AtCHE1, the Arabidopsis homolog of mammalian AATF/Che-1 protein, is involved in safeguarding genome stability

Liu, Fang; Wang, Bingshan; Wang, Xiangyang; Dong, Daofeng; De Veylder, Lieven; Qi, Shengdong; Horvath, Beatrix M.; Palme, Klaus; Li, Xugang

doi:10.1038/s42003-025-08490-1

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Published: 02 September 2025

AtCHE1, the Arabidopsis homolog of mammalian AATF/Che-1 protein, is involved in safeguarding genome stability

Fang Liu ORCID: orcid.org/0009-0003-2922-0410^1,2^na1,
Bingshan Wang²^na1,
Xiangyang Wang^1,3,
Daofeng Dong⁴,
Lieven De Veylder ORCID: orcid.org/0000-0003-1150-4426^5,6,
Shengdong Qi¹,
Beatrix M. Horvath^7,8,
Klaus Palme ORCID: orcid.org/0000-0002-2728-3835^2,9,10 &
…
Xugang Li^1,9^na2

Communications Biology volume 8, Article number: 1329 (2025) Cite this article

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Abstract

Both endogenous and exogenous genotoxins can inflict damage on cellular DNA, leading to reduced genomic stability in plants, which adversely affects development. Apoptosis Antagonizing Transcription Factor (AATF), also referred to as Che-1, has been identified as a binding protein for RNA polymerase II. It plays a crucial role in various cellular processes, including cell proliferation, transcriptional regulation, apoptosis, DNA damage response, and ribosome biogenesis in mammals. In this study, we identify the che1 mutant derived from an ethyl methanesulfonate (EMS)-mutagenized Arabidopsis Col-0 population, characterized by a short root and small leaf phenotype. The underlying mutation is a G-to-A transition located at the boundary of the eighth intron and ninth exon of the AT5G61330 gene, resulting in a misprocessed mRNA transcript. AtCHE1, a homolog of the mammalian AATF/Che-1, contains both the conserved AATF/Che-1 and TRAUB domains in Arabidopsis. Under standard conditions, the che1 mutant exhibits an accumulation of damaged DNA, cell death, and differentiation defects at the root tip. Collectively, these findings underscore the importance of AtChe-1 in meristem maintenance and genome stability.

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Introduction

In higher plants, new organs are generated from stem cells located in the root and shoot meristems. Within the root apical meristem (RAM), stem cells surround the rarely dividing quiescent center (QC) cells, which express the WUSCHEL-RELATED HOMEOBOX5 (WOX5) transcription factor gene^1,2. Stem cell niche maintenance is regulated by several transcription factors, including the AP2/PLT (APETALA2/PLETHORA) transcription factors, providing an apical-basal patterning signal. They are regulated by auxin, and form together a gradient of PLT expression³. On the other hand, the GRAS (derived from GAI: gibberellic acid insensitive, RGA: repressor of GA1-3 mutant, and SCR: scarecrow), SHORTROOT (SHR), and SCARECROW (SCR) transcription factors provide a radial patterning signal^4,5. Besides, meristem maintenance requires genome integrity. Members of the MAIN (MAINTENANCE OF MERISTEMS) and MAIN-like family, the three homologs: MAIN-LIKE1 (MAIL1, AT2G25010), MAIN-LIKE2 (MAIL2, AT2G04865), and MAIN-LIKE3 (MAIL3, AT1G48120) are involved in maintaining genome stability. Their mutants show short primary roots, disorganized RAM, and accumulated DNA breaks^6,7. AtMMS21 (Methyl Methanesulfonate Sensitivity gene 21), a subunit of the STRUCTURAL MAINTENANCE OF CHROMOSOME5/6 complex, is involved in ameliorating DNA double-strand breaks (DSBs) and maintaining the stem cell niche during root development⁸. The m56-1fas2-4 triple mutant lacking the H3/H4 histone chaperone CHROMATIN ASSEMBLY FACTOR-1 (CAF-1) and the H2A/H2B histone chaperone NAP1-RELATED PROTEIN1/2 (NRP1/2), which function synergistically in chromatin maintenance and genome maintenance, exhibit programmed cell death and severe short-root phenotype⁹.

Plants, as any other living organisms, suffer from various endogenous and exogenous stresses, such as ultraviolet radiation, chemical mutagens, and reactive oxygen species, which can damage the integrity of their genomes^10,11. To prevent the transmission of damaged DNA to daughter cells, the ATM/RAD53-RELATED (ATR) and ATAXIA-TELANGIECTASIA MUTATED (ATM) kinases are activated in response to replication defects and DSBs, respectively^12,13, ultimately leading to DNA damage repair, programmed cell death, or endoreduplication. Unlike animals, there is no orthologue of Chk1, Chk2, or CDC25, which act downstream of ATM or ATR. In plants, the p53 functional homolog SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1) and the cell cycle regulatory kinase, WEE1 play a central role in signaling. In response to DNA damage, SOG1 is phosphorylated by ATM^14,15,16. The activated SOG1 transcriptionally regulates hundreds of DNA damage response genes involved in cell cycle regulation, endocycle, cell death, and DNA repair. While the WEE1 controls cell cycle progression via the WEE1-RPL1-CYCDs and the WEE1-FBL17/SKP2-CKI-CDKs axis in response to replication stress^17,18. Furthermore, as recent studies have shown, RETINOBLASTOMA RELATED (RBR), the Arabidopsis retinoblastoma (Rb) homolog, in addition to its well-known cell cycle control and stem cell maintenance functions, also maintains genome integrity of root meristematic cells. It mediates the localization of the repair protein RAD51 to DNA lesions and accumulates together with E2FA and AtBRCA1 at the damaged, γH2AX-labeled foci in Arabidopsis^19,20. Besides cell death, DSBs also induce endoreplication through a series of cell cycle regulators controlled by the ATM-SOG1 and ATR-SOG1 pathways²¹.

The human AATF/Che-1, identified as a subunit of RNA polymerase II, interacts with Rb to repress its function²². In response to DNA damage, Che-1 is stabilized by phosphorylation by ATM/ATR and Chk2²³. Phosphorylated Che-1 activates p53 and p21 transcription to maintain the G2/M checkpoint²³. As a cofactor, phosphorylated Che-1 binds to p53 and forms a ternary complex with Brca1 in the first hours of DNA damage, resulting in transcription of growth arrest genes. Consequently, when cells accumulate an excess of damaged DNA, p53 is released from the Che-1/p53/Brca1 complex to promote transcription of pro-apoptotic genes²⁴. AATF/Che has a wide variety of functions. In general, AATF/Che-1 plays an important role in the regulation of proliferation and survival in the DNA damage response pathway in mammals. Besides this role, AATF/Che-1 is also a key component of ribosome biogenesis. It has been reported to interact with neuroguidin (NGDN) and NOL10 to form a complex called the AATF-NGDN-NOL10 (ANN) complex. And this nucleolar complex is involved in the synthesis of 40S ribosomal subunits²⁵. AATF/Che-1 also interacts with several RNA species and proteins such as 45S pre-rRNA, snoRNAs, ribosome biogenesis mRNAs, rRNA processing proteins, and ribosomal proteins²⁶. In addition, Che-1 affects rDNA transcription by binding to the RNA polymerase I machinery²⁷.

Although AATF/Che-1 has been well studied in animals, little is known about its plant homolog. Here, we describe the isolation of the mutant, named che1, which exhibits severe aberrations in root architecture, root growth inhibition and cell death in the root meristem. Based on the phenotypic changes, we propose that the nuclear-localized AtChe1, the Arabidopsis AATF/Che-1 homolog, is involved in root development and genome stability. Our analysis of sog1-1;che1 double mutant shows that AtCHE1 and SOG1 play a synergistic role in genome safeguarding.

Results

The che1 mutant exhibits a short primary root and a disorganized stem cell niche (SCN)

Isolated from an EMS-mutagenized Arabidopsis Col-0 population, the che1 mutant demonstrates retarded growth, characterized by shorter primary roots and smaller cotyledons compared to Col-0 (Fig. 1A). To rule out potential background mutations, we backcrossed the homozygous che1 mutant with Col-0. Analysis of the backcrossed lines revealed a 3:1 (221:68, Chi-squared test, p = 0.56), confirming that che1 is a recessive allele. At 4 days after germination (DAG), the root meristem length in the backcrossed che1 mutant was already reduced and remained significantly shorter than that in Col-0 at 8 DAG (Fig. 1B, C, and E). The number of transit amplifying cells in the cortex layer was also significantly decreased (P < 0.01) in the che1 mutant at 4 DAG (Fig. 1D). While Col-0 showed a slight increase in cell numbers at 6 DAG, the che1 mutant continued to exhibit a decline (Fig. 1B, D), indicating a progressive retardation of the meristem, and that shortening of the mitotic zone is often observed in roots undergoing genomic stress⁶. These findings suggest that transit-amplifying cells in the che1 mutant differentiate at an earlier developmental stage.

Fig. 1: Primary root growth and SCN maintenance are inhibited in the che1 mutant. — **Fig. 1: Primary root growth and SCN maintenance are inhibited in the *che1* mutant.**

To determine whether the observed phenotypic changes in root meristem development correlate with alterations in the stem cell niche, specifically the QC, we analyzed its structure (Fig. 1F) and monitored the expression of stem cell-specific markers (Fig. 1G, H). Propidium-iodide (PI) staining revealed disorganized QC architecture in the che1 mutant (Fig. 1F), which was further confirmed by altered expression pattern of the WOX5_pro:GFP (Fig. 1G).

We also examined the distal stem cell niche in the che1 mutant using the J2341 enhancer trap line, which indicates expression in the columella initials in Col-0. In the che1 mutant, J2341 expression expanded into additional cells surrounding the QC, unlike in Col-0 (Fig. 1H). To delve into the changes in the proximal meristem, we analyzed the expression of CO2_pro:H2B-YFP, CO3_pro:H2B-YFP, and SCR_pro:GFP, which are specifically expressed in cortical and endodermal layers²⁸. In the che1 root meristem, all three markers exhibited abnormal localization and cell division patterns within the cortex and endodermis (Fig. 1I, J, and K). Collectively, these results indicate that AtCHE1 is essential for maintaining the stem cell niche and proper organization of the root meristem.

AtCHE1 encodes the Arabidopsis homolog of the mammalian AATF/Che-1

To identify the AtCHE1 gene, we conducted map-based cloning. Preliminary mapping indicated that the mutated site is located at the tail of chromosome 5, between markers MTE17 and MSN2²⁹.

Using simple sequence length polymorphism (SSLP) markers and derived cleaved amplified polymorphic sequence (CAPS) markers, we fine-mapped the mutation and sequenced all the genes in this region, comparing them to the wild-type control. The mutated base pair was identified in the gene annotated as AT5G61330, at the junction of its eighth intron and ninth exon, where a G-to-A transition led to RNA mis-splicing (Fig. 2A, Supplementary data 1). To assess the effects of this alternative splicing, we compared the sequences of mutant and wild-type cDNAs via reverse transcription-polymerase chain reaction (RT-PCR) using total RNA extracted from both mutant and wild-type seedlings. The mis-splicing event appears to result in premature translation termination 870 bp from the translation start site (Fig. 2A), likely producing a truncated protein of 290 amino acids that still retains the part of the domain (Fig. 2B).

Fig. 2: AtCHE1 encodes AATF/CHE-1 in Arabidopsis. — **Fig. 2: *AtCHE1* encodes AATF/CHE-1 in Arabidopsis.**

To confirm that the mutation in the AtCHE1 gene was solely responsible for the growth defects observed in the che1 mutants, we transformed homozygous che1 plants with the full-length genomic region of AtCHE1, expressed under its own promoter (The sequence spanning 2197 bp between AtCHE1 and the adjacent gene AT5G61340, along with portions of both exon and the 3’UTR of AT5G61340. This design aims to encompass any potential distal control elements.) and including its 3’-UTR (AtCHE1_pro-AtCHE1_g-AtCHE1_3’-UTR). The wild-type AtCHE1 protein fully complemented the short primary root phenotype of the che1 mutant (Supplementary Fig. 1), confirming that the mutation in the AtCHE1 gene was indeed responsible for the observed phenotypic alterations.

According to the public database (TAIR), AtCHE1 is predicted to encode an rRNA processing protein, with its coding sequence showing 41.7% sequence similarity and 26.5% sequence identity with the human AATF/Che-1 protein, which encodes the Apoptosis Antagonizing Transcription Factor (AATF) (https://www.ebi.ac.uk/Tools/psa/emboss_needle/). AtCHE1 contains two conserved protein domains: the N-terminal leucine zipper region of AATF/Che-1 (from amino acids 142 to 271) and the C-terminal TRAUB domain (from amino acids 350 to 425) (Fig. 2B). Notably, the truncated AtCHE1 protein retains the AATF domain. Our comparative analysis indicates that AtCHE1 is the only AATF/Che-1 homolog identified in Arabidopsis that demonstrates evolutionary conservation between the animal and plant kingdoms (Supplementary Fig. 2 and Supplementary Fig. 3).

AtCHE1 is ubiquitously expressed and localized in the nucleus

To investigate AtCHE1 expression in Arabidopsis, we generated AtCHE1_pro:GUS reporter lines and analyzed three independent transgenic lines. We observed GUS activity in young roots, shoots, leaves, flowers, and siliques (Fig. 3A–G), with particularly high expression in the root apical meristem, aligning with the observed root phenotypes of the che1 mutant.

**Fig. 3: AtCHE1 is widely expressed and localized in nucleus.**

To explore the cellular and subcellular localization of the AtCHE1 protein, we created translational reporter lines (AtCHE1_pro:AtCHE1_g-GFP) expressing the AtCHE1-GFP fusion protein under its own promoter. Consistent with the expression patterns of the AtCHE1_pro:GUS lines, the AtCHE1-GFP fusion protein exhibited strong expression in stem cells and transit amplifying cells, with weaker expression in the QC, columella cells, and the elongation zone, where cell division is less active (Fig. 3H).

The AtCHE1-GFP fusion protein localizes to the nucleus (Fig. 3H). To further examine this localization, we introgressed the nucleoplasm marker 35S:H2B-tdTomato into the AtCHE1_pro:AtCHE1_g-GFP transgenic line. The H2B labeling allowed for a more precise assessment of nuclear morphology, including the nucleolus and surrounding nucleoplasm³⁰. Confocal images revealed that the peaks of tdTomato and GFP signals did not overlap (Fig. 3I, K). Additionally, the fluorescent dye 4’,6-diamidino-2-phenylindole (DAPI) was used to visualize nuclear DNA, highlighting the nucleolus as a darker region due to its low DNA concentration³¹. DAPI staining in the AtCHE1_pro:AtCHE1_g-GFP transgenic line confirmed the nuclear presence of AtCHE1; however, the peak of the GFP signal was located differently than that of DAPI (Fig. 3J, L).

Collectively, these observations demonstrate that AtCHE1 is predominantly localized in the nucleus of dividing cells.

Expression of the root master regulator genes is altered in the che1 roots

Given the retarded root growth and the expression of AtCHE1 in the root meristem, we investigated whether these developmental changes were linked to alterations in the auxin/PLT pathway. To analyze auxin response in the che1 mutant, we examined the expression of the DR5rev:GFP reporter. The auxin response maxima in the QC and the distribution in columella cells in the che1 mutant were comparable to those in Col-0 (Fig. 4A).

Fig. 4: Auxin signaling and expression patterns of root developmental regulator genes are altered in che1. — Fig. 4: Auxin signaling and expression patterns of root developmental regulator genes are altered in *che1.*

We further assessed the transcriptional changes of root master regulators by crossing the PLT1_pro:CFP and PLT2_pro:CFP marker lines into the che1 mutant background. Both PLT1 and PLT2 are essential for maintaining stem cell identity, promoting the mitotic activity of stem cell daughters, and facilitating cell differentiation³². In the Col-0 background, PLT1_pro:CFP and PLT2_pro:CFP displayed high expression in the stem cell niche, intermediate levels in rapidly dividing cells, and low levels in elongated cells. In contrast, the transcriptional expression of PLT1 and PLT2 in the che1 mutant was reduced and restricted primarily to the stem cell niche (Fig. 4B, C; Supplementary Fig. 4A, B). Quantitative RT-PCR (qRT-PCR) analysis of PLT1 and PLT2 transcript levels in che1 root tips confirmed these findings (Fig. 4F). Similarly, expression levels of the GRAS family genes SCR and SHR were also decreased in the che1 mutant compared to Col-0 (Fig. 4D, E, and G; Supplementary Fig. 4C, D). Collectively, these results suggest that the aberrant root formation and stem cell niche maintenance in the che1 mutants are associated with the disrupted distribution of PLT1, PLT2, SCR, and SHR.

che1 mutant shows cell death and DNA damage

Similar to the mutations in genes, such as FAS1 (subunit of the counterpart of chromatin assembly factor-1)³³, MMS21-1⁸ and RBR¹⁹ involved in stem cell niche and meristem maintenance and critical for genome integrity, we also observed occasional cell death response in the che1 mutant (42%, Fig. 5A control and Supplementary Fig. 5A).

Fig. 5: che1 mutant develops DNA damage response. — **Fig. 5: *che1* mutant develops DNA damage response.**

To investigate whether the absence of AtCHE1 leads to genome instability, first we used the neutral comet assay, and afterwards we followed the accumulation of the phosphorylated histone variant, γH2AX to detect DNA damage. The comet assay showed that the level of damaged DNA was higher in the che1 mutant compared to the wild type (Fig. 5B, C). With the use of immunofluorescence staining with the γH2AX antibody, we showed that che1 had increased DNA damage compared to the control under normal growth conditions (Fig. 5D, E). The number of γH2AX foci in the che1 mutant was similar to that of Col-0 treated with 10 µM zeocin, a radiomimetic drug that causes DSBs (Fig. 5A, D, E). The increased expression level of DNA damage marker genes, such as AtBRCA1 and AtPARP2, in the che1 mutant further supported our hypothesis that the absence of AtCHE1 leads to DNA damage (Fig. 5F, G).

In mammals, AATF/Che1 plays an essential role in DNA damage response pathway, contributes to cell cycle arrest and apoptosis depending on the DNA damage level³⁴. To study whether che1 is sensitive to genotoxic agents, we tested its response besides upon hydroxyurea (HU, 2 mM), also upon mitomycin C (MMC, 10 µM) and zeocin (20 µM) treatment (4 DAG seedling, 24 h) to induce replication stress, DNA cross-linking, and DSBs, respectively. PI-stained roots, che1, and Col-0 as a control, were imaged by confocal microscopy. By measuring the area of dead cells in the proximal meristem, we could conclude that after HU, MMC, and zeocin treatments, the che1 mutant was more sensitive to HU and MMC than Col-0 (Fig. 5A; Supplementary Fig. 5A, B). Furthermore, the increased expression levels of the DNA damage marker genes AtBRCA1, AtRAD51, and AtPARP2, induced by HU, were found to be lower in the che1 mutant compared to Col-0 (Fig. 5F, G, and H). Based on these observations, we concluded that AtCHE1 protects genome integrity and is involved in the DNA damage response to replication stress.

Since the DNA damage responses, both cell death and DNA damage response (DDR) gene induction, are different in the AtCHE1 mutant normal growth conditions and upon treatment, we asked whether AtCHE1 gene expression itself is also triggered by genotoxic stress. Upon genotoxic treatment of the transgenic lines AtCHE1_pro:GUS and AtCHE1_pro:AtCHE1_g-GFP (5 DAG, 24 h), to 1/2 MS containing 2 mM HU, 10 µM MMC or 20 µM zeocin and grown for 24 hours. following GUS expression, we did not detect significantly changes upon HU compared to the seedlings grown under control conditions (Supplementary Fig. 6A). In contrast, the presence of GUS was significantly reduced upon zeocin treatment (20 µM) due to severe retardation of the root meristem (Supplementary Fig. 6A). qRT-PCR experiments confirmed these observations (Supplementary Fig. 6B). Meanwhile, AtCHE1_pro:AtCHE1_g-GFP confocal images showed that HU, MMC, and zeocin reduced AtCHE1 fusion protein levels (Supplementary Fig. 6C, D). It is worth to note that under zeocin treatment, the reduction of GFP and GUS expression is likely due to extensive cell death and shrinkage of mitotic zone.

AtCHE1 plays a synergistic role with SOG1

To clarify the role of AtCHE1 in the DDR signaling pathway, we investigated its interaction with SOG1, the master regulator of DDR. The sog1-1;che1 double mutant showed enhanced level of cell death under control conditions compared to either single mutant, che1 and sog1-1, as well as Col-0 (Fig. 6). This elevated response indicates a synergistic interaction, with AtCHE1 and SOG1 maintaining meristematic cell integrity.

Fig. 6: Cell death response in che1 is independent on SOG1. — **Fig. 6: Cell death response in *che1* is independent on SOG1.**

Given the importance of ATR kinase in the cellular response to replication stress, we also explored the relationship between AtCHE1 and ATR functions in DDR. We introgressed atr-2 and che1 mutants for this purpose. The atr-2 mutant treated with hydroxyurea (HU, 2 mM) demonstrated a significant reduction in root growth (Fig. 7A–D, Supplementary Fig. 7). In contrast, the root length and number of meristematic cells in the atr-2;che1 double mutant were comparable to those in the che1 mutant, suggesting that the loss of AtCHE1 partially alleviates the root growth inhibition observed in the atr-2 mutant under replication stress (Fig. 7A–D).

Fig. 7: Loss of AtCHE1 partially rescues impaired root development of atr-2 under HU treatment. — **Fig. 7: Loss of AtCHE1 partially rescues impaired root development of *atr-2* under HU treatment.**

Lastly, we analyzed the ratio of roots exhibiting cell death and measured the cell death area in the atr-2;che1 double mutant compared to the controls. Under normal growth conditions, the ratio of roots with cell death was similar between the atr-2;che1 and che1 mutants, while significantly lower in the atr-2 mutant (Fig. 7E, G and H). However, upon HU treatment (24 hours), all genotypes, regardless of genetic background, displayed a cell death response (Fig. 7F, G). Notably, the extent of cell death varied significantly among the genotypes. The atr-2 mutant displayed a robust cell death response compared to either Col-0 or che1 (Fig. 7F), which was substantially diminished in the absence of AtCHE1 function (Fig. 7F, G and H). These findings suggest that the loss of AtCHE1 partially rescues impaired root development caused by the deficiency of ATR under HU stress.

Discussion

AATF/Che-1, an RNA polymerase II-binding protein, plays a critical role in a wide array of cellular processes, including transcriptional regulation, cell proliferation, DNA damage response, apoptosis, and ribosome biogenesis in mammals. In this study, we demonstrate that AtCHE1, the Arabidopsis homolog of AATF/Che-1, is essential for root development and genome integrity.

Accumulating evidence underscores the importance of genome integrity in maintaining the SCN. Mutants such as teb, fas and mms21, which involve helicase and DNA polymerase domain encoding gene by the TEBICHI, as well as FASCIATA (FAS) gene that encodes subunits of the Arabidopsis counterpart of chromatin assembly factor-1 (CAF-1), and the methyl methanesulfonate sensitivity gene21 functions as a subunit of the STRUCTURAL MAINTENANCE OF CHROMOSOMES5/6 complex, demonstrate severe meristem organization defects alongside cell death at the root tip^8,33,35. Similar effects were induced by the mutations of MAIN-related genes, MEDIATOR18 and MERISTEM DISORGANIZATION 1 genes, lead to analogous defects. These genes encode components involved in plant-specific processes, such as aminotransferase-like plant mobile domain, a subunit of mediator complex, and an essential telomere protein in CTC1 (Cdc13)/STN1/TEN1 complex, respectively^6,7,36,37,38. The RETINOBLASTOMA-RELATED (RBR) protein is also crucial for both stem cell maintenance and the DNA damage response^19,20,39.

In this study, we report that mutations in the AtCHE1 gene result in a disorganized SCN and a cell death response at the root tip, similar to the phenotypes observed in fas or mms21 mutants that lack functional telomeres or chromatin-organizing complexes. In addition to the SCN abnormalities, we observed exhaustion of the root meristem and disorganized cell division in the cortex and endodermal cells of the che1 mutant. Increased DNA content in the comet tail, accumulation of γH2AX foci, and upregulation of DNA damage response genes indicated genomic instability in the che1 mutant. Similar to the effects of genotoxic agents, the absence of AtCHE1 function led to reduced transcription of critical root development genes, including WOX5, SHR, SCR, PLT1, and PLT2^7,8.

At this point in our investigation, it remains difficult to determine whether the disrupted expression of root patterning genes leads to aberrant root formation in the che1 mutant or if genome instability itself drives the DNA damage response, cell death, and premature differentiation. However, given the phenotypic similarities between the che1 mutant and those observed in chromatin-organizing protein mutants, along with the anti-apoptotic role of AtCHE1’s ortholog, AATF/Che-1, in animals, we hypothesize that AtCHE1 plays a primary role in ensuring genome stability, which is critical for maintaining the integrity of the root meristem.

Given the role of AATF/Che-1 in the DNA damage response in animals, we explored whether AtCHE1 serves a similar function in Arabidopsis. Unlike their animal counterparts, the inactivation of plant genes involved in DDR typically results in subtle phenotypes under normal growth conditions, but increases sensitivity to genotoxic stress. The che1 mutant, akin to wee1-1 and atr-2 mutants, exhibits heightened cell death in response to hydroxyurea (HU) treatment⁴⁰. It is noteworthy that the transcription levels of BRCA1, RAD51, and PARP2 are elevated in mutants but remain lower than those observed in wild type. We assume either that the transcription of these genes is affected by AtCHE1 or the accumulation of cell death in the mutant is due to the lack of BRCA1, RAD51 and PARP2 involved in DNA damage repair.

Since the ATR kinase plays a central role in the response to replication stress¹³ we examined the relationship between the AtCHE1 and ATR signaling pathways. Upon HU treatment, the atr-2;che1 double mutant displayed less severe cell death and only moderate growth impairment compared to the atr-2 mutant alone. This phenomenon could be explained by the followings: either, ATR and AtCHE1 act in the same pathway, ATR is upstream or downstream of AtCHE1 or the retarded growth of the che1 mutant partly restores the function of ATR, arresting the cell cycle in response to HU treatment. Additional experimentation is required to provide evidence to verify these options. Moreover, genetic analysis indicates that cell death in the che1 mutant operates independently of SOG1, with AtCHE1 and SOG1 playing synergistic roles in promoting cell survival. In mammals, AATF/Che-1 activity is regulated post-translationally through phosphorylation, ubiquitination, poly(ADP-ribosyl)ation, and conformational changes^23,41,42. Further investigations should provide evidence for post-transcriptional and post-translational regulation of AtCHE1 in response to DNA damage stress. Similar to its homolog AATF, AtCHE1 has a predicted nuclear and a nucleolar localization signal, and is predominantly detected in the nucleus. The cellular localization of AtCHE1 provides important insight into its function, paralleling the role of the mouse AATF/Che-1 homolog Traube (Trb), which is crucial for the growth of preimplantation embryos. trb mutant embryos exhibit reduced total cell numbers, developmental arrest, and a lack of ribosomes, polyribosomes, and rough endoplasmic reticulum⁴³. The absence of ribosomes in the trb mutant and the nucleolar localization of Traube suggested that its involvement in ribosome synthesis. The research for the refined and dynamic subcellular localization of AtCHE1 is important for studying AtCHE1 gene function.

The che1 mutant may retain the complete AATF motif (290 amino acids) while lacking the TRAUB motif, suggesting that it is likely a weak allele. Using a single mutant to study gene function has limitations. For example, if the AtCHE1 knockout causes early lethality, a weak allele might mask its role in embryonic development. To comprehensively investigate gene function, it is essential to obtain a broader variety of mutants, including knockout mutants, knockdown mutants, and single or multiple amino acid substitution mutants.

In conclusion, our results indicate that AtCHE1 plays a critical role in the regulation of genome integrity and stem cell maintenance in Arabidopsis.

Materials and methods

Plant materials and growth conditions

In this study, we utilized the Arabidopsis thaliana Columbia-0 (Col-0) ectopy is used as the wild type. The marker lines employed have been previously characterized: WOX5_pro:GFP⁴⁴, SCR_pro:GFP^4,45, SHR_pro:SHR-GFP⁴⁶, PLT1_pro:CFP³², PLT2_pro:CFP³², J2341⁴⁷, CO2_pro:H2B-YFP²⁸, CO3_pro:H2B-YFP²⁸, and DR5_rev:GFP⁴⁸, all of which are in the Col-0 ecotype background. The atr-2 (SALK_032841) and che1 mutants utilized in this research are also based on the Col-0 ecotype. The sog1-1 mutant has been described previously⁴⁹. Additionally, the 35S:H2B-tdTomato line was generously provided by Prof. Dr. Thomas Laux. Sequence information for the primers used for genotyping is provided in Supplementary Table 1.

Surface-sterilized seeds were planted on 1/2 Murashige and Skoog (MS) (containing 1% sucrose, 1.3% Agar, pH5.7) and incubated at 4 °C in the dark for 2 days. Subsequently, the seeds were transferred to a growth environment maintained at 22 °C with a photoperiod of 16 hours light and 8 hours dark. For growing in soil, seedlings were transferred to soil between 10-14 days and grown under the aforementioned conditions in a growth chamber.

For DNA damage reagents treatments, MMC (Mitomycin C from Streptomyces caespitosus, M0503, Sigma) was prepared by adding 1 vial to 4 ml H₂O as a stock solution, stored stock solution at 4 °C. HU (H8627 Sigma) was dissolved in H₂O to create a 0.5 M stock solution and stored at −20 °C. 100 mg ml⁻¹ Zeocin stock solution was bought from Invitrogen (R25001) and stored at −20 °C. For long time (6 days) and short time (24 h) treatment, diluted 100 mg ml⁻¹ zeocin to 10 μM and 20 μM, 0.5 M HU to 1 mM and 2 mM and 0.5 g l^-1 MMC to 2.5 mg l⁻¹ and 10 μM in 1/2 MS medium, respectively. Seedlings were grown (long-time treatment) or transferred (short-time treatment) to the medium containing DNA damage reagents and grown under the described conditions.

Protein structure prediction

Nuclear localization signals were predicted by NLS mapper (http://nls-mapper.iab.keio.ac.jp)^50,51,52. For detecting nucleolar localization sequence, NOD: NuclOlar localization sequence Detector (dundee.ac.uk) was utilized (http://www.compbio.dundee.ac.uk/www-nod/index.jsp)^53,54. The phosphorylation sites of AtCHE1 protein were predicted via the NetPhos 3.1 server (http://www.cbs.dtu.dk/services/NetPhos/)⁵⁵. Finally, the protein domain structure was generated using DOG software for visualization of the protein domains (version 2.0)⁵⁶.

EMS mutagenesis and map-based cloning of AtCHE1

In this study, we conducted EMS mutagenesis on an Arabidopsis population (ecotype: Col-0) and isolated mutants according to the protocols established by Jander et al. and Yu et al.^57,58. Col-0 seeds were soaked in a 50 ml Falcon tube containing 40 ml of sterile 100 mM phosphate buffer at 4 °C with continuous shaking. Subsequently, the phosphate buffer was removed and replaced with 40 ml of fresh sterile 100 mM phosphate buffer supplemented with 0.4% EMS. The seeds were then incubated at room temperature with gentle shaking. Following incubation, the seeds were washed with sterile water 20 times. Finally, the washed seeds were dried under a hood, resulting in the EMS-mutagenized population (M1). To map the AtCHE1 locus, we employed standard map-based cloning techniques as detailed by Lukowitz et al.²⁹. The che1 mutant, also in the Col-0 background, was crossed with Landsberg erecta to generate an F2 mapping population. Initial coarse mapping localized the AtCHE1 gene to a region on chromosome 5 between MTE17 and MSN2. For fine mapping, we utilized simple sequence length polymorphisms (SSLPs) and derived CAPS (dCAPS) markers. Sequence analysis revealed a G to A mutation in the AT5G61330 gene, which caused RNA mis-splicing. A 5700 bp genomic fragment from the wild type (Col) AT5G61330 locus, which includes an additional 2197 bp upstream, a 2817 bp transcribed region, and a 686 bp downstream sequence, was introduced into the che1 mutant. This fragment effectively complemented the short root and dwarf phenotype of the che1 mutant. The sequence information for the primers used in mapping and the mutation site of the che1 mutant can be found in Supplementary Table 2 and Supplementary Table 3. Additionally, genomic DNA and coding sequence information for both the wild type and the che1 mutant are provided in Supplemental Data 1.

Plasmid construction and generation of transgenic lines

To construct AtCHE1_pro:GUS, the AtCHE1 promoter (comprising 2197 bp upstream and 201 bp transcribed region) was amplified from BAC MFB13 using primers 164-6 and 164-7. The Pvu I and Asc I cleavage sites were introduced using these primers, with Pvu I being compatible with Pac I. The PCR product was digested with Pvu I and Asc I, while the pMDC162 vector was digested by Pac I and Asc I. The AtCHE1 promoter fragment was then inserted into pMDC162.

For phenotypic complementation of the mutant lines, we amplified the native AtCHE1 promoter along with the full-length genomic DNA (comprising 2197 bp upstream, 2817 bp transcribed region, and 686 bp downstream) from BAC MFB13, using primers 164-35 and 164-11. Sbf I and Sac I cleavage sites were introduced with these primers. The pCR-Blunt intermediate vector containing the PCR product was digested with Sbf I and Sac I, as was pMDC85 after the removal of the sequence containing the CaMV35S promoter and GFP tag between these cleavage sites. AtCHE1_pro:AtCHE1_g-AtCHE1_3’-UTR construct was generated following this procedure.

To investigate the subcellular localization of the AtCHE1 protein, the native AtCHE1 promoter and full-length genomic DNA (excluding the stop codon) were amplified from BAC MFB13 using primers 164-35 and 164-12. The resulting PCR product was similarly digested with Sbf I and Asc I, and inserted into pMDC85 after removing the CaMV35S promoter sequence. This generated the AtCHE1_pro:AtCHE1_g-GFP construct.

All constructs were introduced into the Agrobacterium strain GV3101 and transformed into Arabidopsis through the floral dip method⁵⁹. Sequence information for the primers used to construct the vectors is provided in Supplementary Table 4.

Histochemical analysis

The activity of the reporter enzyme β-glucuronidase (GUS) was assessed in transgenic plants using X-Gluc as a substrate for enzyme activity. Seedlings were first fixed in 90% acetone for 20 min. Then, rinsed with GUS staining buffer comprised of 50 mg l⁻¹ X-Gluc; 100 mM NaH₂PO₄; 10 mM Na₂EDTA; 0.5 mM K ferrocyanide; 0.5 mM K ferricyanide; 1‰ Triton X-100 pH = 7.0. After vacuum infiltration in GUS staining solution for 2~3 min the seedlings were incubated in the solution at 37 °C. Following incubation, they were washed twice with PBS buffer and subsequently in 70% Ethanol. The seedlings were then mounted in 40% glycerol. Tissue samples were photographed using an AxioImager A1 microscope (Carl Zeiss Microimaging) and a SteREO Discovery V20 stereomicroscope (Carl Zeiss Microimaging) connected to an AxionCam MRc digital camera (Carl Zeiss Microimaging).

Phenotypic analysis

For assessing primary root length, seedlings were scanned, and root lengths were measured using Image J (National Institutes of Health; http://rsb.info.nih.gov/ij). For the root meristem, roots were mounted in a chloral hydrate solution (chloral hydrate: water: glycerol=4:3:1) and photographed with an AxioImager A1 microscope (Carl Zeiss Microimaging). Meristem length and cell number were calculated using Image J, based on the region between the QC and the first elongated cell in the cortical cell file^60,61. For PI staining, seedlings were rinsed in a 10 µg ml^-1 PI for 2 min. After a thorough rinse in water, roots were mounted in water for analysis via confocal laser scanning microscopy.

qRT-PCR

Gene expression was evaluated using quantitative RT-PCR (qRT-PCR). Total RNA was extracted from 2 mm root tips of seedlings at 5 DAG using the RNeasy Plus Micro Kit (cat. No. 74034 QIAGEN). Prior to cDNA synthesis, the RNA samples were incubated with 1 μl DNase I at 37 °C for 30 min (Thermo Scientific), followed by the addition of 1 μl 50 mM EDTA at 65 °C for 10 min to eliminate any residual DNA. RNA concentration was measured using a NanoDrop 1000 spectrophotometer (PRQlab), and RNA integrity was assessed via agarose gel electrophoresis. Reverse transcription was conducted using Thermo Scientific ReverAid Reverse Transcriptase (EP0041). qRT-PCR was performed with Maxima SYBR Green qPCR Mastermix (Thermo Scientific K0222) on an ABI 7300 real-time PCR system (Applied Biosystems). The primers utilized for qRT-PCR are listed in Supplementary Table 5, with UBIQUITIN10 (UBQ10) serving as the reference gene.

Confocal microscopy

Fluorescence imaging was conducted using a Zeiss LSM 510 microscope (Carl Zeiss Microimaging) and an A1 HD25/A1R HD25 confocal microscope (Nikon). A 488 nm line laser was employed for the excitation of GFP and Alexa Fluor 488 picolyl azide, with emission detected from 505 to 530 nm. For CFP, a 458 nm line laser was used, and emission were collected from 465 to 520 nm. PI and tdTomato fluorescene were excited at 561 nm, with emissions recorded between 590 and 653 nm. DAPI was excited at 408 nm, and emissions were detected at 480 nm. Images were processed using ZEN lite (Carl Zeiss Microimaging) and NIS-Elements Viewer (Nikon). Fluorescence intensity and plot profiles were analyzed using Image J software (National Institutes of Health; http://rsb.info.nih.gov/ij) and Fiji⁶².

Comet assay

The comet assay was performed following established methods^63,64, and ¹⁶. Briefly, 7-day-old seedlings were dissected using a fresh razor blade on ice in PBS buffer containing 50 mM EDTA. The resulting nuclear suspensions were filtered through a 40 µm sieve. This nuclear suspension was mixed with 1% low-melting-point agarose at a 1:10 (v/v) ratio. Two drops, each containing 50 µl of the nuclear suspension, were placed separately on precoated slides and covered with 22 mm × 22 mm coverslips. The slides were incubated at 4 °C for 60 minutes, followed by treatment with a neutral lysis solution (high salt: 2.5 M NaCl, 10 mM Tris-HCl, pH 7.5, 100 mM EDTA) for 20 minutes at room temperature (N/N protocol). The slides were then equilibrated in 1× TBE buffer for 5 minutes and subjected to electrophoresis in the same buffer for 6 minutes at 25 V (1 V cm⁻¹). Excess electrophoresis solution was drained from the slides and the slides were gently rinsed twice in dH₂O for 5 minutes each, followed by a 5-minute rinse in 70% ethanol. The slides were then dried on a sterile bench. Dried agarose gels were stained with 10 µg ml⁻¹ PI and covered with coverslips. Comets were observed using an AxioImager A1 fluorescence microscope (ZEISS). Comet analysis was conducted using TriTek Comet Score software, measuring at least 100 comet images from each slide while avoiding doublets or comets at the slide edges.

Immunofluorescence staining

5 DAG plants were incubated for 30 min in fixation buffer (4% paraformaldehyde in 100 ml MTSB, MTSB buffer: 50 mM PIPES, 5 mM MgSO₄, 5 mM EGTA, pH 6.9) in the cold room (4°C). Cell wall digestion, blocking, primary antibody incubation, secondary antibody incubation, and nuclear co-staining of the nucleus were performed as previously described⁶⁵ and ²⁰. For cell wall digestion, 600 μl enzyme mix (2.5% pectinase (Mazerozym R10), 2.5% cellulase (Onozuka RS), and 2.5% pectolyase (Y-23) in buffer MTSB) was added to new wells. Seedlings were then transferred into the enzyme mix and incubated at 37 °C for 8 minutes. Root tips were excised under a binocular microscope and placed into MTSB buffer. Subsequently, root tips were transferred onto the slides and dried on a sterile bench. For blocking, 60 μl of blocking solution (2% BSA in MTSB.) was applied to the area containing the root tips, followed by incubation at room temperature for 1 hour. For primary antibody incubation, the blocking solution was replaced with 60 μl of primary antibody solution (γH2AX antibody), which was incubated for 1 hour at 37 °C. The root tips were washed 3 times with 100 μl MTSB buffer. For secondary antibody incubation, 60 μl of the secondary antibody solution (Goat anti-rabbit Alexa Fluor 488) was added to the root tips and incubated for 1 hour at 37 °C. The root tips underwent 3 washes with MTSB buffer. To co-staining the nucleus, 100 μl of the DAPI solution (0.2 mg l⁻¹) was added to the root tips and incubated for 10 minutes before washing 3 times with dH₂O for 5 minutes each time. A rabbit anti-plant γH2AX antibody⁶⁶ and a goat anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 488 (Invitrogen, A-11034), were utilized at a working concentration diluted to 1:500 in an antibody dilution buffer (1% BSA in MTSB).

Accession numbers

Sequence data from this article is available from the Arabidopsis Genome initiative under the following accession numbers: AT5G61330 (AtCHE1), AT3G11260 (WOX5), AT4G05320 (UBQ10), AT3G20840 (PLT1), AT1G51190 (PLT2), AT3G54220 (SCR), AT4G37650 (SHR), AT5G40820 (ATR), AT4G21070 (BRCA1), AT4G02390 (PARP2), AT1G25580 (SOG1), and AT5G20850 (RAD51). And the accession numbers of protein in phylogenetic analysis (Supplementary Fig. 2 and Supplementary Fig. 3) are provided as follow: NP_001331141.1 (Arabidopsis thaliana), XP_003552502 (Glycine max), NP_036270 (Homo sapiens), NP_062790 XP_919127 (Mus musculus), XP_015623660 (Oryza sativa), AJU67199 (Saccharomyces cerevisiae), NP_001150275 (Zea mays).

Statistics and reproducibility

Statistical analysis was conducted using IBM SPSS statistics 21 and Microsoft Excel. Data are presented as the mean ± standard deviation (SD). The two-tailed Student’s t-test and one-way ANOVA test were used for comparing two groups and more than two groups, respectively. For the purpose of reproducibility, three independent biological replicates were utilized in qRT-PCR, comet assays, immunofluorescence staining, and the analysis of double mutant phenotypes. Additionally, at least three independent experiments were conducted to compare the responses to DNA damage treatments in che1 and Col-0. The sample size and the number of experimental repetitions were detailed in the figure legend.

Data availability

All data supporting the findings of this study are available from the corresponding author upon reasonable request. The source data underlying the graphs presented in this paper can be found in Supplementary Data 2.

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Acknowledgements

We thank Prof. Dr. Yan Zhang for comments on the manuscript. This work was supported by the initiation fund from the Shandong Agricultural University, National Natural Science Foundation of China (31570291); Shandong “Foreign experts double hundred” Program (WST2017008), and Natural Science Foundation of Shandong Province (ZR2021MC175). In additional the work was supported by Bundesministerium für Bildung und Forschung [BMBF FKZ 031B0556]; the Excellence Initiative of the German Federal and State Governments [EXC 294, SFB746]; and the Deutsches Zentrum für Luft und Raumfahrt [DLR 50WB1022]. Beatrix. M. Horvath was funded by a Marie-Curie IEF fellowship (FP7-PEOPLE-2012-IEF-330789).

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Author information

These authors contributed equally: Fang Liu, Bingshan Wang.
Deceased.

Authors and Affiliations

State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’an, China
Fang Liu, Xiangyang Wang, Shengdong Qi & Xugang Li
Institute of Biology II, Faculty of Biology, Albert-Ludwigs-University of Freiburg, Schänzlestrasse 1, Freiburg, Germany
Fang Liu, Bingshan Wang & Klaus Palme
Key Laboratory of Herbage & Endemic Crop Biology of Ministry of Education, Inner Mongolia Key Laboratory of Herbage & Endemic Crop Biotechnology, School of Life Sciences, Inner Mongolia University, Hohhot, China
Xiangyang Wang
Shandong Key Laboratory of Greenhouse Vegetable Biology, Shandong Branch of National Vegetable Improvement Center, Institute of Vegetables, Shandong Academy of Agricultural Sciences, Jinan, China
Daofeng Dong
Department of Plant Biotechnology and Bioinformatics, Ghent University, Gent, Belgium
Lieven De Veylder
Center for Plant Systems Biology, VIB, Gent, Belgium
Lieven De Veylder
School of Biological Science, Centre for Systems and Synthetic Biology, Royal Holloway, University of London, Egham Hill, Egham, UK
Beatrix M. Horvath
Molecular Life Sciences, Wageningen University, Stippenweg 2, Wageningen, The Netherlands
Beatrix M. Horvath
Sino-German Joint Research Center on Agricultural Biology, College of Life Sciences, Shandong Agricultural University, Tai’an, China
Klaus Palme & Xugang Li
ScreenSYS GmbH, Biotechpark, Engesserstr. 4a, Freiburg, Germany
Klaus Palme

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Contributions

X.L. and K.P. designed the research and supervised the experiments; X.L., F.L., and K.P. wrote the manuscript; B.M.H. improved the preliminary version of the manuscript; F.L., B.W., and X.W. performed most of the experiments; B.M.H., L.D.V., D.D., and S.Q. contributed reagents, materials, analytical tools, and suggestions.

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Correspondence to Fang Liu or Klaus Palme.

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Liu, F., Wang, B., Wang, X. et al. AtCHE1, the Arabidopsis homolog of mammalian AATF/Che-1 protein, is involved in safeguarding genome stability. Commun Biol 8, 1329 (2025). https://doi.org/10.1038/s42003-025-08490-1

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Received: 02 July 2024
Accepted: 07 July 2025
Published: 02 September 2025
Version of record: 02 September 2025
DOI: https://doi.org/10.1038/s42003-025-08490-1