which was similar to the response to Zeocin treatment. We then examined the phenotypes of QC Similarly, expression of WOX5 exhibited reduced intensity, and showed a more diffuse expression pattern as the concentrations of BL increased. WOX5 was reported to inhibit cell division We further tested how genotoxic reagents affect QC development.

When the concentrations of these chemicals further increased, the expression of WOX5 decreased significantly. The GUS signal of the QC46 marker genes did not show an increased expression domain, but became undetectable once the WOX5 expression domain increased, suggesting that QC identity may be lost before the complete disappearance of the WOX5 signal.

In summary, the above results suggested that prolonged exposure to genotoxic reagents may promote QC cell division through the activation of PSK5 , which eventually leads to the loss of their QC identity. Similarly, defects in QC development in sav6 may result from a constitutively active DNA stress response. In this study, we isolated an Arabidopsis sav6 mutant that contains a point mutation in a putative At FEN1 gene. It was also suggested to be the preferred cellular substrate of FEN1 GEN activity on the bubble DNA substrates can be translated into the ability to repair stalled replication forks In addition, in some of the cells, SAV6 localizes to specific nuclear foci.

The nucleolus is not stained with DAPI, which appears as a dark circle inside of the blue nucleus. The patterns of these speckles look similar to those of the chromocenters. It would be interesting to see if the formation of these speckles is regulated or correlates with DNA repair. Furthermore, as the EXO or GEN activity of FEN1 can be greatly stimulated by its interacting proteins 27 , 45 , 46 , it would be of interest to identify proteins that may interact with SAV6 and regulate its activity in vivo.

Null mutations in Rad27 , a yeast FEN1 homologue, result in slow growth, hypersensitivity to DNA damaging reagents and genome instability, whereas homozygous Fen1 knock-out in mice is embryonic lethal 11— When grown under normal conditions, the most obvious phenotypes of sav6 are the short hypocotyls and primary roots, which were both completely rescued by genomic SAV6.

In sav6 , the mutation reduces the splicing efficiency of SAV6 , and it is therefore a knock-down mutant of SAV6 , which explains the rather mild phenotype of this mutant.

Expression pattern analysis revealed that SAV6 is highly expressed in tissues with rapidly dividing cells, such as root and shoot apical meristems, lateral root primordia, and progenitor cells of the stomatal lineage. It is also expressed in cells that undergo endoreplication, such as trichomes. We observed that primary roots of sav6 stopped growing a few days after germination.

A lateral root emerged to replace the primary root Supplementary Figure S1A.

As this lateral root can grow much longer compared to the primary root Supplementary Figure S1B , its defect in RAM may not be as severe as that observed in the primary root. We speculate that the severity of the defect may be associated with the cell division rates.

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Alternatively, at different developmental stages, different degrees of DNA stresses may be encountered, which affects the severity of the phenotype. After being transferred to soil, sav6 mutants grow normally Supplementary Figure S10A , indicating that there are other mechanisms to compensate for the developmental defects associated with sav6. Finally, seedlings treated with DSB-inducing reagents, but not Aphidicolin, exhibited cell death in meristem cells and their early descendents 7 and we also observed cell death in sav6 roots without any treatment.

Together, the above data indicate that SAV6 is required for both the maintenance of genome integrity and response to DNA damage. Consistent with the above hypothesis, we found no obvious defect in endoreplication-regulated processes, such as trichome development Supplementary Figure S10B.

We speculate that the original sav6 mutant may contain other mutations that affect the response to DNA damage, as the SAV6 pro: Alternatively, the presence of enhancers outside of the transgene we used could explain the partial rescue phenotype. Because the described phenotypes were partially suppressed by introduction of a genomic SAV6 transgene and the characterization of the sav6 mutant was performed using the sav6 mutant that was backcrossed to Col-0 for three generations, we believe our conclusions should still be reliable.

Instead, it may maintain telomere stability by facilitating replication through the G-rich lagging strand and ensuring high fidelity telomere replication Telomeres protect chromosome ends from being recognized as DSBs and help to solve the end replication problem associated with linear genomic DNA. We therefore speculate that the root defects of sav6 may result from defects in telomere maintenance, which may generate DSB stress and subsequently affect RAM development.

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In support of this hypothesis, it is required for recovery from UV-C induced replication inhibition 11 , Using the yeast system, it was demonstrated that the FEN activity of FEN1 is not required for this recovery process They demonstrated that these cyclin-dependent kinase inhibitors possess cell cycle inhibitory potential. PSK5 promotes QC division. We hypothesize that long-term exposure to DSB stress may activate QC cell division in order to replenish dead stem cells. You can't quite label it a red or brown hue, but the rich auburn tone glistening from Pont Neuf's lights is to die for.

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