SUPPRESSOR OF GAMMA RESPONSE1 Links DNADamage Response to Organ Regeneration1[OPEN]

Ross A. Johnson,2 Phillip A. Conklin,2 Michelle Tjahjadi, Victor Missirian, Ted Toal, Siobhan M. Brady,and Anne B. Britt3

Department of Plant Biology, University of California Davis, 1 Shields Avenue, Davis, California 95616

ORCID IDs: 0000-0002-0024-6700 (R.A.J.); 0000-0003-2159-4912 (P.A.C.); 0000-0002-0967-4998 (T.T.); 0000-0001-9424-8055 (S.M.B.);0000-0003-2244-1790 (A.B.B.).

In Arabidopsis, DNA damage-induced programmed cell death is limited to the meristematic stem cell niche and its earlydescendants. The significance of this cell-type-specific programmed cell death is unclear. Here, we demonstrate in roots that it isthe programmed destruction of the mitotically compromised stem cell niche that triggers its regeneration, enabling growthrecovery. In contrast to wild-type plants, sog1 plants, which are defective in damage-induced programmed cell death, maintainthe cell identities and stereotypical structure of the stem cell niche after irradiation, but these cells fail to undergo cell division,terminating root growth. We propose DNA damage-induced programmed cell death is employed by plants as a developmentalresponse, contrasting with its role as an anticarcinogenic response in animals. This role in plants may have evolved to restore thegrowth of embryos after the accumulation of DNA damage in seeds.

DNA damage can cause cytostatic and cytotoxic ef-fects, and can potentially lead to heritable mutations(Waterworth et al., 2015). Double-strand DNA breaks(DSBs) are particularly growth disruptive, leading tochromosome aberrations and mutations if incorrectlyrepaired and cell death if mitosis occurs before therepair of broken chromosomes (Hu et al., 2016;Waterworth et al., 2015). DSBs are also potent inducersof checkpoint response, where cell cycle progression istransiently inhibited to allow for DNA repair beforemitotic M phase (Hu et al., 2016). These arrest and re-pair processes, together with programmed cell death(Curtis and Hays, 2007) and the early induction of theendoreduplicative cell cycle (Adachi et al., 2011), arecollectively known as the DNA damage response(DDR), which safeguards the genomic integrity of theorganism as a whole (Yoshiyama, 2016). In plants, DDRhas a key role in the germination of seeds that have

accumulated DNA damage during aging from desic-cation/rehydration cycles, as repair is limited in thedesiccated state (Waterworth et al., 2015, 2016). Here,we demonstrate the role of DDR genes in seedling re-covery from growth-disruptive levels of DNA damage,which we have artificially induced by exposure toionizing radiation (IR). In this work, we have usedgamma irradiation for a ubiquitous induction of DNAdamage throughout the seedling, with DSBs being themost cytotoxic lesion triggered (Tounekti et al., 2001;Moiseenko et al., 2001). We have typically used anacute, transiently growth-inhibiting 150-Gy dose,which is less than that triggering a permanent growtharrest (e.g. 500 Gy) but greater than that resolvableby constitutively expressed DNA repair processes(e.g. 5 Gy; Einset and Collins, 2015); eukaryotic ge-nomes routinely encounter a benign level of DSBs(such as from collapsed DNA replication forks) thatdo not induce the DDR (Sanchez et al., 1999).

A plant cell’s response to DNA damage first involvesthe DSB-detecting protein kinase ATM (Ataxia-Telangiectasia-Mutated) or the detector of stalled rep-lication forks ATR (Ataxia Telangiectasia Mutated andRad3-related protein; Culligan et al., 2006; Furukawaet al., 2010). ATM (Yoshiyama et al., 2013) and inferablyATR (Furukawa et al., 2010; Yoshiyama et al., 2009) canphosphoactivate the transcription factor SOG1, as wellas other proteins, in plant cells (Roitinger et al., 2015;Yoshiyama et al., 2013). Once activated, SOG1 tran-scriptionally induces various functional classes of DDRgenes (Yoshiyama et al., 2009; Missirian et al., 2014;Ricaud et al., 2007); SOG1 induces a robust set of .100transcripts by $4-fold within 1.5 h in response to100 Gy IR (Culligan et al., 2006; Furukawa et al., 2010).SOG1 has a known role in transcriptionally inhibiting

1 This work was supported by a grant to A.B.B. from the NationalScience Foundation Division of Molecular Biosciences (award#1158443) and to P.A.C. from the Elsie Taylor Stocking Fellowship.

2 These authors contributed equally to the article.3 Address correspondence to abbritt@ucdavis.edu.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Anne B. Britt (abbritt@ucdavis.edu).

P.A.C., R.A.J., S.M.B., and A.B.B. designed the experiments; R.A.J.and P.A.C. performed experiments and produced figures; A.B.B.,R.A.J., and P.A.C. wrote the manuscript; A.B.B., R.A.J., P.A.C.,M.T., and S.M.B. edited the manuscript; M.T. developed novel sog1lines; T.T. wrote the read-trimming script; and V.M. processed thetranscriptomics data.

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cell cycle progression in response to DNA damage(Preuss and Britt, 2003; Yoshiyama et al., 2009). Thesog1-1 mutant was originally isolated by its lacking the.6-d growth delay in true leaf development observedduring germination in a repair-defective xpf back-ground, after a 100 Gy IR dose was applied to imbibedseeds. Under these conditions, the sog1-1mutant lackedthe DNA damage-induced G2-phase cell cycle arrestobserved in its xpf background, but the plants weregenetically unstable (Preuss and Britt, 2003; Huefneret al., 2014). The SOG1 transcriptional induction of cellcycle arrest involves down-regulating certain factors(e.g. CDKB1;2, CDKB2;1, and KNOLLE [Yoshiyamaet al., 2009; Missirian et al., 2014]), while directlyup-regulating other factors (e.g. CYCB1;1, SMR-5,7[Weimer et al., 2016a; Yi et al., 2014], and WEE1 indi-rectly [De Schutter et al., 2007, Cools et al., 2011]).IR-induced SOG1 also transcriptionally activates DNArepair genes, including BRCA1 and RAD51 (Yoshiyamaet al., 2009), which function in homologous recombi-nation (HR)-based repair in S and G2 phase duringnormal growth (Shrivastav et al., 2008; Menges et al.,2003). SOG1 appears to mediate (via upregulatedCYCB1;1, in complex with CDKB1) damage-localizedHR repair by BRCA1 and RAD51, in conjunction withRBR1 (Biedermann et al., 2017; Weimer et al., 2016a;Horvath et al., 2017). HR repair can help to restore adamaged cell’s genomic integrity in addition to thefaster (Mao et al., 2008), predominating canonicalnonhomologous end-joining repair pathway (Čermáket al., 2017). SOG1’s induction of HR repair, in combi-nation with cell cycle arrest, can prevent cell death frommitosis occurring prematurely in the presence of unre-paired DSBs (Furukawa et al., 2010; Cools et al., 2011;Léguillier et al., 2012; Yi et al., 2014).

During normal growth, the stem cell niche (SCN) ofroot and shoot meristems contains stem cells that aremaintained in an undifferentiated state. Each stem cellcan self-renew and produce a transit-amplifyingdaughter cell in a specialized “asymmetric” cell divi-sion. The transit-amplifying daughter cells can prolif-erate through conventional mitotic (symmetrical) celldivisions and subsequently differentiate into special-ized cell types. The aforementioned processes are reg-ulated by positional signals (Heidstra and Sabatini,2014). Cell-type-specific programmed cell death (PCD)has been observed in root meristems of the model plantArabidopsis (Arabidopsis thaliana) after exposure to IR,radiomimetic chemicals, UV (Curtis and Hays, 2007;Furukawa et al., 2010; Fulcher and Sablowski, 2009),and chilling stress (Hong et al., 2017). This programmedresponse requires SOG1, ATM, and (to a lesser degree)ATR as well as de novo protein synthesis (Furukawaet al., 2010). This PCD is focused in the stele cell initialsand their immediate daughters, as well as to a lesserextent in columella initials; contrastingly, in SOG1-deficient lines, cell death is observed 1 d later and isdistributed randomly throughout the mitotic popula-tion (Furukawa et al., 2010). DDR-induced PCD re-duces the accumulation of cells with compromised

genomic integrity in a multicellular organism (Hu et al.,2016) and acts to prevent tumor formation inmammals.Most plant species are not susceptible to neoplasia, ascells are immobilized by the cell wall and have multi-faceted hormone regulation by neighboring cells(Doonan and Sablowski, 2010). The relevance of thisdevelopmentally specific DNA damage-induced PCDin the primary root has been somewhat unclear, giventhat the primary root’s function can be effectivelyreplaced by a lateral root, and the root does not con-tribute to the next generation.

Here, we report the role of SOG1 in the recovery ofthe root tip after DNA damage induced by an acutedose of IR (150 Gy). We demonstrate that SOG1-mediated PCD (Furukawa et al., 2010) triggers theremoval of a subset of stem cells, with the resultingcell death triggering a regeneration response in thesurrounding root apical meristem (RAM). We demon-strate that SOG1 mediates an arrest to proliferative(anticlinal) cell division, which, in combination withSOG1’s known role in transcriptionally inducing HR(Yoshiyama et al., 2009), likely supports the mitotic-competency of remnant cells. We also demonstratethat the regeneration response, which involves a partialloss of cellular identities and the induction of regener-ative (periclinal) cell divisions, facilitates the rebuildingof a functional stem cell niche able to resume prolifer-ative cell division (and thus root growth). As SOG1 isunique to seed-bearing plants (Yoshiyama, 2016), thisdevelopmental response may have evolved to rescuethe growth of seed-borne embryos from DNA damageaccumulated during aging (Waterworth et al., 2015).

RESULTS

Recovery of the Primary Root after IR Involves Dissolutionand Reconstruction of the RAM

To understand the recovery of roots after acute DNAdamage, we followed the long-term effects of an acutedose of IR (150 Gy) on 5-d-old Arabidopsis roots. Afterirradiation, growth in wild type slowed to less than amillimeter per day for several days (2–5+ d), withgrowth recovering after about 1 week; sog1 mutants, incontrast, had stopped all growth by 3 d after IR and didnot recover (Fig. 1, A and B). The difference in growthrate between wild type versus sog1-1 after IR was muchmore noticeable in roots than in shoots (Fig. 1A versusSupplemental Fig. S1, note that sog1 grows similarly towild type in unstressed conditions). Irradiated wild-type roots, but not sog1 roots, undergo short term(6+ h after IR), cell-type-specific induction of PCD in thestele precursor cells and columella initials (Fig. 1C). Inthe days (24+ h) following IR, we observed (SOG1-independent) death across the mitotic zone of sog1roots in all cell types (Fig. 1C), whereas such death wasnot observed across the mitotic zone of wild-type roots.These root cell death patterns are consistent with pre-vious observations of the short-term (#2 d) effects ofSOG1 deficiency after 100Gy IR (Furukawa et al., 2010).

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IR also affected the structure, as well as the growthrate, of the RAM. The quiescent center (QC) is a clusterof four cells that rarely divide, with the stem cells di-viding proximal to it driving root elongation and thecells dividing distal to it producing the continuallysloughing root cap (Heidstra and Sabatini, 2014). Con-current with the aforementioned growth arrest in wildtype (2–5+ days post IR), the stereotypical pattern ofcells in the wild-type RAM became disorganized, andthe QC became impossible to identify morphologically(Fig. 1C). Over the next 5 d, we observed that aQC-specific marker, pWOX5:ERGFP (ten Hove et al.,2010), transiently expanded its expression in an irreg-ular pattern that included the former position of the QC(Fig. 2A) but later refocused as the stereotypical RAM(and root growth) was reestablished. This pWOX5:ERGFP expansion was previously observed after achronic (24 h) exposure to a radiomimetic compound

(0.6 mg/mL bleomycin). This WOX5 expansion wasthought to reflect QC cell division (Heyman et al., 2013),consistent with the long-established hypothesis that theQC can divide to replace adjacent stem cells if they die(Clowes, 1959).

If the expansion of WOX5 expression simply reflectsan enlargement of the QC, one would expect tosee additional QC-specific markers expressed in thesame pattern. We followed the QC and SCN marker,pAGL42:ERGFP (Nawy et al., 2005), and observed atransient expansion of expression, then refocusing asRAM organization and root growth were restored(Supplemental Fig. S2A), similar to the changes ob-served for pWOX5:ERGFP. We then followed the ex-pression of two enhancer-trap GUS markers: QC25(which is expressed in both the QC and the more distalcolumella cells) and QC46 (Sabatini et al., 2003). Theexpression of these markers transiently diminished

Figure 1. Growth recovery after IR. A,Root growth rate per day (growth sinceprevious day) after 150 Gy IR, as a fractionof root growth rate for the mock-irradiatedcontrol, in wild type (WT), sog1-1, andsog1-1 + tgSOG1. ***sog1-1 samples aresignificantly different from wild-type(P, 0.001); *wild-type samples at 7 d afterIR are significantly different from those 6 dafter IR (P , 0.05). B, Wild-type (left) andsog1-1 (right) roots at 8 d after IR. Arrow-head indicates root tip position at time ofIR. C, Five-day-old wild-type, sog1-1, orsog1-1 + SOG1 root tips were stained withpropidium iodide and imaged up to 7 dafter 150 Gy IR.

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(in contrast to the expansion observed for WOX5 andAGL42), before being reestablished along with RAMorganization and root growth. Put together, thesefindings suggest that some aspects of QC identity werelost (Sabatini et al., 2003), and/or that some aspects ofQC identity were ectopically acquired by neighboringcells (Heyman et al., 2013). It is unlikely that the loss ofthe QC markers was due to the death of QC cells, asthey are particularly resistant to IR-induced cell death(Curtis andHays, 2007; Heyman et al., 2013), in contrastto the cells that surround the QC (Fig. 1C; Furukawaet al., 2010; Yoshiyama et al., 2013).

Further support for the premise that RAM cells un-dergo partial loss of identity during the restoration ofSCN mitotic-competency came from the rapid anddramatic expansion, then refocusing, of the pCYCD6:ERGFP (Sozzani et al., 2010) cortex/endodermis initial

(CEI) cell marker (Supplemental Fig. S2B). This gene isinvolved in promoting the periclinal division of the CEIcell during normal growth in order to form the cortexand endodermal cell files (Heidstra and Sabatini, 2014;Sozzani et al., 2010). The identity of the stele precursorcells as characterized by pWOL:ERGFP expression(Birnbaum et al., 2003; Supplemental Fig. S2C)remained unchanged, despite the expansion of pWOX5:ERGFP expression into this stele tissue. Localization ofthe auxin maximum in the QC is crucial for root SCNfunction (Heidstra and Sabatini, 2014) and was sur-veyed based on pDR5:ERGFP expression (Ottenschlägeret al., 2003). We did not observe noticeable changes inthis auxin maximum (Supplemental Fig. S3), such asthose that were recently described in response to chill-ing stress (Hong et al., 2017). Put together, the changeswe observed in cell typemarker expression suggest that

Figure 2. Effects of IR on QC marker ex-pression. A, Five-day-old seedlings carryingpWOX5:ERGFP in wild-type (WT), sog1-1, orsog1-9 backgrounds were irradiated andimaged up to 8 d after 150Gy IR. False color:black, propidium iodide; purple, GFP. B,Five-day-old seedlings carrying QC25 (top)or QC46 (bottom) GUS enhancer trap lineswere stained and imaged up to 8 d after150 Gy IR.

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both the QC and its surrounding cells undergo a partialloss of identity during the recovery process.

Dissolution of RAM Organization Is a ProgrammedResponse Requiring SOG1

The QC of wild-type root tips became morphologi-cally unrecognizable within 48 h of IR, whereas thestereotypical organization of the RAM was stable insog1 (Fig. 1C). In addition, expansion of the WOX5 ex-pression domain (2–6 d) was observed in wild-typeroots, but not in sog1 roots (Fig. 2A). We observedthat recovery of normal root growth rate and mor-phology occurred in wild type 7 d after IR, whereas thesog1 root tip failed to recover and showed cellular en-largements normally only observed in the cells of theelongation zone (Fig. 1C). These cellular enlargementsin sog1 probably reflected the progression of differen-tiation and endoreduplication (which occurs normallyduring root development) down to the tip of thearrested root. The conservation of root tip structure andcell identity in sog1 suggests that SOG1-dependent PCD(and/or another unknown SOG1-dependent response)is associated with the disrupted RAM identity dis-played in irradiated wild-type plants. Root growth re-covery is thus associated with both the PCD and partialloss of cell type identities observed in wild-type roottips; both of these processes are absent in sog1 root tips.

SOG1 Is Required for Cell Cycle Arrest Immediatelyafter IR

In sog1 mutants, we observed (SOG1-independent)death in the days following IR, including more celldeath across the mitotic zone (in all cell types, without aSCN-focus) comparedwith wild type (e.g. Fig. 2A, wildtype versus sog1 at 32 h after IR). We hypothesized thatthis death in sog1 may be due to mitosis proceeding inthe presence of unrepaired DSBs, based on the distri-bution across the mitotic zone of the root and thedelayed onset with respect to PCD (32 h versus 8 h; Yiet al., 2014; Furukawa et al., 2010; Cools et al., 2011). Toinvestigate the role of SOG1 (and hence the transcrip-tional response) in cell cycle arrest after IR, we mea-sured DNA replication (as 5-ethynyl-29deoxyribose[EdU] incorporation during S phase) and cell division(by visualizing metaphase cells using a fluorescenthistone marker) in wild-type and sog1 lines. We foundthat DNA replication (Fig. 3A) and cell division(Fig. 3B) were largely inhibited in the mitotic zone6 to 10 h after IR in wild-type seedlings, but not in sog1.The lack of cell cycle control in sog1, coupled withthe defective induction of HR repair transcripts(Yoshiyama et al., 2009), may be responsible for thepresumably unprogrammed cell death in the mitoticzone (Furukawa et al., 2010). We also wished to analyzethe frequency of regenerative periclinal (sideways) celldivisions, which establish new cell files and thus are

essential for the restoration of a functional root meri-stem (Heyman et al., 2016) during growth recovery. Aspart of visualizing metaphase cells, we found an aver-age of 1 to 2 periclinal metaphase cells per wild-typeroot but essentially none per sog1 root from 5 to 7 d afterIR (Fig. 3B). Furthermore, by 5 d after IR, wild-type roots had restored their proliferative anticlinal(lengthwise) cell divisions, which contribute to rootelongation (Heidstra and Sabatini, 2014), to levels ob-served in mock-irradiated roots.

ERF115 Is Induced Ectopically, Outside of the QC, inResponse to Cell Death

ETHYLENE RESPONSE FACTOR115 (ERF115;AT5G07310) is a gene recently demonstrated to beinduced in cells that are near dead cells in the roottip (Heyman et al., 2016; Zhang et al., 2016); Alongwith PHYTOCHROME A SIGNAL TRANSDUCTION1(PAT1), ERF115 has a key role in promoting regenera-tive divisions of neighboring cells and thus is importantin the maintenance and recovery of a damaged SCN(Heyman et al., 2013, 2016). We sought to investigatethe induction of ERF115, alongside PCD, after IR. Ac-cordingly, we visualized the pERF115:NLS(-GUS/)GFPmarker line (Heyman et al., 2013) hourly during theonset of PCD (3–7 h after IR), while staining for celldeath. We found that ERF115 was induced in the same5+ h time frame and cell types as PCD in wild-typeroots (Fig. 4). We found that ERF115 expression wasinduced in an average of 2 to 3 remnant cells per eachdead cell, as observable in these two-dimensional im-ages (6 and 7 h after IR; Fig. 4).

To determine the duration of ERF115 induction afterIR, we visualized the pERF115:GFP:ERF115marker linethat encodes the ERF115 protein (Heyman et al., 2013),which is particularly subject to degradation in the un-perturbed cell (Heyman et al., 2013). We found thatpERF115:GFP:ERF115 expression peaked a few daysafter IR and disappeared once root growthwas restored(Supplemental Fig. S4). In these visualizations, wecould clearly observe ERF115 expression focused in thestele precursor cells.

We then sought to determine the dependency ofERF115 induction on SOG1. At 8 h after IR, we foundthat ERF115was specifically induced in wild-type rootsin a SCN-focused pattern similar to the induction ofPCD in wild type (Fig. 5, A and B). In sog1 roots, thefocused expression in the SCN is lost; there is insteadERF115 induction across the mitotic zone at 32 h afterIR, locatedwithin the stele, and to a limited extent in theendodermis (Fig. 5A). Cell death is similarly presentin these stele and endodermal tissues in sog1 lines(Fig. 5B). The cortical and epidermal cells in the mitoticzone of sog1 lines also exhibit cell death yet no ERF115expression; this finding is in line with a previous studythat found these cell types did not induce ERF115 orregenerative periclinal cell divisions in response todead cells nearby (Heyman et al., 2016). This ERF115

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marker was also induced in the shoot, similar to thatseen in the root, with a SOG1-dependent 8-h onset andfocus in the shoot apical meristem for wild type(Supplemental Fig. S5). In the sog1 mutant, the 8-h in-duction focused in the shoot apical meristem was lost,and ERF115 expression was observed at 32 h after IR,throughout the growing leaflet.

The results described above for the root, in which celldeath is easily assayed, indicate that the induction ofERF115 can be induced by cell death in the absenceSOG1, and that it roughly follows the pattern of celldeath. It has been previously shown that ERF115 ex-pression has been induced inwild type uponwoundingroots by excising their tips (Heyman et al., 2016).To determine whether this ERF115 induction was alsoSOG1 independent, we compared wound-inducedpERF115:GUS expression in wild-type versus sog1roots. We found that pERF115:GUS was indeed in-duced by death, regardless of the presence or absence ofSOG1 (Fig. 5C). In the decapitated root, we observedERF115 induction primarily in stele cells, which play animportant role in root tip regeneration (Efroni et al.,2016). Similar to a previous study (Heyman et al., 2016),

we did not see ERF115 induction in epidermal andcortical cells bordering the stump of the decapitatedroot. Taken together, these observations indicate thatpERF115:GUS expression is driven by cell death, be itfrom SOG1-dependent PCD, from SOG1-independentmitosis-linked cell death, or from wounding-induceddeath. It is possible that the induction of ERF115 byIR in the SCN is the one exception to this rule and doesrequire transcriptional induction by SOG1, but thesimpler hypothesis is that SOG1 is instead inducingdeath, and death per se is inducing ERF115.

The transcriptional induction of ERF115 has not beenpreviously observed in IR-response transcriptomes(Ricaud et al., 2007; Yoshiyama et al., 2009; Missirianet al., 2014), possibly due to its localized expression in asmall subset of cells. Nonetheless, this discrepancy didraise the question of whether its IR induction is an ar-tifact of the GUS or GFP fusion transgenes, rather thanthe native gene. To follow expression of the native gene,we employed cell-type-specific transcriptomics. ApWOL:ERGFP line (Birnbaum et al., 2003) was used topurify GFP+ protoplasts from the stele precursor cells.Wewould not expect to captureQC cellswhen purifying

Figure 3. Effects of SOG1 on cell cycle arrest inthe RAM after irradiation. A, DNA replication inthe mitotic zone measured as EdU-labeled nuclei.Five-day-old seedlings were irradiated to 150 Gyand labeledwith EdU for 4 h beginning at 6 h afterthe completion of IR. Error bars are SE (wild-type[WT], n = 9; sog1-1, n = 6; sog1-1 + tgSOG1,n = 6; wild type + IR, n = 11; sog1-1 + IR, n = 10;sog1-1 + tgSOG1 + IR, n = 10). *+ IR samplesare significantly different frommock-IR controls(P , 0.05). B, The number of metaphase cellsper root tip observed in anticlinal versus peri-clinal orientations, up to 7 d after 5-d-old wild-type or sog1-9 seedlings, both carrying pRPS5a::H2B:eCFP, were irradiated with 150 Gy IR. Errorbars are SE (wild type, n = 8; sog1-9, n = 8). * and**sog1-9 samples are significantly different fromwild type (P values are , 0.05 and , 0.01, re-spectively).

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for cells expressing this construct (Efroni et al., 2015,2016). We could thus compare the transcriptomes of ir-radiated stele precursor cells (during the onset of PCD,8.5 h after 100 Gy; Supplemental Figs. S6 and S7) versus

entire root tips. ERF115 was indeed transcriptionallyinduced in the stele precursors (Fig. 6). We found thatERF114 (ERF115’s closest homolog, AT5G61890) andPAT1 (ERF115’s partner) were also induced in steleprecursors during the onset of PCD (Fig. 6).

We investigated the role of ERF115 in root growthrecovery. We found that erf115 mutant seedlings couldrecover their growth after IR, albeit with a delay ofseveral days with respect to wild type (SupplementalFig. S8). Considering the apparent functional redun-dancy of ERF115 in its initially reported role in pro-moting QC cell division (Heyman et al., 2013), we thenanalyzed the p35S:ERF115-SRDX transgenic line(Heyman et al., 2013, 2016). Using this line, whichexpresses a dominant transcriptional repressor ofERF115’s targets, we found that the majority of rootsfailed to recover their growth (Supplemental Fig. S9),even when analyzed over a longer (15 d) time course. Itwas previously reported that p35S:ERF115-SRDXtransgenics failed to recover growth over a shorter (5 d)time course, which was done after an acute (24 h) ex-posure to (0.6 mg/mL) bleomycin (Heyman et al., 2013).

Figure 5. pERF115:GUS expression aftercell death. A, Five-day-old seedlings car-rying pERF115:NLS-GUS/GFP were irradi-ated and imaged 8 and 32 h after 150 Gy IR(or mock irradiation). B, Five-day-oldseedlings were irradiated with 150 Gy.Seedlings were stained with propidium io-dide to visualize PCD up to 32 h after IR. C,Five-day-old wild-type (WT) or sog1-1seedlings carrying pERF115:NLS-GUS/GFPwere either cut in the meristematic zone orleft intact, then stained with propidiumiodide (top) up to 8 h after cutting, andimmediately following visualization; thesame seedlings were stained for GUS ac-tivity (bottom).

Figure 4. Effects of IR on ERF115 expression and PCD. Five-day-oldseedlings carrying pERF115:NLS-GUS/GFP in wild-type backgroundswere irradiated and imaged up to 7 h after 150Gy IR. False color: black,propidium iodide; purple, GFP.

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DISCUSSION

SOG1 Is Required to Direct an ERF115-DependentRegeneration Pathway and an Immediate Cell Cycle Arrestin Response to IR

Here, we describe key roles of SOG1 in the long-termgrowth recovery of the Arabidopsis seedling’s primaryroot after acute DNAdamage.Wild-type primary roots,but not those of sog1mutants, can recover their growthafter a severe (150 Gy) IR dose after 7 d. This recoveryrequires an initial SOG1-mediated DDR, which servesin the long-term regeneration of a mitotically compe-tent SCN. Our prior understanding of SOG1 in theDDR has been essentially limited to short-termeffects, namely the transcriptional induction of PCD(Furukawa et al., 2010; Yoshiyama et al., 2009), cellcycle inhibitors (Culligan et al., 2006; Yoshiyama et al.,2009; Yi et al., 2014), and DNA repair factors(Yoshiyama et al., 2009).

SOG1 transcriptionally regulates a variety of short-term (#2 d) processes in the DDR, as described fol-lowing. SOG1 induces PCD in many of the IR-damagedstem cells (and their daughters) in the first 6 to 10 h afterIR (Furukawa et al., 2010), thereby rapidly but indirectlyinducing an ERF115-mediated regeneration responsenear the damaged SCN. sog1’s defect in PCD in theIR-compromised SCN (6–10 h after IR) results in thepersistence of damaged cells, whichmight then act as ananatomical block to regeneration. We also demonstratethat SOG1 is required for the arrest of the cell cycle inthese surviving cells, also within 6 to 10 h after IR. Thisarrest, along with the previously reported role of SOG1in the induction DNA repair transcripts (Culligan et al.,2006; Yoshiyama et al., 2013), can support the mitoticcompetency of remnant cells that are needed to replen-ish the IR-compromised SCNandhence resume growth.sog1mutants’ defect in cell cycle arrest (8 h), along withtheir failed induction of DNA repair transcripts(Yoshiyama et al., 2013), predisposes them to the (32 honset) cell death seen across the root’s mitotic zone in allcell types. A similar (patchy) pattern of IR-induced celldeath across the mitotic zone was also demonstrated inatr, atm double mutants (Furukawa et al., 2010). ERF115induction is associated with the cell death across themitotic zone (primarily in the stele tissue) in sog1 roots

(Fig. 5, A and B); however, ERF115 induction in the SCNis weak in sog1 relative to wild type. ERF115 inductionis associated with cell death induced by wounding inwild type (Heyman et al., 2016) and in sog1 (Fig. 5C).Both of these experiments show that ERF115 is inducedby cell death (independently of SOG1) and hencestrongly suggest that the SCN-focused ERF115 induc-tion in wild-type roots after IR is due to SOG1-dependent PCD, rather than due to a more directtranscriptional induction by SOG1.

We have observed two long-term processes associ-ated with SOG1 activity and root growth recovery afterIR. The first process is the transient loss of the stereo-typical RAM structure, including some loss in iden-tity for its constituent cell types (2–5 d after IR),which subsequently reform along with the restora-tion of proliferative cell divisions responsible for growth(5+ d after IR). The second process we have observedin root growth recovery is the occurrence of regenera-tive periclinal cell division in most wild-type roots(5–7 d after IR). Such cell division may function inreplacing the (re)growth-enabling SCN cells from mi-totically competent remnant cells nearby (Heymanet al., 2016), such as the transit-amplifying cells (Efroniet al., 2016) and/or surviving SCN cells. We reasonthat these changes in cytoarchitecture occur as a re-sponse to SOG1-dependent PCD at the SCN; as such,cell identity changes have been previously observedduring regeneration after cell ablation (van den Berget al., 1997) or root-tip excision (Efroni et al., 2016). Theprocess of SCN regeneration after SOG1-dependentPCD may, like SCN regeneration after excision, fol-low the plant’s endogenous positional patterning thatis established in the embryo (Efroni et al., 2016). sog1mutants, in contrast, maintain a stereotypical RAM,including a well-defined QC surrounded by a mitoti-cally compromised SCN, rather than undergoing thepartial loss of cellular identities (2–5 d) observed inwild type. Moreover, sog1 mutants fail to induce theregenerative periclinal cell divisions (5–7 d after IR)that seem necessary to reform a mitotically competentSCN; only one periclinal cell division was observedacross 24 sog1 roots, whereas 30 such divisions wereobserved across 24 wild-type roots. The failure of theaforementioned SOG1-dependent processes likelycontributes to the permanent growth arrest and ter-minal differentiation observed in sog1 roots. Put to-gether, we conclude that SOG1 functions in salvagingthe overall mitotic competency of the primary root afterIR; both by removing damage-compromised SCN cellsto stimulate (normally very rare) periclinal cell divi-sions for replacing these dead cells, as well as inducingcell cycle arrest and DNA repair in remnant cells.

What Role Might This SOG1-Dependent PCD Haveduring Normal Plant Development?

We have observed that the Arabidopsis primary rootwill undergo aweek-long process of growth restoration

Figure 6. WOX5, ERF115 induction during the onset of PCD. WOX5,ERF115, ERF114, PAT1, ACT1 (ACTIN1) fold change in GFP+ (stele-specific) or whole-root-tip protoplasts during the onset of PCD after100 Gy IR. Error bars are SE. *Irradiated samples are significantly dif-ferentially expressed from mock-irradiated controls (P , 0.05).

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in response to acute DNA damage, even though theroot’s growth might be restored via the production of alateral root. During a plant’s normal growth in the soil,a growing root tip might encounter a persistent sourceof DNA damage, such as from the presence of a toxicheavy metal (Hu et al., 2016; Sjogren et al., 2015). In thisscenario, the preferential growth of lateral roots (ratherthan a futile cycle of primary root DDR-induced PCDand regeneration) could successfully redirect rootgrowth away from the chronic DNA-damaging agent.In contrast, the restoration of primary root tip growthseems most cost beneficial (and evolutionarily favor-able) in the case of an acute and transient exposure toDNA damage, which is the case for seeds upon imbi-bition, where they acutely experience the DNA damagethey accumulated during aging (Waterworth et al.,2015, 2016). Regeneration of the embryonic root in re-sponse to DNA damage is critical for the viability of agerminating seed, as the linear-growing embryonic rootcarries no additional root primordia (Van Normanet al., 2013). We propose that SOG1, a gene unique toseed-bearing plants (Yoshiyama, 2016), may haveevolved to salvage the overall mitotic competency(prevent permanent mitotic arrest) of the embryonicroot during the germination of aged seeds.

What Are the Relative Contributions of Individual DDRand Regeneration Processes to the Overall RootGrowth Recovery?

Some stem cells and some of their early descendants aremoreprone to IR-inducedPCDthanothers. The factors thatspecifically regulate cell-type-specific PCD, downstream ofSOG1, are unknown (Hu et al., 2016). It is possible thatdamaged cells begin PCD after reaching a critical thresholdof damage, and that threshold may depend on both thecell type and its position in cell cycle (Hu et al., 2016).Due to the absence of lines uniquely defective in PCD

(but not in other SOG1-influenced processes; Hu et al.,2016), we have not been able to characterize the uniquecontribution of PCD to the growth recovery of thedamaged root, relative to the contribution of otherSOG1-induced processes (e.g. cell cycle arrest andDNArepair). Nonetheless, it is likely that various SOG1-induced genes contribute to root growth recovery af-ter DNA damage, which can be appreciated by thehypersensitivity of relevant mutants to DNA damage.For example, the growth of cycb1;1 and rad51mutants iscompromised in response to cisplatin, a DNA crosslinker and DSB-inducer (Weimer et al., 2016a). Simi-larly, brca1 mutants show enhanced cell death to Mi-tomycin C, a DNA cross linker (Horvath et al., 2017).DNA damage-induced hypersensitivity was also ob-served for mutants in PARP1,2 (Song et al., 2015) andRAD17 (Heitzeberg et al., 2004) in response to bleomycinand Mitomycin C; these genes are also damage-inducedby SOG1 (Culligan et al., 2006), with a function in analternative, microhomology-mediated nonhomologousend-joining repair pathway, and in ssDNA-sensing for

checkpoint control, respectively (Shrivastav et al., 2008;Hu et al., 2016).

It is possible that the transient enlargement of the zoneof expression for the pCYCD6:ERGFP (Sozzani et al.,2010) marker of cortex/endodermis initial cells(Supplemental Fig. S2B; 8 h–5 d) is due to its role in thepromotion of periclinal cell division, which CEI cellsundergo during normal growth to form the cortex andendodermal cell files (Heidstra and Sabatini, 2014;Sozzani et al., 2010). The expansion of this marker into avariety of cells within the RAM may reflect the replace-ment of dead cells by replenishing periclinal divisions ofneighboring cells (Heyman et al., 2016). The finding thatp35S:ERF115-SRDX plants generally failed to recovertheir growth after damage (Heyman et al., 2013),whereas erf115 knockout mutants merely exhibited adelayed restoration of growth, suggests that there is analternate “backup” pathway for SCN regeneration. It ispossible that ERF114 plays a role in this alternativepathway for regeneration, as it is the closest homolog ofERF115 (Heyman et al., 2016) and is also IR inducible inthe stele progenitor cells (Fig. 6). It is also possible that analternate regeneration pathway may be involved.

MATERIALS AND METHODS

Growth and Irradiation of Seedlings

Arabidopsis (Arabidopsis thaliana) seeds were sterilized in 20% Clorox bleachand 0.1%TritonX-100 and sownon 13MSsalts, 0.3%Suc (Sigma), 0.8%Phytoagar(BioWorld), pH 5.7. Seedlings were grown on vertical plates in 16-h days undercool-white lamps (photon flux density of 100 mmol m22 s21), 20°C for 5 d afterhaving stratified for 48 h at 4°C. Plantswere restored to these conditions after IR orcutting. Any time point specified in text is the amount of time that has passed aftercompletion of IR or cutting. The seedlings were gamma-irradiated in the dark,using a Cs137 source with a dose rate of either 5.95 Gy/min (for the tissue-specifictranscriptomics experiment) or 1.8 Gy/min for all other experiments. Irradiationswere performed between 8 and 10 a.m., with typical experiments requiring a 1.5-hexposure. The growth chamber lamps were on from 8 a.m. to midnight.

Plant Material and Transgenic Lines

We employed Arabidopsis Col-0 (Columbia) as wild type. The sog1-1 linewas derived from our previously reported xpf-2 sog1-1 cycB1;1:GUS line, aLandsberg erecta/Col hybrid (Preuss and Britt, 2003), by backcrossing to Col-0twice and then self-pollinated to generate homozygous sog1-1 lines. sog1-9 wasderived from a tetraploid Col-0 TILLING population (Tsai et al., 2013) andhaploidized via GFP-tailswap(-CENH3) (Ravi et al., 2014). The resulting diploidwas backcrossed three times to Col-0 and then self-pollinated to generate ho-mozygotes. sog1-9 carries a nonsense C-to-T mutation 826 bp downstream ofthe ATG. The pERF115:NLS-GUS/GFP (Heyman et al., 2013) and pWOX5:

ERGFP (ten Hove et al., 2010; Blilou et al., 2005) markers were each crossedwith sog1-1 and sog1-9 lines; plants that were homozygous for the sog1 alleleswere identified in the F2. Experiments were performedwith sog1 lines that weresegregating for the markers, scoring only those seedlings expressing themarkerin lateral and/or primary roots. Similarly, the pRPS5a::H2B:eCFP marker linewas crossed with sog1-9, and mutant homozygotes were identified in the F2,then used for mitotic figure experiments (along with the pRPS5a::H2B:eCFPmarker line). A Student’s t test was applied to the comparisons shown in Fig-ures 1A and 3B, with two tails and the assumption of unequal variance.

GUS Staining and Visualization

Seedlingswereharvested into0.4mLof ice-cold 80%acetone ina48-well,flat-bottomCostarmicrotitre plate (Corning) and incubated at room temperature for

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20min. Following removal of the acetone, the sampleswere incubated in 0.2mLof GUS staining buffer (25 mM NaH2PO4/Na2HPO4 buffer; pH 7.0, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 0.25% Triton, 0.25 mM EDTA, 1 mg/mL X-Gluc GoldBiotechnology) at 37°C for 1 h. The samples were mounted on slides in an 8:3:1mixture of chloral hydrate:water:glycerol and analyzed using an Axioskop2 plus microscope (Zeiss) under DIC optics using the Axiovision program(version 4.8).

Tissue-Specific Transcriptomics of Cell Death

Five-day-old pWOL:ERGFP (Birnbaum et al., 2003) seedlings grown on 0.8%Murashige and Skoog + 1% Suc media were irradiated to 100 Gy at a dose rateof 5.94 Gy/min in the dark. Fifteen minutes after IR, plates were returned to thegrowth chamber. Five-and-a-half hours after IR, root tips were chopped andprotoplasted as described (Birnbaum et al., 2005). Protoplasts were sorted witha Cytomation MoFlo Cell Sorter and frozen in liquid nitrogen ;3 h afterchopping. RNA was isolated using Trizol, and mRNA was captured usingOligodT Dynabeads using the manufacturer’s protocol (Invitrogen). RNA-seqlibraries were created as described (Kumar et al., 2012), with the modificationthat the total RNAwas extracted and purified from the protoplasts using Trizol.The libraries were multiplexed and sequenced using Illumina’s GAII andHiSEquation 2000. Reads were quality trimmed with a custom script (writtenby Ted Toal, UCDavis). Alignment to TAIR10 was performedwith BWA-MEM(Li, 2013), transcript abundance was counted using the HTSeq library (Anderset al., 2015), and differential expression calls were calculatedwith DEseq2 (Loveet al., 2014), v1.4.5. Raw data are available in the SRA with the BioProject Ac-cession: PRJNA380494.

Visualization of Cell Death

Seedlings were incubated with 5 mg/mL propidium iodide in water for 5 mon a microscope slide, before confocal imaging with a Zeiss LSM710.

EdU Incorporation

Using the Click-iT EdU Alexa Fluor 488 Imaging Kit (Invitrogen), seedlingswere incubatedwith 10mMEdU for 4 h tomeasure S phase entry beginning at 6,24, and 48 h after IR or mock treatment, before confocal imaging. Nuclei werecounted in the mitotic zone using ImageJ, with the plugin ITCN (width 15, mindistance 7.5, threshold 0.1)

Tip Excision

pERF115:NLS-GUS/GFP primary roots were cut by hand with an 18 Ga3 1”Monoject 200 needle (Medtronic) in the approximate QC position. After incu-bation, the seedlings were stained for GUS activity and visualized.

Accession Numbers

ERF114 (AT5G61890), ERF115 (AT5G07310), PAT1 (AT5G48150), and SOG1(AT1G25580).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. True leaf production after IR.

Supplemental Figure S2. Effects of IR on cell type-specific marker expres-sion.

Supplemental Figure S3. Effects of IR on pDR5:ERGFP expression.

Supplemental Figure S4. Effects of IR on pERF115:GFP:ERF115 expression.

Supplemental Figure S5. pERF115:GUS expression in the shoot apical mer-istem.

Supplemental Figure S6. Representation of roots used to obtain WOLmarker-expressing cells, versus whole-root-tip cells, for stele-specifictranscriptomics.

Supplemental Figure S7. Timing of PCD after 100 Gy IR.

Supplemental Figure S8. Growth recovery after IR in erf1152/2.

Supplemental Figure S9. Growth recovery after IR in p35S:ERF115-SRDX.

ACKNOWLEDGMENTS

The authors thank Lieven De Veylder (VIB and University of Ghent) forpERF115:NLS-GUS/GFP, pERF115:ERF115:GFP, erf115 (KO, SALK_021981),and p35S:ERF115-SRDX; Philip Benfey (Duke University) for pWOL:ERGFP;Mohan Marimuthu and Luca Comai (University of California Davis) for devel-oping and sharing their unpublished pRPS5a::H2B:eCFP marker line; ElliotMeyerowitz (California Institute of Technology) for pWOX5:ERGFP; RenzeHeidstra (Wageningen University) for the GUS enhancer trap lines QC25 andQC46; Klaus Palme (University of Freiburg) for the pDR5:ERGFP marker line;Natalie Clark and Ross Sozzani (North Carolina State University) for other GFPlines, and; Idan Efroni (the Hebrew University) for root cutting advice.

Received September 11, 2017; accepted December 2, 2017; published December8, 2017.

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