Redox-Mediated Endocytosis of a Receptor-Like Kinase ... · observed DSC layers using Lugol’s...

Redox-Mediated Endocytosis of a Receptor-Like Kinaseduring Distal Stem Cell Differentiation Depends on ItsTumor Necrosis Factor Receptor Domain1[OPEN]

Yingying Qin,a,2 Li Yang,b,2 Zhihui Sun,a Xiangfeng Wang,a Yu Wang,a Jing Zhang,a Amin Ur Rehman,a

Zhizhong Chen,a Junsheng Qi,a Baoshan Wang,c Chunpeng Song,d Shuhua Yang,a and Zhizhong Gonga,3,4

aState Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China AgriculturalUniversity, Beijing 100193, ChinabKey Laboratory of Horticultural Plant Biology, Ministry of Education, College of Horticulture and ForestrySciences, Huazhong Agricultural University, Wuhan 430070, ChinacKey Laboratory of Plant Stress Research, College of Life Science, Shandong Normal University, Ji’nan 250014,ChinadCollaborative Innovation Center of Crop Stress Biology, Henan Province, Institute of Plant Stress Biology,Henan University, Kaifeng 475001, China

ORCID IDs: 0000-0003-3230-1839 (Y.Q.); 0000-0001-7255-7906 (Z.S.); 0000-0002-3808-433X (J.Z.); 0000-0002-0991-9190 (B.W.);0000-0001-8774-4309 (C.S.); 0000-0003-1229-7166 (S.Y.); 0000-0001-6551-6014 (Z.G.).

Cellular redox status plays critical roles in cell division and differentiation, but the underlying mechanism is unclear. Here weexplored the effect of redox status on stem cell identity in distal stem cells (DSCs) of Arabidopsis (Arabidopsis thaliana) roots.Treatment with the reductive reagent glutathione and the oxidative reagent H2O2 inhibited DSC differentiation, as didendogenously altering reactive oxygen species production via various mutations. This suggests that both highly reductiveand oxidative environments inhibit specification of stem cell identity. In our observations of mutant components of theCLAVATA3/ENDOSPERM SURROUNDING REGION 40 (CLE40)-ARABIDOPSIS CRINKLY4 (ACR4)/CLAVATA1(CLV1)-WUSCHEL RELATED HOMEOBOX5 (WOX5) module, both reductive and oxidative reagents influenced DSCdifferentiation in wox5-1 and clv1-1, but not in acr4-2 or cle40 mutant plants. The stability of the receptor-like kinase ACR4is modulated by redox status through endocytosis in root tips. ACR4 with multiple Cys mutations in the tumor necrosis factorreceptor (TNFR) extracellular domain failed to undergo endocytosis. ACR4 with a complete deletion of the TNFR domain waslocalized directly to endosomes, bypassing the plasma membrane. Both mutations affected DSC differentiation, but not seedfilling. Conversely, the intracellular domain of the ACR4 protein is partially required for seed filling, but not for DSCdifferentiation. Our study uncovers an important biological role of the TNFR domain in redox-mediated endocytosis ofACR4 in root DSC differentiation.

Reactive oxygen species (ROS) are produced inthe plant apoplast and in various organelles, such asmitochondria, chloroplasts, and peroxisomes, undernormal and unfavorable environmental conditions

(Nanda et al., 2010; Qi et al., 2018). High concentra-tions of ROS are toxic to macromolecules such asDNA, RNA, lipids, and proteins. Plant cells havedeveloped an astonishing ability to adjust internaland external ROS levels using enzymatic and non-enzymatic scavenging mechanisms (Mittler et al.,2004; Baxter et al., 2014; Schmidt and Schippers,2015; Qi et al., 2018). Nevertheless, ROS are impor-tant redox signals that regulate diverse processesincluding cell division and differentiation in bothanimals and plants (Owusu-Ansah and Banerjee,2009; Wang et al., 2013; Paul et al., 2014; Schmidt andSchippers, 2015; Qi et al., 2018). In the Arabidopsis(Arabidopsis thaliana) root tip, the redox potential (asquantified using the redox-sensitive GFP reporter) ismore negative in the quiescent center (QC) and sur-rounding cells than in other cells of the root meri-stem (Jiang et al., 2016). Glutathione (GSH)/thioredoxin and ascorbate, which function as keyregulators of redox homeostasis, are required for cell

1This work was supported by the National Key Scientific ResearchProject (2015CB910203).

2These authors contributed equally to the article.3Author for contact: [email protected] author.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:Zhizhong Gong ([email protected]).

Z.G., Y.Q., and L.Y. conceived and designed the experiments. Y.Q.,L.Y., Z.S., and J.Z. performed the experiments. Z.G., Y.Q., and L.Y.wrote the manuscript. All other authors participated in the discussionof the results and commented on the manuscript.

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http://orcid.org/0000-0003-1229-7166

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http://orcid.org/0000-0003-3230-1839

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http://orcid.org/0000-0002-3808-433X

http://orcid.org/0000-0002-0991-9190

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http://orcid.org/0000-0003-1229-7166

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cycle progression and function in various oxida-tive stress-sensitive pathways (Potters et al., 2000;Vernoux et al., 2000; Reichheld et al., 2007; De Tullioet al., 2010; Correa-Aragunde et al., 2013; Ding et al.,2016). Mutants of ROOT MERISTEMLESS1 (RML1),encoding a g-glutamyl-Cys synthetase (GSH1) local-ized in plastids (Vernoux et al., 2000); MIAO, en-coding GLUTATHIONE REDUCTASE2 (GR2; Yuet al., 2013); the cytosolic H2O2-scavenging enzymeASCORBATE PEROXIDASE1 (APX1; Correa-Aragunde et al., 2013); and NADPH-dependent thi-oredoxin reductase (NTRA and NTRB; Reichheldet al., 2007; Bashandy et al., 2010) show growth de-fects of the root meristem.

Redox signaling is closely associated with the planthormone auxin (Tognetti et al., 2017). For example, themiaomutant shows reduced expression of PLETHORA(PLT) and the auxin transporter gene PIN. PLT genesare auxin-inducible, key regulators that maintainmeristem cell niche activity in Arabidopsis (Aidaet al., 2004). Superoxide anion (O2

.2) activatesWUSCHEL and maintains stem cell identity inshoots, whereas H2O2 promotes stem cell differentiationin the shoot meristem (Zeng et al., 2017). In Arabidopsisroots, O2

.2 primarily accumulates in dividing meristemcells to regulate cell proliferation, whereas H2O2 ismainly produced in the elongation zone and root hairs topromote cell differentiation (Dunand et al., 2007;Tsukagoshi et al., 2010). The distribution of O2

.2 andH2O2 is regulated by the basic helix-loop-helix tran-scription factor UPBEAT1 (UPB1), which directly regu-lates the expression of a set of peroxidases (Tsukagoshiet al., 2010). These findings indicate that cellular redoxplays crucial roles in maintaining meristem activity.

Root growth is maintained by the stem cell niche,which provides all types of root cells. The mitoti-cally inactive QC in the root stem cell niche is requiredfor maintaining the surrounding stem cells, includ-ing distal stem cells (DSCs; Wierzba and Tax,2013). DSCs differentiate into starch-containingcolumella cells, which can easily be observed afterLugol’s staining. Auxin promotes DSC differentia-tion, which requires the involvement of IAA17/AXR3, a well-characterized transcriptional repres-sor of the TRANSPORT INHIBITOR RESPONSE1(TIR1)-mediated auxin-signaling pathway (Dingand Friml, 2010). The homeodomain transcriptionfactor WUSCHEL RELATED HOMEOBOX5 (WOX5),which is specifically expressed in QC cells, is requiredfor maintaining the undifferentiated status of the QCand DSCs (Sarkar et al., 2007). The loss of WOX5promotes DSC differentiation, whereas the over-expression of WOX5 represses DSC differentiation,resulting in increased numbers of DSCs (Sarkar et al.,2007).

Auxin acts upstream of WOX5 and represses itsexpression, which partially depends on the auxin re-sponse factors ARF10 and ARF16 (Ding and Friml,2010). REPRESSOR OF WUSCHEL1 (ROW1) directlyrepressesWOX5 expression through linkingH3K4me3

to its promoter region (Zhang et al., 2015). IAA17 andWOX5 form a feedback circuit required for auxin-guided patterning of root DSC niches (Tian et al.,2014). The receptor-like kinase ARABIDOPSIS CRIN-KLY4 (ACR4), which forms a complex with thereceptor-like kinase CLAVATA1 (CLV1), plays a cru-cial role in repressing formative columella cell divi-sion. Mutations in ACR4 and CLV1 lead to theproduction of additional DSCs (De Smet et al., 2008;Stahl et al., 2013). The ACR4-ACR4 homodimer andACR4-CLV1 heterodimer perceive CLVATA3/EM-BRYO SURROUNDING REGION40 (CLE40) from theDSCs and some differentiated columella cells to limitWOX5 expression (Stahl et al., 2009, 2013). CLE40-ACR4/CLV1-WOX5 form a feedback module thatcontrols DSC fate (Stahl et al., 2009, 2013; Stahl andSimon, 2012). The balance between cell division anddifferentiation is important for stem cell maintenanceand proper root growth.

Drought stress induces abscisic acid (ABA) accumu-lation and promotes ROS production via mitochondriaand the NADPH oxidases AtRBOHD and AtRBOHFthat function in the plasma membrane (Kwak et al.,2003; He et al., 2012; Yang et al., 2014). Geneticscreening for ABA overly sensitive (ABO) mutantsrevealed several genes that function in mitochondria(Liu et al., 2010; He et al., 2012; Yang et al., 2014). ABO6encodes a DEXH Box RNA helicase responsible for thesplicing of several complex I genes in mitochondria (Heet al., 2012). Both ABO5 and ABO8 encode P-typepentatricopeptide repeat (PPR) domain proteins in-volved in the splicing of mitochondrial NADH dehy-drogenase subunit 2 (Nad2) intron 3 and subunit 4(Nad4), respectively (Liu et al., 2010; Yang et al., 2014).The abo5, abo6, and abo8 mutants have shorter meri-stems and higher ROS levels in the root meristemcompared to the wild type. Both abo6 and abo8 exhibitreduced expression of auxin transporter genes (Heet al., 2012; Yang et al., 2014). Furthermore, abo8 ex-hibits delayed DSC differentiation and reduced ex-pression of PLT1 and PLT2, which can be reversed bythe addition of GSH (Yang et al., 2014).

In this study, we explored the effects of redox po-tential on DSC differentiation. Interestingly, we foundthat both positive and negative redox potential delayDSC differentiation. The stability of ACR4 is regulatedby redox status. The Cys residues in the extracellulartumor necrosis factor receptor (TNFR) domain of ACR4play crucial roles in promoting ACR4 degradationthrough the endocytotic pathway, which is required forDSC differentiation but is only partially required forseed filling in siliques. Interestingly, ACR4 with a de-letion of the TNFR domain (ACR4DTNFR) no longerlocalized to the plasma membrane in root tips, whichcomplemented the seed filling defect of acr4-2, but notits defective DSC differentiation. Our study suggeststhat both highly reductive and oxidative environmentsare unfavorable for DSC identity, and uncovers theunique role of the TNFR domain in ROS-mediated en-docytosis of ACR4 in DSC differentiation, which occurs

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independently of WOX5 and CLV1. To our knowl-edge, the redox-mediated endocytosis of a plasmamembrane-localized receptor-like protein has not beenreported in plants.

RESULTS

Redox Affects DSC Differentiation in the Root Meristem

To investigate whether ROS homeostasis affects DSCidentity in the Arabidopsis root meristem, we germi-nated wild-type Arabidopsis (Columbia [Col]) seeds onMurashige and Skoog (MS) medium containing 300,600, or 800 mM of the reducing reagent GSH or 0.5, 1.0,or 1.5 mM of the oxidizing reagent H2O2. After 5 d, weobserved DSC layers using Lugol’s staining, whichdoes not stain the DSC layer but stains differentiatedcolumella cells containing starch grains. In the wildtype, the ratio of root tips with two DSC layers in-creased to 40% in response to 800 mM GSH and 46% inresponse to 1.5 mM H2O2. By contrast, the addition of300 or 600mMGSH or 0.5 or 1.0mMH2O2 had little effecton the DSC layer (Fig. 1, A and B; Supplemental Fig. S1,A and B). We then investigated the distribution of GFPin the DSC marker line J2341 (in the C24 accessionbackground; Fig. 1, C and D). Given that C24 seeds arevery sensitive to 800mMGSHor 1.5mMH2O2 during thegermination stage, in this experiment, we transferred 4-d-old J2341 seedlings from MS medium to MS mediumcontaining 800 mM GSH or 1.5 mM H2O2 and grew themfor 2 more days. Both treatments induced an increase inDSC layer formation compared to the untreated control(Fig. 1, C and D; Supplemental Fig. S1, C and D).We then compared the number of DSC layers inwild-

type plants to that in mutants whose redox status isdisturbed, including abo8-1 and pad2-1 (a GSH1 mu-tant), both of which have higher oxidative potentialthan the wild type (Parisy et al., 2007; Yang et al., 2014),and the atrbohD/F double mutant, which has higherreductive potential than the wild type (Kwak et al.,2003). The ratio of plants with two DSC layers wasmarkedly higher in abo8-1 and pad2-1 plants, as well asin atrbohD/F, than in the wild type, as revealed byLugol’s staining (Fig. 1, E and F) and observation of theJ2341 marker line (Fig. 1, G and H). The number of DSClayers was not clearly altered in response to a 300 mM

GSH treatment in the wild type. However, the ratio ofplants with two DSC layers in abo8-1 and pad2-1 seed-lings was restored to wild-type levels when they weretreated with 300 mM GSH (Fig. 1, I and J). Consistently,the addition of 1.5mMH2O2 increased the ratio of plantswith two DSC layers in the wild type but decreased thisratio from 47% to 24% in the atrbohD/F double mutant(Fig. 1, K and L). These results demonstrate that bothpositive and negative redox potentials delay DSC dif-ferentiation in the root meristem. Nevertheless, thepad2-1 mutant showed strong columella differentiationand the absence of DSCs and even QCs when treatedwith 1.5 mM H2O2 (Fig. 1, I and J), which was not

observed in abo8-1. Treatment with 800 mMGSH furtherreduced the number of DSC layers in atrbohD/F (Fig. 1,K and L). These results suggest that pad2-1 and atr-bohD/F have a greatly impaired ability to maintain re-dox homeostasis, which is further disturbed bytreatment of the plants with oxidative or reductive re-agents, respectively, as these treatments may destroystem cell identity around the QC in roots.We noticed that in some previous studies, for exam-

ple, the GR2 mutant (miao) accumulated high levels ofoxidized glutathione and exhibited increased glutathi-one oxidation, which accelerated DSC differentiation(Yu et al., 2013). Recently, Yu et al. (2016) also reportedthat both an increase and a decrease in the ROS levelenhanced the root DSC differentiation. It seems that ourresults are contradictory to these reports, which ispossibly due to different plant growth conditions, es-pecially the photoperiod and whether the experimentwas performed under dark treatment or in light. In ourprevious study, the differentiation of DSCs wasdelayed in the abo8-1mutant, and this phenotype couldbe restored by adding GSH under the 22-h light/2-hdark photoperiod (observed in light at ;17 h; Yanget al., 2014). In previous studies of ACR4, long-termlight was also used (Stahl et al., 2009), so we used the22-h light/2-h dark photoperiod as the plant growthphotoperiod in this study. We also found that under a22-h light/2-h dark cycle condition, the app1-1 andapp1-2 mutants had more DSCs than the wild type,which could be restored to the wild-type level bytreatment with 1.5 mM H2O2 (Supplemental Fig. S2A),consistent with the observation in the atrbohD/F doublemutant (Fig. 1, K and L). However, as in the case ofatrbohD/F (Fig. 1, K and L), treatment with 800 mM

GSH further reduced the number of DSC layers in app1-1 and app1-2 (Supplemental Fig. S2A). Nevertheless,under a 16-h light/8-h dark photoperiod (observed inlight at ;1 h), the differentiation of DSCs was acceler-ated in both mutants abo8-1 and pad2-1, with high ROSaccumulation, and mutants atrbohD/F, app1-1, andapp1-2, with low ROS accumulation (Supplemental Fig.S2B). The increased DSC differentiation in abo8-1 andpad2-1 or atrbohD/F, app1-1, and app1-2 could be re-versed by exogenous treatment with either GSH orH2O2, respectively (Supplemental Fig. S2B). Nitrobluetetrazolium and 29,79-dichlorodihydrofluorescin diace-tate (DCFH-DA) staining indicated that the wild type(Col) grown under the 22-h light/2-h dark photoperiodhad increased H2O2 levels and decreased O2

.2 levels inthe root tip compared with that grown under the 16-hlight/8-h dark cycle (Supplemental Fig. S2, C–E). Theseresults suggest that photoperiod is involved in ROSaccumulation in root tips for DSC differentiation.

Delayed DSC Differentiation Caused by Defects in AuxinSignaling Can Be Reversed under A Reduced State

Auxin promotes DSC differentiation in Arabidopsisroots (Ding and Friml, 2010). We therefore selected

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Figure 1. Disruption of redox homeostasis delays the differentiation of DSCs in the root meristem. A, DSC status could bechanged by GSH and H2O2 treatment in the wild-type (Col) roots. The seeds were grown on MS medium or MS medium sup-plemented with 800 mM GSH or 1.5 mM H2O2 for 5 d before observation. Bar5 20 mm. B, Quantitative analyses of DSC layers inA. C, The expression pattern of DSC marker J2341 (shown in green) in the wild type growing on MS medium or MS mediumsupplementedwith 800mMGSH or 1.5mMH2O2 for 6 d. Bar5 20mm. D,Quantitative analyses of DSC layers in C. E, DSC statusin the roots of abo8-1, pad2-1, and atrbohD/Fmutants growing on MS medium for 5 d. Bar5 20 mm. F, Quantitative analyses ofDSC layers in E. G, The expression pattern of DSCmarker J2341 (shown in green) in roots of thewild type and abo8-1, pad2-1, andatrbohD/Fmutants growing on MS medium for 5 d.. Bar5 20 mm. H, Quantitative analyses of DSC layers in G. I, GSH treatmentcould recover the delayed differentiation of DSCs in the roots of abo8-1 and pad2-1. H2O2 treatment could further promote theDSC differentiation in the pad2-1 mutant. Asterisks indicate ectopic starch granule accumulation in the QC and DSCs. Theseedlings were grown on MS medium or MS medium supplemented with 300 mM GSH or 1.5 mM H2O2 for 5 d. Bar5 20 mm. J,Quantitative analyses of the DSC layers in I. K, H2O2 treatment could recover the delayed differentiation of DSCs in the roots ofatrbohD/F. GSH treatment could further promote the differentiation of DSCs in the atrbohD/F mutant. The asterisk indicatesectopic starch granule accumulation in DSCs. The seedlings were grown on MS medium or MS medium supplemented with 800mM GSH or 1.5 mM H2O2 for 5 d. Bar5 20 mm. L, Quantitative analyses of the DSC layers in K. For quantitative analyses of DSClayers in B, D, F, H, J, and L, three different experiments were performedwith similar results, and about 20 roots were analyzed foreach experiment. Data are the means 6 SE of three biological repetitions. ns, No significant differences; *P , 0.05, **P , 0.01(Student’s t test; B, D, F, H, J, and L). Blue arrows indicate DSCs and yellow arrowheads the QC. Asterisks in I and K indicateectopic starch granule accumulation in QC or DSC.


Qin et al.



several auxin signaling-related mutants to investi-gate the effects of redox on DSC identity. WEAKETHYLENE INSENSITIVE2/ANTHRANILATE SYN-THASEa1 (WEI2/ASA1) encodes the a-subunit of an-thranilate synthase, a rate-limiting enzyme of Trpbiosynthesis that is required for auxin biosynthesis(Stepanova et al., 2005). The auxin efflux carrierPINFORMED3 (PIN3) is localized to the columella cellsof the root apicalmeristem (Friml et al., 2002). The auxinreceptor TIR1 and its homologs, AUXIN SIGNALINGF-BOX 1 (AFB1), AFB2, and AFB3 interact with anddegrade the negative regulatory factors IAA/AUXsafter binding to auxin, thus activating ARFs (Mockaitisand Estelle, 2008). The asa1-1, pin3-4, and tir1-1 afb1,2,3mutants show delayed DSC differentiation comparedto the wild type (Ding and Friml, 2010). Interestingly,we found that the DSC differentiation defects of allmutants were recovered after treatment with 800 mMGSH or introduction of the atrbohF mutation into eachof these mutants (Fig. 2). However, treatment with1.5 mM H2O2 or introduction of abo8-1 into the asa1-1 and pin3-4 mutants did not alter the DSC layer(Fig. 2). The DCFH-DA staining assay showed that theasa1-1 and tir1-1 afb1,2,3 mutants accumulated moreH2O2 in root tips than the wild type but not the pin3-4mutant (Supplemental Fig. S3, A–C), likely due to thelimited expression zone of PIN3 and/or the functionalredundancy of other PIN genes, such as PIN4 and PIN7.These results suggest that defects in auxin biosynthe-sis, transport, or early signaling increase the oxidative

potential, consistent with the previous observation of amore negative redox potential in the QC and sur-rounding cells than in other cells (Jiang et al., 2016),which delays DSC differentiation and can be reversedby treatment with the reductive reagent GSH or intro-duction of the atrbohF mutation.

ACR4 and CLE40 Play Crucial Roles in Redox-MediatedDSC Differentiation

The CLE40-ACR4/CLV1 module negatively medi-ates DSC fate through both WOX5-dependent andWOX5-independent pathways in the root tip meristem(Stahl et al., 2009; Stahl et al., 2013). The acr4-2 mutanthad more two-layer DSCs than the wild type (Fig. 3A).Unlike the wild type and the above-mentioned mu-tants, although the acr4-2 mutant accumulated moresuperoxide in root tips than did the wild type(Supplemental Fig. S4, A and B), the acr4-2DSCnumberdid not appear to be altered by treatment with 800 mM

GSH or 1.5 mM H2O2 (Fig. 3, A–C). Introducing atrbohFinto the abo8-1 or pad2-1 background restored the DSCnumber to a level similar to that of atrbohF and the wildtype (Fig. 3, D and E). However, the number of DSCs inacr4-2 atrobhF, acr4-2 pad2-1, and acr4-2 abo8-1 wassimilar to that in acr4-2 (Fig. 3, D and E). We failed toobtain any acr4-2 atrobhD/F triple mutant, possiblybecause the triple mutant is lethal or dies at the earlydevelopmental stage. Nevertheless, the DSC number of

Figure 2. Reducing conditions could recover the delayed differentiation of distal stem cell (DSC) in auxin-related mutants. A,GSH could recover the delayed differentiation of DSC in the roots of asa1-1, pin3-4 and tir1-1 afb1,2,3 mutants. The seedlingswere grown on MS medium or MS medium supplemented with 800 mM GSH or 1.5 mM H2O2 for 5 d. Bar5 20 mm. Col5 wildtype. B, Quantitative analyses of DSC layers in A. C, The delayed differentiation of DSC in the roots of asa1-1 and pin3-4 could berecovered by atrbohF, but not by abo8-1mutation. Different mutant seedlings were grown on MS medium for 5 d. Bar5 20 mm.D, Quantitative analyses of DSC layers in C. For quantitative analyses of DSC layers in B and D, three different experiments wereperformed with similar results, and ;20 roots were analyzed for each experiment. Values are means 6 SE of three biologicalrepetitions. ns: no significant differences, **P, 0.01 (Student’s t test). Blue arrows indicate DSCs and yellow arrowheads the QC.








pad2-1 acr4-2 and atrobhF acr4-2, like those of pad2-1 andatrbohF, was reduced by treatment with 1.5 mMH2O2 or800 mM GSH (Fig. 3F), respectively. Here we noticedthat as with the wild type, 1.5 mM H2O2 treatment in-creased the DSC number in atrobhF, suggesting thatatrobhF has low reductive potential that can be dis-turbed by high oxidative potential (Fig. 3F). Interest-ingly, treatment with 800 mM GSH promoted DSCdifferentiation in acr4-2 pad2-1 (Fig. 3F), suggesting thatthe ability to maintain redox homeostasis is reduced inacr4-2 pad2-1 compared to pad2-1. These results suggestthat ACR4 is important but not essential for redox-mediated DSC differentiation.

CLE40 is a ligand of the ACR4-CLV1 heteromericcomplex and the ACR4-ACR4 homomeric complex(Stahl et al., 2009, 2013). In addition to ACR4 and CLV1,CLE40 can also be perceived by CRN/CLV2 receptor-like protein kinases (Pallakies and Simon, 2014). To in-vestigate the biological roles of CLE40 in ROS-mediatedDSC differentiation, we produced cle40 knockout mu-tants using an egg cell-specific promoter via CRISPR/Cas9 (Wang et al., 2015b; Supplemental Fig. S5, A–J).Consistent with previous results (Stahl et al., 2009), thecle40mutants hadmoreDSCs than the wild type (Fig. 4);

this phenotype was rescued by adding 1 mM of a syn-thetic CLE40 peptide (CLE40p) to the medium(Supplemental Fig. S6). Previous studies have shownthat CLE40 induced differentiation of distal cells (Stahlet al., 2009). We also observed ectopic starch granuleaccumulation in the QC and DSCs of the wild type andacr4-2 when treated with CLE40p (Supplemental Fig.S6). Like the acr4-2 mutant, the cle40 mutant accumu-lated more superoxide than did the wild type in roottips (Supplemental Fig. S4A). However, treatment with800 mM GSH or 1.5 mM H2O2 did not alter the DSCnumber in the cle40 mutants (Fig. 4). Furthermore, theDSC number in the cle40 acr4-2 double mutant did notchange in response to treatment with GSH or H2O2(Fig. 4). These results indicate that CLE40, together withACR4, modulates ROS-mediated DSC differentiation.

As ACR4 can form a homomeric complex and/or aheteromeric complex with CLV1, we reasoned thatACR4 could transduce signals in both a CLV1-dependent and a CLV1-independent manner. The clv1-1 mutant had more DSCs than the wild type, as previ-ously reported (Stahl et al., 2013; Supplemental Fig. S7, Aand B). Treatment with 300 or 800 mM GSH, but not1.5 mM H2O2, reduced the DSC number in clv1-1 to a

Figure 3. ACR4 participates in redox-mediated DSC differentiation. A, Exogenous addition of GSH and H2O2 did not affect DSCdifferentiation in the acr4-2mutant. acr4-2 seedlings were grown onMSmedium orMSmedium supplementedwith 800mMGSHor 1.5mMH2O2 for 5 d. Thewild-type (Col) and abo8-1 plantswere used as controls. Bar5 20mm. B and C,Quantitative analysisof DSC layers in MS medium containing different concentrations of GSH (B) or H2O2 (C). D, The delayed DSC differentiation ofthe acr4-2 mutant is not changed by abo8-1, pad2-1, or atrobhF. As a control, the delayed DSC differentiation of abo8-1 andpad2-1 could be recovered by atrbohF. The different seedlings were grown on MS medium for 5 d. Bar5 20 mm. E, Quantitativeanalysis of the DSC layers in D; the mean for letter a is significantly lower (P , 0.05, Student’s t test) than that for letter b. F,Exogenous addition of GSHor H2O2 could disturb theDSC status of acr4-2 atrobhF or acr4-2 pad2-1 doublemutant. Quantitativeanalysis of DSC layers in the roots of different plants on MS medium or MS medium with 800 mM GSH or 1.5 mM H2O2. Forquantitative analysis of DSC layers in B, C, E, and F, three different experiments were performed with similar results, and ;20roots were analyzed for each experiment. Values are means 6 SE of three biological repetitions. ns, No significant differences;*P , 0.05, **P , 0.01 (Student’s t test). Blue arrows indicate DSCs and yellow arrowheads the QC.


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level similar to that of untreated wild-type plants(Supplemental Fig. S7, A and B). Consistently, theDSC number of clv1-1 atrbohF was similar to those ofthe wild type and atrbohF, and the DSC number ofclv1-1 abo8-1 was similar to those of clv1-1 and abo8-1 (Supplemental Fig. S7, C and D). These resultssuggest that clv1-1 may have a greater oxidative po-tential in DSCs than the wild type, which can becompromised by the addition of the reductive rea-gent GSH. They also suggest that CLV1 acts in par-allel with or upstream of ROS signaling to mediateDSC differentiation.ACR4 interacts with and phosphorylates PP2A-3 (a

catalytic subunit of the PP2A holoenzyme), and PP2Adephosphorylates ACR4 to positively mediate itsmembrane localization (Yue et al., 2016). PP2A-3 andPP2A-4 redundantly mediate DSC differentiation andprimary root growth (Yue et al., 2016). We thereforeinvestigated whether these PP2As are involved in ROS-mediated DSC differentiation. The pp2a-3-1 and pp2a-3-2mutants had more DSCs than the wild type, and DSCnumber was significantly reduced by adding 1.5 mM

H2O2 and appeared to be reduced in response to 800mM

GSH. By contrast, the DSC number in pp2a-4 was re-duced by the addition of 800 mM GSH but not 1.5 mM

H2O2 (Supplemental Fig. S8A). Like the pp2a-4 singlemutant, the DSC number in the pp2a-3 pp2a-4 doublemutant was reduced to wild-type levels in response to800 mM GSH but was not substantially altered in re-sponse to 1.5 mM H2O2 (Supplemental Fig. S8A).Nitroblue tetrazolium and DCFH-DA staining indi-cated that pp2a-4 increased the H2O2 level, and pp2a-3pp2a-4 increased both H2O2 and superoxide levels, butpp2a-3 did not change the redox status (SupplementalFig. S8, B and C). These results suggest that althoughPP2A-3 and PP2A-4 have redundant functions, pp2a-4

has a more highly altered oxidative status than the wildtype (Col) or pp2a-3, which mediates DSC differentia-tion, whereas PP2A-3 itself may play a small role inredox-mediated DSC differentiation.

The Internalization and Turnover of ACR4 Are Mediatedby Changes in Redox Status

Our analyses suggest that ACR4 is a key componentinvolved in redox-mediated DSC differentiation. ACR4becomes internalized and undergoes rapid turnoverthrough the endocytotic pathway (Gifford et al., 2005;Stahl et al., 2013; Czyzewicz et al., 2016). We wantedto know whether the localization of ACR4 is modu-lated by ROS homeostasis. We investigated thestrength of ACR4-GFP signals in the ProACR4:ACR4-GFP transgenic line and found that they were sig-nificantly reduced in plants treated with GSH orH2O2 (Fig. 5, A and B; Supplemental Fig. S9, A and B).We also found that some mobile patches colocalizedwith LysoTracker lytic vacuoles in DSCs with GSH orH2O2 treatment, but not in the wild type (Fig. 5, A andE). As a control, the strength of PIN3-GFP signals inDSCs was significantly reduced in the ProPIN3:PIN3-GFP line treated with H2O2 only, and no puncta orsome relatively large patches were observed inDSCs (Fig. 5, C and D), probably because the expressionof PIN3 is regulated by auxin that is downregulated byROS-reduced PLTs (Yang et al., 2014; Wang et al., 2015a;Santuari et al., 2016). We also introduced the atrbohF,pad2-1, or abo8-1 mutation into the ProACR4:ACR4-GFPand Pro-35S:ACR4-GFP backgrounds. Again, ACR4-GFP signals were significantly lower in ProACR4/Pro-35S:ACR4-GFP/atrbohF, ProACR4/Pro-35S:ACR4-GFP/pad2-1, and ProACR4/Pro-35S:ACR4-GFP/abo8-1 plants

Figure 4. CLE40 participates in redox-mediated DSC differentiation. A, Exogenous addition of GSH and H2O2 do not affectdelayedDSC differentiation in the roots of cle40 and cle40 acr4-2mutants comparedwith the wild type (Col). The seedlingsweregrown for 5 d onMSmedium orMSmedium supplementedwith 800mMGSHor 1.5mMH2O2 before taking pictures. Blue arrowsindicate DSCs and yellow arrowheads the QC. Bar 5 20 mm. B, Quantitative analyses of the DSC layers in A. Three differentexperiments were performed with similar results, and ;20 roots were analyzed for each experiment. Values are means 6 SE ofthree biological repetitions. ns, No significant differences; **P , 0.01 (Student’s t test).












than in the control (ProACR4/Pro-35S:ACR4-GFP)plants (Fig. 5, F, G, and I). Darkness promotes the in-ternalization of ACR4-GFP due to increased vacuolarpH. In the light, we detected only very few mobilepuncta or some relatively large patches showingGFP fluorescence that overlapped with LysoTrackerlytic vacuoles in ProACR4/Pro-35S:ACR4-GFPplants (Fig. 5, A, E, and F), which is consistent with theprevious results (Stahl et al., 2013). However, Pro-ACR4/Pro-35S:ACR4-GFP/atrbohF, ProACR4/Pro-35S:ACR4-GFP/pad2-1, and ProACR4/Pro-35-S:ACR4-GFP/abo8-1 plants contained more mobilepuncta and some relatively large patches comparedto control plants (ProACR4/Pro-35S:ACR4-GFP)under normal light conditions (Fig. 5, F, H, and J).

These results suggest that the internalization andturnover of ACR4 are enhanced by an imbalance ofredox homeostasis.

TNFR Domain Is Required for Redox-MediatedEndocytosis of ACR4 in DSC Differentiation

The extracellular domain of ACR4 contains sevencrinkly repeats, each with regularly spaced Cys resi-dues, followed by the Cys-rich regions of the TNFRdomain (Gifford et al., 2005; Czyzewicz et al., 2016).The kinase and TNFR domains are dispensable for thebiological roles of ACR4 in ovule and seed develop-ment (Gifford et al., 2005). As the internalization and

Figure 5. The membrane localization and stability of ACR4 protein are mediated by redox homeostasis. A, GSH and H2O2

treatment reducedGFP fluorescence in the root tips of ProACR4:ACR4-GFP transgenic seedlings that were grown onMSmediumorMSmedium supplementedwith 800mMGSHor 1.5mMH2O2 for 5 d. The seedlings were stainedwith propidium iodide for redcolor to show the outline of root tips. The top row shows the GFP fluorescence in the root tips of ProACR4:ACR4-GFP and thebottom row shows a combination of red and green fluorescence. Asterisks indicate the QC. Arrows indicate the GFP puncta. Bar5 10 mm. B, Relative GFP fluorescence in A quantified with Image J software. C, GFP fluorescence in the root tips of Pro-PIN3:PIN3-GFP transgenic seedlings that were grown onMSmedium or MSmedium supplementedwith 800 mM GSH or 1.5 mM

H2O2 for 5 d. The seedlings were stained with propidium iodide for red color to show the outline of root tips. The top row showsGFP fluorescence in the root tips of ProPIN3:PIN3-GFP and the bottom row shows a combination of red and green fluorescence.Asterisks indicate the QC. Bar 5 10 mm. D, Relative GFP fluorescence in C quantified with Image J software. E, Colocalization(merge) of GFP fluorescencewith LysoTracker Red in the roots of ProACR4:ACR4-GFP transgenic seedlings grown onMSmediumor MS medium supplemented with 800 mM GSH or 1.5 mM H2O2 for 5 d. Asterisks indicate the QC. Arrows indicate the colo-calized puncta. Bar 5 10 mm. F, GFP fluorescence analyses of the wild type (Col) and abo8-1, pad2-1, and atrbohF mutantscarrying the same ProACR4:ACR4-GFP. The seeds were germinated and grown onMSmedium for 5 d. Asterisks indicate the QC.Arrows indicate the GFP puncta. Bar5 10 mm. G, Relative GFP fluorescence in F analyzedwith Image J software. H, The averagenumber of GFP puncta in F. I, Relative GFP fluorescence of the wild type (Col) and abo8-1, pad2-1, and atrbohFmutants carryingthe same Pro-35S:ACR4-GFP analyzed with Image J software. J, The average number of GFP puncta of the wild type (Col) andabo8-1, pad2-1, and atrbohFmutants carrying the same Pro-35S:ACR4-GFP. For quantitative analyses in B, D, G, H, I, and J, threedifferent experimentswere performedwith similar results, and;15 rootswere analyzed for each experiment. Values aremeans6SE of three biological repetitions. ns, No significant differences; *P , 0.05, **P , 0.01 (Student’s t test).


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turnover of ACR4-GFP are influenced by redox status,we reasoned that the Cys residues in the extracellulardomain of this protein might contribute to its struc-tural stability in a redox environment (Czyzewiczet al., 2016). We investigated the status of the extra-cellular domain with the transmembrane domain (ET)under treatment with a reductive or oxidative reagentin stably transgenic Pro-35S:ACR4 (ET)-HA-Flag plants(Fig. 6A). We also examined Pro-35S:ACR4-HA-Flagtransgenic plants with and without the ACR4 intra-cellular domain [ID; Pro-35S:ACR4 (ID)-HA-Flag;Fig. 6A]. ACR4 (ET)-HA-Flag and full-length ACR4proteinmovedmore slowly on the SDS-PAGE gel aftertreatment with the reductive reagent dithiothreitol(DTT) compared with H2O2 treatment and no treat-ment; this effect was reversed by the addition of H2O2(Fig. 6B). By contrast, the position of the ACR4 kinasedomain on the gel did not change in response to DTTtreatment (Fig. 6B). Moreover, the movement of ACR4(ET)-HA-Flag in the gel decreased with increasingconcentration of DTT, which is similar to the resultsobtained with ACR4 (Fig. 6C). We noticed that theamount of ACR4 increased when the samples weretreated with increasing concentrations of DTT. It islikely that DTT could protect ACR4 from degradationby some proteases, for example. These results suggestthat ACR4 (ET) forms a stable structure under nor-mal/oxidative conditions, which can be disrupted bythe addition of DTT.We then performed a transient assay in protoplasts to

identify which part of ACR4 (ET) contributes to thestability of this protein. The position of the seven crin-kly repeats on the SDS-PAGE gel did not change inresponse to the addition of DTT (Fig. 6D). However,when we mutated 12 Cys residues (all Cys residuesafter the seventh repeat in the extracellular region) or 11Cys residues (not including the first Cys after the sev-enth repeat in the extracellular region) to Ala in theTNFR domain or deleted the TNFR domain, the rate ofthe shift in position of ET (12C-A), ET (11C-A), or ET(DTNFR) did not change with the addition of DTT(Fig. 6D). These results suggest that the Cys residues inthe TNFR domain are responsible for the formation of astable ACR4 structure that affects its position on theSDS-PAGE gel in the presence of DTT.Among the seven crinkly repeats, the Cys-180-to-Tyr

mutation in the acr4-7 mutant prevents ACR4 fromfunctioning in seed filling, which may disrupt the for-mation of the disulfide bond and/or the correct foldingof this repeat within the b-propeller structure (Giffordet al., 2005). To investigate whether the Cys residues inother repeats play roles similar to that of Cys-180(Gifford et al., 2005), we mutated two putative Cysresidues to Ala in the Cys pair of each repeat, placedthem under the control of theACR4 promoter, and usedthese constructs to transform acr4-2. We also con-structed ProACR4:ACR4 (ET)-GFP and ProAC-R4:ACR4(ID)-GFP to transform acr4-2. Consistent witha previous study (Gifford et al., 2005), ProACR4:ACR4-GFP complemented the phenotype of acr4-2 in terms of

DSC number (Fig. 6E; Supplemental Fig. S10A). Dif-ferent from the result that ProACR4:ACR4 (ET)-GFPcould not complement the seed filling phenotype(Gifford et al., 2005), in our study, ProACR4:ACR4 (ET)-GFP complemented the DSC phenotype of acr4-2 in;50% of transgenic plants (Supplemental Fig. S10B),but ProACR4:ACR4 (ID)-GFP did not do so in anytransgenic plants (Supplemental Fig. S10C). As ex-pected, ProACR4:ACR4 (C180-A)-GFP (encoding ACR4with a Cys-180-Ala mutation) did not complement theDSC phenotype of acr4-2 in any of the tested transgenicplants (Fig. 6E; Supplemental Fig. S10J). However,constructs harboring mutations of the Cys pairs in allsix of the remaining repeats and the Cys-191 mutation(which was thought to form a disulfide bridge withCys-180 in repeat 4) complemented the DSC phenotypeof acr4-2 in most transgenic plants (Supplemental Fig.S10, D–I and K–Q). These results suggest that thefolding structure mediated by Cys-180 in repeat 4 isimportant for either ligand binding or protein-proteininteractions or both, but none of the other Cys residues(six pairs and Cys-191) in the seven crinkly repeats isrequired for ACR4 function, and Cys-180 plays differ-ent roles in DSC identity and seed filling.We also introduced ProACR4:ACR4 (harboring

TNFR12C-A mutations, i.e. all 12 Cys residues weremutated to Ala)-GFP or ProACR4:ACR4 (DTNFR, i.e.without the TNFR domain)-GFP into acr4-2 and foundthat they could not complement the DSC phenotype ofacr4-2 (Fig. 6E; Supplemental Fig. S10, R and S), but didcomplement its seed filling phenotype (SupplementalFig. S11). ACR4 with a deletion of the TNFR domainwas previously found to complement the siliquefilling and seed phenotypes of acr4-2 (Gifford et al.,2005), which was confirmed in our study using Pro-ACR4:ACR4 (DTNFR)-GFP/acr4-2 plants. Furthermore,constructs with all Cys-Ala mutations, including Cys-180-Ala, complemented the seed filling defect of acr4-2(Supplemental Fig. S11). We then examined whetherthe C-Amutations affect the intracellular localization ofACR4. Under both reductive and oxidative conditions,the level of GFP fluorescence was reduced to a muchlesser extent in the DSCs of plants harboring ProAC-R4:ACR4 (TNFR12C-A)-GFP versus those harboringProACR4:ACR4-GFP (compare Fig. 6, F and G, to Fig. 5,A and B). Interestingly, when treated with GSH orH2O2, mobile puncta and some relatively large patcheswere clearly detected in the DSCs of ProACR4:ACR4-GFP (Fig. 5E) but not in those of ProACR4:ACR4(TNFR12C-A)-GFP plants (Fig. 6H). These results indi-cate that the Cys residues in the TNFR play a positiverole in redox-mediated internalization and turnover ofmembrane-localizedACR4,which are important for thefunction of ACR4.Compared to the wild type carrying ProACR4:ACR4-

GFP (Fig. 7A, left), in the root tips of ProACR4:ACR4(DTNFR)-GFP/acr4-2 plants, no clear GFP signalswere observed on the plasma membrane, and only afew mobile puncta with GFP fluorescence colocalizedwith LysoTracker around QC cells (Fig. 7B, left). These

















Figure 6. The cysteines in the TNFR domain play key roles in ACR4-mediated DSC differentiation. A, Derivatives of ACR4 forprotein analysis and complementation analysis. TM, Transmembrane domain; 12C→12A, 12 cysteines (all Cys residues after theseventh repeat in the extracellular region) mutated to alanines in the TNFR domain; 11C→11A, 11 cysteines (not including thefirst Cys after the seventh repeat in the extracellular region) mutated to alanines in the TNFR domain. B, The gel shift of ACR4 orthe ACR4 extracellular domain, but not the ID, was delayed by DTT treatment. Total proteins extracted from Pro-35S:ACR4 (ET)-HA-Flag, Pro-35S:ACR4 (ID)-HA-Flag, and Pro-35S:ACR4-HA-Flag transgenic plants were treated with (1) or without (2) 25 mM

DTTand/or 50 mM H2O2 in the sample buffer and detected by immunoblot using anti-HA or anti-ACTIN monoclonal antibodiesor Rubisco. The ETand ACR4 show the shift delayed byDTT treatment, which could be recovered by addition of H2O2. The ID didnot show the difference of shift. Three different experiments were performed with similar results. C, The mobility of the ET andACR4, but not the ID, was delayed with an increase in DTT concentration. Total protein extracted from Pro-35S:ACR4 (ET)-HA-Flag, Pro-35S:ACR4 (ID)-HA-Flag, and Pro-35S:ACR4-HA-Flag transgenic plantswas treatedwith different concentrations of DTTin the sample buffer and detected by immunoblot using anti-HA or anti-ACTIN monoclonal antibodies. ACTIN was used as acontrol. Three different experiments were performedwith similar results. D, ACR4with cysteinesmutated to alanines in the TNFRdomain or deletion of the TNFR domain lost gel-shift difference upon DTT treatment. Total proteins extracted from protoplaststransiently expressing plasmids Pro-35S:ACR4 (ET)-HA-Flag (ET), Pro-35S:ACR4 (ID)-HA-Flag (ID), Pro-35S:ACR4ET (12C-A)-HA-Flag [ET(12C-A)], Pro-35S:ACR4Repeats-HA-Flag (Repeats), Pro-35S:ACR4ET (DTNFR)-HA-Flag [ET(DTNFR)], or Pro-35S:ACR4ET (11C-A)-HA-Flag [ET(11C-A)] were treated with (1) or without (2) 25 mM DTT in the sample buffer and detected byimmunoblot using anti-HA or anti-ACTIN monoclonal antibodies. ACTIN was used as a control. Please note that Repeats did not


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results are in contrast to the previous observation thatACR4 (DTNFR)-GFP signals were slightly strongerthan ACR4-GFP signals in the ovule (Gifford et al.,2005). Perhaps ACR4 (DTNFR)-GFP undergoes pro-tein turnover more quickly than ACR4-GFP, or perhapsless ACR4 (DTNFR)-GFP enters the plasma membranein root tips than in ovules.E-64d is a membrane-permeable Cys protease in-

hibitor that inhibits protein degradation in plant vacu-oles (Oh-ye et al., 2011). In response to E-64d treatment,more mobile puncta and some relatively large patcheswith GFP fluorescence were detected (Fig. 7, A–C),which were colocalized with LysoTracker in ProAC-R4:ACR4-GFP and ProACR4:ACR4 (DTNFR)-GFP/acr4-2 plants (Fig. 7D). Tyrphostin A23 is an endocytosisinhibitor that causes proteins to be retained on theplasma membrane (Luu et al., 2012). GFP signals weresignificantly enhanced in ProACR4:ACR4-GFP in re-sponse to various concentrations of tyrphostin A23(Fig. 7, E and F). However, in ProACR4:ACR4 (DTNFR)-GFP/acr4-2 plants treated with tyrphostin A23, GFPsignals exhibited little change compared to the un-treated control, suggesting that less ACR4 (DTNFR)-GFP traveled to the plasma membrane compared toACR4-GFP (Fig. 7G). Brefeldin A (BFA) is a chemi-cal that interferes with the activity of H1-ATPase,causing both the Golgi apparatus and the trans-Golginetwork/early endosome to form visible aggregatesand also to inhibiting recycling pathways (Lam et al.,2009; Jelínková et al., 2010; Naramoto et al., 2014).BFA treatment induced visible aggregates of ACR4-GFP in Pro-35S:ACR4-GFP, but not in ProACR4:ACR4(DTNFR)-GFP/acr4-2 (Supplemental Fig. S12), sug-gesting that ACR4 (DTNFR)-GFP synthesized in theendoplasmic reticulum is not trafficked to the plasmamembrane through the Golgi apparatus, but enters themultivesicular body (MVB)/late endosome (LE) orsome unknown compartments for its function in redoxsignaling or the vacuole for degradation. Consistently,E-64d treatment did not affect DSC number in wild-type (Col) or acr4-2 plants, but it restored DSC num-ber to wild-type levels in ProACR4:ACR4 (DTNFR)-GFP/acr4-2 (Fig. 7, H and I), suggesting that E-64dtreatment stabilizes ACR4 (DTNFR), which may play

a crucial role in lytic vacuoles or MVB/LE or someunknown compartments to mediate DSC differentia-tion. Tyrphostin A23 treatment did not affect DSCnumber in acr4-2 or ProACR4:ACR4 (DTNFR)-GFP/acr4-2 plants, but it increased DSC number in the wildtype (Fig. 7, H and I), likely because more ACR4 wasretained on the plasma membrane and failed to inter-nalize to perform its function, which is very similar toProACR4:ACR4 (TNFR12C-A)-GFP without restoringacr4-2 phenotype. Treatment with 800 mM GSH or1.5 mM H2O2 increased DSC number in the wild type,likely due to the high turnover rate of ACR4 protein(Fig. 5, A and B), and this trend was reversed by E-64dtreatment, but not in acr4-2 (Fig. 7J). These resultssuggest that the TNFR domain of ACR4 determines itstrafficking from the endoplasmic reticulum to theplasma membrane, as well as its stability in the endo-cytotic pathway. However, ACR4 lacking the TNFRdomain still maintains its normal function in siliquefilling. Finally, inhibiting the degradation of ACR4(DTNFR)-GFP through the endocytotic pathwaylargely restores its activity in the root stem cell niche.

Redox Mediates DSC Differentiation Independentlyof WOX5

WOX5 expressed in the QC is important for main-taining the undifferentiated status of DSCs (Sarkaret al., 2007). Consistent with a previous study (Sarkaret al., 2007), wox5-1 has fewer DSCs than the wild type(Supplemental Fig. S13, A and B). However, treatmentwith 800 mM GSH or 1.5 mM H2O2 increased DSCnumber in both the wild type and wox5-1 (Supplemental Fig. S13, A and B). The abo8-1 wox5-1 double mutant and the atrbohD/F wox5-1 triple mu-tant had more DSCs than wox5-1 (Supplemental Fig.S13, C and D). We examined the effect of redox on DSCdifferentiation in dexamethasone (DEX)-inducible Pro-35S:WOX5-GR transgenic plants (Ding and Friml,2010). Pro-35S:WOX5-GR plants had more DSCs inthe presence of DEX than in its absence (SupplementalFig. S13, E and F). However, the addition of 800 mM

GSH or 1.5 mM H2O2 greatly reduced the effect of DEX

Figure 6. (Continued.)show the difference of shift. Three different experiments were performed with similar results. Asterisks indicate the nonspecificbands. E, ProACR4:ACR4 (TNFR12C-A)-GFP/acr4-2, ProACR4:ACR4 (180C-A)-GFP/acr4-2, and ProACR4:ACR4 (DTNFR)-GFP/acr4-2were not able to complement the delayed DSC differentiation phenotype in acr4-2 roots. Different seeds were germinatedand grown on MS medium for 5 d before staining the differentiated columella cells with Lugol’s solution. Blue arrows indicateDSCs and yellow arrowheads the QC. Bar 5 20 mm. F, GFP fluorescence in the ProACR4:ACR4 (TNFR12C-A)-GFP/acr4-2transgenic line was not reduced much by GSH and H2O2 treatment compared with the wild type (see A and B). Seeds weregerminated and grown on MS medium or MS medium supplemented with 800 mM GSH or 1.5 mM H2O2 for 5 d. The seedlingswere stained with propidium iodide for red color to show the outline of root tips. The top row shows the GFP fluorescence in theroot tips of ProACR4:ACR4 (TNFR12C-A)-GFP/acr4-2 and the bottom row shows a combination of red and green fluorescence.Asterisks indicate the QC. Bar 5 10 mm. G, Relative GFP fluorescence in F analyzed with Image J software. Three differentexperiments were performed with similar results, and;15 roots were analyzed for each experiment. Values are means6 SE fromthree experiments. ns, No significant differences; *P, 0.05 (Student’s t test). H, No GFP fluorescence puncta could be found tocolocalize with LysoTracker Red in the roots of ProACR4:ACR4 (TNFR12C-A)-GFP/acr4-2 seedlings grown onMSmedium or MSmedium supplemented with 800 mM GSH or 1.5 mM H2O2 for 5 d. Asterisks indicate the QC. Bar 5 10 mm.












Figure 7. ACR4 with deletion of the TNFR domain is unstable and no longer targeted to the plasma membrane. A and B, E-64dtreatment increased the GFP fluorescence in ProACR4:ACR4-GFP (A) and ProACR4:ACR4 (DTNFR)-GFP/acr4-2 (B) transgeniclines. Seeds were germinated and grown on MS medium or MS medium supplemented with 10 mM E-64d for 6 d before obser-vation. The seedlings were stainedwith propidium iodide for red color to show the outline of root tips. The top rows show theGFPfluorescence in the root tips of ProACR4:ACR4-GFP (A) and ProACR4:ACR4 (DTNFR)-GFP/acr4-2 (B) and the bottom rows showa


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on DSC formation (Supplemental Fig. S13, E and F).Furthermore, Pro-35S:WOX5-GR/abo8-1 and Pro-35S:WOX5-GR/atrbohD/F plants had fewer DSC layersthan Pro-35S:WOX5-GR plants after DEX induction(Supplemental Fig. S13, G andH). These results suggestthat redox mediates DSC differentiation independentlyof WOX5.

PLTs Act in Parallel or Downstream of Redox Signaling toMaintain DSC Identity

PLT1, an AP2-type transcription factor, functionsdownstream of auxin to maintain DSC identity (Dingand Friml, 2010). plt1-4 mutants have fewer DSCs thanthe wild type, and plt1-4 plt2-2 plants lack a clear DSClayer (Ding and Friml, 2010). Treatment with 800 mM

GSH or 1.5 mM H2O2 increased the ratio of plants withtwo DSC layers in the wild type but did not alter theDSC number in plt1-4 or plt1-4 plt2-2 (Fig. 8, A and B).We also crossed abo8-1 and atrbohD/F with plt1-4 andplt1-4 plt2-2 and did not observe a change in DSCnumber in plt1-4 abo8-1, plt1-4 plt2-2 abo8-1, plt1-4 atr-bohD/F, or plt1-4 plt2-2 atrbohD/F compared to plt1-4and plt1-4 plt2-2 (Fig. 8, C and D). In acr4-2, the ex-pression levels of ProPLT1:CFP and ProPLT2:CFPwere reduced to ;43% and 52%, respectively, of thoseof the wild type (Fig. 8, E–H), suggesting that ACR4affects the transcriptional level of PLT1 and PLT2.However, PLT1-YFP and PLT2-YFP protein levels inProPLT1:PLT1-YFP/acr4-2 and ProPLT2:PLT2-YFP/acr4-2 plants were reduced to only ;91% and ;83%,

respectively, of wild-type levels (Fig. 8, I–L), suggestingthat the loss of ACR4 activity also influences PLT1 andPLT2 expression at the posttranscriptional level. H2O2and GSH treatment reduced the fluorescent signals inboth PLT1-YFP and PLT2-YFP more strongly in thewild type than in acr4-2 (Fig. 8, M and N). The acr4-2plt1-4, acr4-2 plt2-2, and acr4-2 plt1-4 plt2-2 mutantsshowed the same DSC phenotypes as plt1-4, plt2-2, andplt1-4 plt2-2, respectively (Supplemental Fig. S14, A andB). Therefore, our genetic analysis suggested that PLTsact in parallel or downstream of ACR4 and redox toregulate DSC differentiation.Based on these results, we proposed a working

model for redox-mediated DSC differentiation in Ara-bidopsis roots (Fig. 9A). According to this model, auxinsignaling negatively regulates ROS accumulation inroot tips, as well as WOX5 expression. The stability ofACR4 is negatively regulated by abnormal redox status(Fig. 9B), which regulates PLT1 and PLT2 expression atthe transcriptional and posttranscriptional levels. PLT1and PLT2 are negative regulators of DSC differentiation(Fig. 9A).

DISCUSSION

ROS produced under various stress conditions aredetrimental to cells, especially affecting cell prolifera-tion. However, redox homeostasis is required for theself-renewal of stem cells in animals (Le Belle et al.,2011; Morimoto et al., 2013; Wang et al., 2013). ROSlevels must be kept at a moderate level, not too high or

Figure 7. (Continued.)combination of red and green fluorescence. Asterisks indicate the QC. Bars 5 10 mm. C, Relative GFP fluorescence of theProACR4:ACR4-GFP transgenic lines in A. Three different experiments were performed with similar results, and;15 roots wereanalyzed for each experiment. Values aremeans6 SE of three biological repeats. **P, 0.01 (Student’s t test).We did not measurethe GFP fluorescence of ProACR4:ACR4 (DTNFR)-GFP/acr4-2, as it did not show any clear GFP fluorescence without E-64dtreatment. D, Colocalization of GFP fluorescence with LysoTracker Red in ProACR4:ACR4-GFP and ProACR4:ACR4 (DTNFR)-GFP/acr4-2 roots. The 7-d-old roots were incubated with 100 mM E-64d for 3 h, then treated with LysoTracker Red for 30 min andincubated with (1E-64d) or without (2E-64d) 100 mM E-64d for another 18 h before observation. Asterisks indicate the QC.Arrows indicate the colocalized puncta. Bars 5 10 mm. E and G, GFP fluorescence in ProACR4:ACR4-GFP (E) and ProAC-R4:ACR4 (DTNFR)-GFP/acr4-2 (G) treated with Tyrphostin A23. The seeds were germinated and grown on MS medium or MSmedium supplemented with 2 mM, 3 mM, and 5 mM Tyrphostin A23 for 6 d before observation. The seedlings were stained withpropidium iodide for red color to show the outline of root tips. The top rows show the GFP fluorescence in the root tips ofProACR4:ACR4-GFP (E) and ProACR4:ACR4 (DTNFR)-GFP/acr4-2 (G) and the bottom rows show a combination of red and greenfluorescence. No clear GFP fluorescence could be detected in ProACR4:ACR4 (DTNFR)-GFP/acr4-2. Asterisks indicate the QC.Bars 5 10 mm. F, Relative GFP fluorescence of ProACR4:ACR4-GFP transgenic lines in E. Three different experiments wereperformed with similar results, and ;15 roots were analyzed for each experiment. Values are means 6 SE of three biologicalrepeats. **P, 0.01 (Student’s t test). We did not measure the GFP fluorescence of ProACR4:ACR4 (DTNFR)-GFP/acr4-2, as it didnot show any clear GFP fluorescence with or without Tyrphostin A23 treatment. H, Comparison of DSC differentiation changes inthe roots of the wild type (Col), acr4-2, and ProACR4:ACR4 (DTNFR)-GFP/acr4-2 (lines 11 and 15) seedlings treatedwith E-64d orTyrphostin A23 (A23). The seedswere germinated and grown onMSmedium orMSmedium supplementedwith 10mM E-64d or 3mM Tyrphostin A23 for 6 d before staining the differentiated columella cells with Lugol’s solution. Blue arrows indicate DSCs andyellow arrowheads the QC. Blue arrows indicate the DSCs and yellow arrowheads theQC. Bar5 20mm. I, Quantitative analysesof the DSC layers in H. J, E-64d treatment could recover the delayed DSC differentiation in the roots of the wild type (Col), but notin those of acr4-2 mutant. The seeds of the wild type and acr4-2 were germinated and grown on MS medium or MS mediumsupplemented with 800 mM GSH, 800 mM GSH1 10 mM E-64d, 1.5 mM H2O2, or 1.5 mM H2O2 1 10 mM E-64d. For quantitativeanalyses of DSC layers in I and J, three different experimentswere performedwith similar results, and;20 rootswere analyzed foreach experiment. Values are means 6 SE of three biological repetitions. ns, No significant differences; *P , 0.05, **P , 0.01(Student’s t test).









Figure 8. PLTs act downstream of ACR4 for redox-mediated homeostasis. A, Exogenous addition of GSH and H2O2 did notchange the DSC number in the plt1-4 and plt1-4 plt2-2 mutants. The seeds were germinated and grown on MS medium or MSmedium supplementedwith 800mMGSH or 1.5 mMH2O2 for 5 d before observation. DSCs (blue arrows) are characterized as thecells below the QC (yellow arrowheads) and above the starch-containing differentiated columella cells. Bar 5 20 mm. B,Quantitative analyses of the DSC layers in A. Three different experimentswere performedwith similar results, and;20 rootswereanalyzed for each experiment. Values are means 6 SE of three biological repetitions. ns, No significant differences; **P , 0.01(Student’s t test). C, DSC numbers in plt1-4 and plt1-4 plt2-2 were not changed by abo8-1 and atrbohD/F mutations in 5-d-oldseedlings growing on MS medium. DSCs (blue arrows) are characterized as the cells below the QC (yellow arrowheads) andabove the starch-containing differentiated columella cells. Bar5 20 mm. D, Quantitative analyses of the DSC layers in C. Threedifferent experimentswere performedwith similar results, and;20 rootswere analyzed for each experiment. Values aremeans6SE of three biological repetitions. ns, No significant differences; **P , 0.01 (Student’s t test). E and G, The expression ofProPLT1:CFP (E) and ProPLT2:CFP (G) in the wild type (Col) and the acr4-2 mutant on MS medium. Bars 5 50 mm. F and H,Relative fluorescence of ProPLT1:CFP (F) and ProPLT2:CFP (H) in E and G. Three different experiments were performedwith similar results, and ;15 roots were analyzed for each experiment. Values are means 6 SE of three biological repetitions.**P, 0.01 (Student’s t test). I and K, The expression of ProPLT1:PLT1-YFP (I) and ProPLT2:PLT2-YFP (K) in the wild type and the


Qin et al.



too low, in order to properly regulate self-renewal anddifferentiation of animal stem cells (Owusu-Ansah andBanerjee, 2009; Liu and Finkel, 2013; Paul et al., 2014;Zhou et al., 2016). In plants, O2

.2 and H2O2 play op-posite roles in determining stem cell identity and dif-ferentiation (Tsukagoshi et al., 2010; Zeng et al., 2017).In the shoot meristem, high O2

.2 levels determine stemcell fate through positive regulation ofWUS expression,while increasing H2O2 levels promotes stem cell dif-ferentiation by negatively regulating O2

.2 and/or WUSlevels (Zeng et al., 2017). In the root, O2

.2 accumulatesin the meristem and H2O2 in the elongation zone(Dunand et al., 2007; Tsukagoshi et al., 2010). Theproper O2

.2/H2O2 balance is mediated by the tran-scription factor UPB1, which defines the boundary ofmeristematic cell fate and differentiation in the elon-gation zone (Tsukagoshi et al., 2010). In this study, wefound that the exogenous addition of the oxidative re-agent H2O2 and the reductive reagent GSH inhibitedDSC differentiation in roots, as did changes in redox invarious ROS-related mutants, suggesting that redoxhomeostasis plays crucial roles in determining DSCdestiny.ROS are produced in the cytosol, in various organ-

elles, and by plasma membrane-localized NADPH ox-idases (Qi et al., 2018). To investigate the effects ofendogenous ROS on DSC differentiation, we used threedifferent mutants that accumulate different levels ofROS in different sites of the cell: abo8-1, which accu-mulates high ROS levels in mitochondria (Yang et al.,2014); pad2-1, which accumulates high ROS levels in theplastids (Vernoux et al., 2000; Parisy et al., 2007); andatrbohD/F, which accumulates reduced levels of ROS inthe extracellular spaces (Kwak et al., 2003). All threemutants exhibit defective DSC differentiation, sug-gesting that ROS levels that are too high or too low arenot beneficial to DSC differentiation. By contrast, wild-type plants maintain a moderate level of ROS to pre-cisely adjust DSC fate in the root meristem.Auxin plays crucial roles in regulating root growth

(Petricka et al., 2012), including promotion of DSCdifferentiation in the root meristem (Ding and Friml,2010). DSC differentiation is delayed in asa1-1, pin3-4,and tir1-1 afb1,2,3 plants (Ding and Friml, 2010).Treating these mutants with GSH or crossing themwithatrbohF restored the delayed DSC differentiation phe-notype to wild-type levels, whereas H2O2 treatment orcrossing with abo8-1 did not. The DCFH-DA stainingassay showed that reduction of the auxin signal in-creases local H2O2 levels. These results suggest that

auxin signaling negatively mediates local ROS levels inthe root tips (Jiang et al., 2016), thereby promoting DSCdifferentiation (Fig. 9).DSC differentiation in the root meristem is strictly

regulated by the CLE40-ACR4/CLV1 receptor kinasesignaling pathway (Stahl et al., 2009, 2013; Stahl andSimon, 2012; Czyzewicz et al., 2016). WOX5 is specif-ically expressed in the QC, where it represses the ex-pression of CYCD3;3 and cell division, therebymaintaining DSCs (Sarkar et al., 2007; Forzani et al.,2014). CLE40 produced from differentiated colu-mella cells is perceived by the ACR4/CLV1 complex(expressed below the QC), which in turn repressesWOX5 activity and promotes DSC differentiation.WOX5 can move to DSCs to suppress the activity ofthe differentiation factor CDF4 in regulating stem cellmaintenance (Pi et al., 2015). Interestingly, neithercle40 nor acr4-2 responded to a general level of changein endogenous or exogenous ROS levels in terms ofDSC differentiation. Although clv1-1 contained moreDSCs than the wild type, the DSC number was re-stored to wild-type levels in response to GSH treat-ment, indicating that clv1-1 plants have a greateroxidative potential than thewild type. However, DSCsin wox5-1 and Pro-35S:WOX5-GR plants responded tochanges in both oxidative and reductive potential,suggesting that WOX5 functions in parallel with ROSsignaling tomediate DSC fate. Given that DSC numberis affected by both positive and negative redox po-tential in pp2a-3 but only by negative redox potential(GSH treatment) in pp2a-4, these results suggest thatPP2A-3 and PP2A-4 do not work together with ACR4during redox-mediated DSC differentiation, which isconsistent with the results obtained in this study thatthe extracellular domain rather than the ID of ACR4 isinvolved in redox-mediated DSC differentiation.PP2A-3, PP2A-4, and ACR4 may function differentlyin redox-mediated DSC differentiation. ACR4 canform a homomeric complex and/or a heteromericcomplex with CLV1 to regulate DSC differentiation(Stahl et al., 2013). Our results suggest that ACR4 ho-mopolymers rather than heteropolymers play a majorrole in the regulation of redox-mediated DSC differ-entiation. Together, these findings suggest that CLE40is perceived by ACR4 to mediate DSC differentiationvia the ROS signaling pathway, which functions in-dependently of WOX5, and that CLV1 functions inparallel with or upstream of ROS signaling (Fig. 9).Given that both CLE40 and ACR4 are required for

determining redox-mediated DSC fate in roots, these

Figure 8. (Continued.)acr4-2mutant on MS medium. Bars5 50 mm. J and L, The relative fluorescence of ProPLT1:PLT1-YFP (J) and ProPLT2:PLT2-YFP(L) in I and K. Three different experiments were performedwith similar results, and;15 roots were analyzed for each experiment.Values are means 6 SE of three biological repetitions. *P , 0.05 (Student’s t test). M and N, The relative fluorescence ofProPLT1:PLT1-YFP (M) and ProPLT2:PLT2-YFP (N) in the wild type and the acr4-2 mutant on MS medium or MS medium sup-plemented with 800 mM GSH or 1.5 mM H2O2. Three different experiments were performed with similar results, and ;15 rootswere analyzed for each experiment. Values are means6 SE of three biological repetitions. Bars marked with letters b and c havemeans significantly lower (P , 0.05, Student’s t test) than those marked with letter a, and the mean for those marked with b issignificantly lower (P , 0.05, Student’s t test) than that of those marked with c.





findings suggest that CLE40 and ACR4 work togetherto transduce ROS signals to mediate DSC fate.However, how these proteins transduce ROS signalsremains a mystery. ACR4 is a plasma membrane-localized receptor-like kinase whose extracellulardomain contains seven crinkly repeats followed by aTNFR-like domain. Each repeat contains a pair of Cysresidues that might form disulfide bonds to stabilizeprotein structure under oxidizing environmentalconditions, suggesting that these Cys residues mightbe important for protein function (Gifford et al.,2005). However, our mutational analyses of each ofthese Cys residues indicated that except for Cys-180,mutations in the other Cys residues, including Cys-191, did not affect ACR4 function in terms of DSCdifferentiation or seed filling. These findings suggestthat Cys-180, but not Cys-191, plays an importantrole in ACR4 activity (Gifford et al., 2005), and thatthe proposed disulfide bond is not functioning or not

formed in the ACR4 tertiary structure. Furthermore,treatment with DTT did not alter the position of theseven repeats on a PAGE gel, suggesting that anydisulfide bonds, if formed in these repeats, did notgreatly affect the protein structure of ACR4. The Cys-180-Ala mutation affected DSC differentiation, butnot seed filling, suggesting that Cys-180 plays dif-ferent roles in these two processes.

Plasma membrane receptors in plants usually un-dergo internalization prior to degradation after per-ceiving ligands, in order to attenuate signaling (Clauset al., 2018; Li, 2018). ACR4 follows the endocytoticpathway for protein internalization and degradation(Gifford et al., 2005; Stahl et al., 2013; Czyzewiczet al., 2016; Claus et al., 2018). ACR4 turnover issubstantially accelerated under both negative andpositive redox potentials imposed by the exogenousaddition of H2O2 or GSH or via endogenously pro-duced redox. Interestingly, we found that the shift of

Figure 9. The proposed model for redox-mediated DSC differentiation. A, Auxin signaling negatively modulates ROS accu-mulation in root tips and WOX5 expression. ACR4 stability is negatively mediated by abnormal redox status, which positivelymodulates the expression of PLT1 and PLT2. PLT1 and PLT2 are negative regulators of DSC differentiation. Blue arrows indicatepositive regulation, red bars indicate inhibition, and dotted lines indicate indirect regulation. B, Redox-mediated trafficking routesof ACR4. Redox signal promotes the endocytosis of ACR4 through the trans-Golgi network/early endosome (TGN/EE) andMVB/LE. ACR4 (TNFR12C-A) does not respond to redox signals for protein endocytosis. ACR4 (DTNFR) cannot be trafficked fromthe endoplasmic reticulum (ER) to the Golgi and plasma membrane by secretion pathway, but might be transported into theMVB/LE or some unknown compartments, where it plays a role in redox signaling, and finally be degraded in vacuoles.


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peptides containing the TNFR-like domain on PAGEgels was altered in response to DTT treatment.However, when the Cys residues in the TNFR do-main were mutated to Ala, DTT treatment no longerled to a shift in protein position, suggesting thatTNFR forms disulfide bonds with itself or with otherCys residues, which should be determined in afuture study.GFP signals in plants harboring ACR4-GFP with the

mutated Cys residues in the TNFR domain were mainlyfound on the plasmamembrane, not inmobile puncta orsome relatively large patches; this localization was notclearly altered under different redox potentials (Fig. 6,F–H). Again, ACR4with themutated Cys residues in theTNFR domain failed to complement the DSC phenotypeof acr4-2, but it did complement the seed filling pheno-type. Decreasing endocytosis of plasma membrane-localized ACR4 via tyrphostin A23 treatment delayedDSC differentiation in the wild type, pointing to a pos-sible role for endocytosis in redox signaling. These re-sults suggest that the Cys residues in the TNFR domainare major determinants of the endocytic degradation ofACR4 in response to changes in redox and that the TNFRitself plays a negative role in this process in root tips.Consistently, ACR4 without the TNFR domain or

with the Cys-mutated TNFR domain failed to comple-ment the DSC phenotype of acr4-2. Gifford et al. (2005)found that ACR4 without the TNFR domain com-plemented the silique filling and seed phenotypes ofacr4-2, although this protein failed to complement themutant DSC phenotype in the current study. We usedthe nativeACR4 promoter in all of our vectors, allowingus to compare ACR4 protein levels based on thestrength of GFP fluorescence. Unlike the other trans-genic plants, the roots of ProACR4 (DTNFR)-GFP/acr4-2 plants showed no GFP signals on the plasma mem-brane, and only some mobile puncta were observed inthe cytosol around the QC in these plants, which sub-stantially increased in response to the inhibition ofprotein degradation via E-64d, but not with BFAtreatment (Fig. 7D; Supplemental Fig. S12). Given thatBFA is an inhibitor of protein trafficking from Golgiapparatus to plasmamembrane, BFA treatment did notcause the accumulation of visible aggregates, suggest-ing that ACR4 (DTNFR)-GFP does not enter the proteintrafficking pathway to the plasma membrane (Fig. 9B).These results suggest that ACR4 (DTNFR)-GFP is un-stable in the root tip, and might enter directly to theMVB/LE or some unknown compartments (Fig. 9B).The finding that E-64d treatment could restore the DSCphenotype of ProACR4 (DTNFR)-GFP/acr4-2 to thewild-type level due to ACR4 (DTNFR)-GFP accumula-tion in MVB/LE suggests that ACR4 (DTNFR)-GFPmay play a role in DSC identity in theMVB/LE or someunknown compartments. Interestingly, ACR4 withoutthe intracellular domain still complemented the defec-tive DSC phenotype of the acr4-2 mutant but onlypartially complemented its seed filling phenotype(Supplemental Figs. S10B and S11), suggesting that theextracellular region of ACR4 must interact with an

unknown protein, most likely a receptor-like kinase, inorder to transduce signals to a cytosolic protein(s) in theroot tip. These findings suggest that a module com-prising CLE40-ACR4 and an unknown factor plays aunique role in redox-mediated DSC differentiation andthat the process involved in maintaining the root stemcell niche is more complex than previously thought(Kong et al., 2015; Pi et al., 2015; Czyzewicz et al., 2016).Based on these observations, we hypothesize that theproCLE40 peptide is processed during its movement(with ACR4) from the endoplasmic reticulum to earlyendosomes, is subsequently targeted to the plasmamembrane and/or a MVB for its function in redox-mediated DSC identity, and finally enters the degra-dation pathway in vacuoles (Fig. 9B). Several studieshave indicated that endocytosis of receptor-like proteinkinases such as LeEix2 and FLS2 is required or partiallyrequired for proper ligand-induced plant defense re-sponses (Bar and Avni, 2009; Sharfman et al., 2011;Mbengue et al., 2016; Claus et al., 2018). Similarly, ourstudy suggests that ACR4/CLE40may transduce redoxsignals from LEs.Interestingly, the silique filling and seed develop-

ment phenotypes of acr4-2were complemented in bothProACR4(DTNFR)-GFP/acr4-2 and ProACR4:ACR4(TNFR12C-A)-GFP/acr4-2; the phenotype of Pro-ACR4(DTNFR)-GFP/acr4-2 is consistent with the re-sults of Gifford et al. (2005). Perhaps ACR4 is expressedat a higher level during seed development than in theroot meristem (Gifford et al., 2005) and the expressionlevel of ACR4 (DTNFR)-GFP is high enough to com-plement the mutant seed phenotype; alternatively,perhaps the TNFR domain of ACR4 plays differentroles in seed development and DSC differentiation.PLT1 and PLT2 are major regulators of DSC identity

and determinants of the meristematic boundary. PLT1and PLT2 expression is regulated by various factors(Aida et al., 2004; Chen et al., 2011a, 2011b; Yang et al.,2014; Wang et al., 2015a; Shinohara et al., 2016;Promchuea et al., 2017). Increasing L-Cys levels andoxidation reduces PLT1 and PLT2 levels (Yu et al., 2013;Yang et al., 2014; Wang et al., 2015a). Genetic analysisindicated that WOX5 is required for PLT1 expression(Ding and Friml, 2010), while PLT-TCP (for teosinte-branched cycloidea PCNA)-SCARECROW directlyregulates the promoter activity of WOX5 (Shimotohnoet al., 2018). Here we found that ACR4 regulates PLT1and PLT2 expression at the transcriptional and post-transcriptional levels. ProPLT1:CFP and ProPLT2:CFPwere expressed at much lower levels in acr4-2 than inthe wild type (Col). PLT1-YFP and PLT2-YFP levelswere also slightly reduced in acr4-2. Interestingly, PLT1and PLT2 protein levels were less affected by H2O2 andGSH in acr4-2 than in the wild type (Col), suggestingthat ACR4 modulates the redox-mediated protein sta-bility of PLT1 and PLT2. Genetic analysis placed PLT1and PLT2 downstream of ACR4 and redox signaling.However, since ACR4 is a membrane-localized protein,it would be interesting to explore how this proteinmodulates PLT1 and PLT2 expression.







MATERIALS AND METHODS

Plant Materials and Growth Conditions

The following mutants and/or transgenic plants were employed in thisstudy: abo8-1 (Yang et al., 2014), pad2-1 (Parisy et al., 2007), atrbohD, atrbohF(Torres et al., 2002), asa1-1 (Sun et al., 2009), pin3-4 (Friml et al., 2003), tir1-1 afb1,2,3 (Parry et al., 2009), acr4-2, ProACR4:ACR4-GFP (Gifford et al., 2005),clv1-1 (Müller et al., 2008), wox5-1, Pro-35S:WOX5-GR, plt1-4, plt2-2, plt1-4 plt2-2, J2341 (De Tullio et al., 2010), ProPLT1:CFP, ProPLT2:CFP, ProPLT1:PLT1-YFP,ProPLT2:PLT2-YFP (Zhou et al., 2010), pp2a-3-1 (SALK_069250), pp2a-4(SALK_035009), pp2a-3pp2a-4 (Yue et al., 2016), and pp2a-3-2 (SALK_115680),app1-1, app1-2 (Yu et al., 2016).

The atrbohD/F double mutant was generated by crossing atrbohD with atr-bohF. The J2341/abo8-1 and J2341/pad2-1 double mutants and the J2341/atr-bohD/F triple mutant were generated by crossing J2341 with pad2-1, abo8-1, andatrbohD/F, respectively. The asa1-1 atrbohF, pin3-4 atrbohF, abo8-1 atrbohF, pad2-1 atrbohF, acr4-2 atrbohF, clv1-1 atrbohF, and ProACR4:ACR4-GFP/atrbohFdouble mutants were generated by crossing atrbohF with asa1-1, pin3-4, abo8-1,pad2-1, acr4-2, clv1-1, and ProACR4:ACR4-GFP, respectively. The wox5-1 atr-bohD/F, Pro-35S:WOX5-GR/atrbohD/F, and plt1-4 atrbohD/F triple mutantsand the plt1-4 plt2-2 atrbohD/F quadruple mutant were generated by crossingatrbohD/F with wox5-1, Pro-35S:WOX5-GR, plt1-4, and plt1-4 plt2-2, respec-tively. The asa1-1 abo8-1, pin3-4 abo8-1, wox5-1 abo8-1, Pro-35S:WOX5-GR/abo8-1, acr4-2 abo8-1, clv1-1 abo8-1, plt1-4 abo8-1, and ProACR4:ACR4-GFP/abo8-1 double mutants and the plt1-4 plt2-2 abo8-1 triple mutant were generated bycrossing abo8-1 with asa1-1, pin3-4, wox5-1, Pro-35S:WOX5-GR, acr4-2, clv1-1,plt1-4, ProACR4:ACR4-GFP, and plt1-4 plt2-2, respectively. The acr4-2 pad2-1 and ProACR4:ACR4-GFP/pad2-1 double mutants were generated by cross-ing pad2-1 with acr4-2 and ProACR4:ACR4-GFP. The ProPLT1:CFP/acr4-2,ProPLT2:CFP/acr4-2, ProPLT1:PLT1-YFP/acr4-2, and ProPLT2:PLT2-YFP/acr4-2 lines were generated by crossing acr4-2 with ProPLT1:CFP, ProPLT2:CFP,ProPLT1:PLT1-YFP, and ProPLT2:PLT2-YFP, respectively. The acr4-2 plt1-4 andacr4-2 plt2-2 double mutants and the acr4-2 plt1-4 plt2-2 triple mutant weregenerated by crossing acr4-2 with plt1-4, plt2-2, and plt1-4 plt2-2, respectively.The cle40 single mutant and cle40 acr4-2 double mutant were generated bytransforming Col and acr4-2 with the CLE40-CRISPR/Cas9 construct, respec-tively. Primers are shown in Supplemental Table S1.

Surface-sterilized seeds were plated onto MS medium containing 2% (w/v)Suc and 0.8% or 1% (w/v) agar, incubated for 2 d at 4°C, and transferred to agrowth chamber with a 22-h light/2-h dark cycle or a 16-h light/8-h dark cycleat 22°C for 4–6 d prior to phenotypic analysis. We analyzed the samples at 17 hin light for the 22-h light/2-h dark cycle and at 1 h in light for the 16-h light/8-hdark cycle.

Phenotypic Analyses of Different Plants

To observe GFP signals in the J2341 DSC marker line under different redoxconditions, 4-d-old seedlings were transferred to MS medium containing dif-ferent concentrations of GSH (Sigma, G6013) or H2O2 (Sigma, 88597-100ML-F)and grown for 2more days. For peptide treatments, MSmedium supplementedwith 1 mM CLE40 (RQVPTGSDPLHHK, where the bold P is hydroxylated) wasused. The cle40 single mutants and cle40 acr4-2 double mutant were grown onMS medium containing 1 mM CLE40p for 5–6 d before observation. To observethe phenotypes of the DSCs in transgenic plants harboring pointmutations, 4-d-old seedlings that survived on MS medium containing hygromycin weretransferred to MS medium and grown for 2 more days. To observe the phe-notypes of Pro-35S:WOX5-GR plants under different redox conditions and indifferent mutant backgrounds, 5-d-old seedlings were transferred to MS me-dium containing 0.1mMDEX (WAKO, 041-18861) and various concentrations ofGSH or H2O2 and grown for an additional day before being photographed. Theother plant materials were grown on the corresponding media for 5–6 d andobserved.

To observe the phenotypes of siliques of T2 transgenic plants with differentmutations, the seedlings were grown in pots containing a mixture of forestsoil:vermiculite (1:1) under a 16-h-light/8-h-dark cycle at 22°C in a greenhouse.

Construction of Different DNA Vectors

To generate various mutations of ACR4, the plasmid pMD18T-ACR4 con-taining the ACR4 open reading frame (ORF) was constructed, and pCAM-BIA1300 was altered by deleting the 35S promoter and adding the GFP andNos

sequences, yielding pMD18T-ACR4 and pCAMBIA1300-GFP, respectively. ThepMD18T-ACR4 (TNFR12C-A) construct was altered using primer pair C342A-F1 and C342A-R1 to amplify pMD18T-ACR4 (TNFR12C-A), yielding pMD18T-ACR4 (TNFR11C-A). The primer pair ACR4-DTNFR-Circle-F and ACR4-DTNFR-Circle-R was used to amplify pMD18T-ACR4, generating pMD18T-ACR4 (DTNFR). The primer pair CXA-F (X:48, 60, 88, 99, 131, 154, 180, 191,221, 233, 273, 284, 312, 324, 342, these numbers mean the location of Cys resides)and CXA-R was used to amplify pMD18T-ACR4, yielding pMD18T-ACR4(CXA). Finally, the ACR4 promoter was amplified with primer pair ProACR4-1300-F and ProACR4-1300-R and cloned into pCAMBIA1300-GFP to obtainpCAMBIA1300-ProACR4-GFP (pACR4-GFP).

To produce the ProACR4:ACR4-GFP (ACR4-GFP) construct, the ACR4 ORFwas amplified from pMD18T-ACR4 with primer pair ACR4-1300-F and ACR4-1300-R and cloned into pACR4-GFP. To produce the ProACR4:ACR4 (ET)-GFP(ACR4ET-GFP) and ProACR4:ACR4 (ID)-GFP (ACR4ID-GFP) constructs,ACR4ET or ACR4ID was amplified from pMD18T-ACR4 with primer pairACR4-ET-1300-F and ACR4-ET-1300-R or ACR4-ID-1300-F and ACR4-ID-1300-R and cloned into pACR4-GFP. To generate the ProACR4:ACR4 (TNFR12C-A)-GFP [ACR4 (TNFR12C-A)-GFP], ProACR4:ACR4 (TNFR11C-A)-GFP[ACR4 (TNFR11C-A)-GFP], ProACR4:ACR4 (DTNFR)-GFP [ACR4(DTNFR)-GFP], and ProACR4:ACR4 (CXA)-GFP [ACR4 (CXA)-GFP] constructs, theACR4ORF was amplified from pMD18T-ACR4 (TNFR12C-A), pMD18T-ACR4(TNFR11C-A), pMD18T-ACR4 (DTNFR), and pMD18T-ACR4 (CXA) usingprimer pair ACR4-1300-F and ACR4-1300-R and cloned into pACR4-GFP.

To generate the Pro-35S:ACR4-HA-Flag construct, the ACR4 ORF was am-plified from pMD18T-ACR4 with primer pair ACR4-1307-F and ACR4-1307-Rand cloned into pCAMBIA1307-HA-Flag. To obtain the Pro-35S:ACR4ID-HA-Flag construct, ACR4ID was amplified from pMD18T-ACR4 with primer pairACR4-ID-1307-F and ACR4-ID-1307-R and cloned into pCAMBIA1307-HA-Flag. To obtain the Pro-35S:ACR4Repeats-HA-Flag construct, ACR4Repeats wasamplified from pMD18T-ACR4 with primer pair ACR4-Repeats-1307-F andACR4-Repeats-1307-R and cloned into pCAMBIA1307-HA-Flag. To generate thePro-35S:ACR4 (ET)-HA-Flag, Pro-35S:ACR4ET (12C-A)-HA-Flag [ET (12C-A)],Pro-35S:ACR4ET (11C-A)-HA-Flag [ET (11C-A)], and Pro-35S:ACR4ET(DTNFR)-HA-Flag [ET (DTNFR)] constructs, ET, ET (12C-A), ET (11C-A), and ET(DTNFR) were amplified from pMD18T-ACR4, pMD18T-ACR4 (TNFR12C-A),pMD18T-ACR4 (TNFR11C-A), and pMD18T-ACR4 (DTNFR) with primer pairACR4-ET-1307-F and ACR4-ET-1307-R and cloned into pCAMBIA1307-HA-Flag. To obtain the construct Pro-35S:ACR4Repeats-HA-Flag, the Repeats se-quence was amplified from pMD18T-ACR4 with primer pair ACR4-Repeats-1307-F and ACR4-Repeats-1307-R and cloned into pCAMBIA1307-HA-Flag.

The CLE40-CRISPR/Cas9 construct was designed as described by Wanget al. (2015b). Primers are shown in Supplemental Table S1.

Plant Transformation and Transgenic Plant Selection

Various transgenic plants were generated by the floral dip method (Cloughand Bent, 1998). The ProACR4:ACR4-GFP, ProACR4:ACR4 (ET)-GFP, ProAC-R4:ACR4 (ID)-GFP, ProACR4:ACR4 (TNFR12C-A)-GFP, ProACR4:ACR4(TNFR11C-A)-GFP, ProACR4:ACR4 (DTNFR)-GFP, and ProACR4:ACR4 (CXA)-GFP constructs in Agrobacterium tumefaciens strain GV3101 were transformedinto the acr4-2 null allele. Col plants were transformed with the Pro-35S:ACR4-HA-Flag, Pro-35S:ACR4 (ET)-HA-Flag, and Pro-35S:ACR4 (ID)-HA-Flag con-structs, and Col and acr4-2 were transformed with CLE40-CRISPR/Cas9. Pos-itive transgenic plants were obtained by screening on MS medium containinghygromycin. For the cle40 single mutant and cle40 acr4-2 double mutant, CLE40was amplified and sequenced to ensure that the mutation site was homozy-gous, and the CRISPR/Cas9 vector was removed as described in Wang et al.(2015b).

Root Tip Staining

Starch staining in root tips was performed as described in Yang et al. (2014).Propidium iodide (Sigma, P4170) staining and LysoTracker Red (Invitrogen,L7528) staining in root tips were performed as described in Stahl et al. (2013).

Measurement of ROS in Plants

Measurement of ROS in root tips was performed as described in Yang et al.(2014).


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Fluorescence Microscopy Observation

TransgenicArabidopsis plants harboring J2341, ProPLT1:CFP,ProPLT2:CFP,ProPLT1:PLT1-YFP, and ProPLT2:PLT2-YFP were observed using an OlympusDual-CCDDP80 fluorescence microscope. To select homozygous lines from theF3 generation, plants were observed using a Carl Zeiss LSM710 microscope.Roots were imaged using a Leica SP5 confocal microscope. GFP was excited at488 nm and emission was detected at 490–530 nm. Propidium iodide andLysoTracker Red were excited at 561 nm and emission was detected at 575–620nm. Fluorescent signal intensities were analyzed as described (Stahl et al., 2013).

Chemical Treatment of Different Plants

The seedlings were treated with E-64d (Abcam, ab144048) as described (Oh-ye et al., 2011). After growing transgenic ProACR4:ACR4 (DTNFR)-GFP andProACR4:ACR4-GFP seedlings on MS medium for 7 d,;5-mm root tip sectionswere excised from the root apices and cultured in one-half strength MS liquidmedium containing 3% (w/v) Suc and 100 mM E-64d or 1% (v/v) ethanol as acontrol. After shaking at 50 rpm for 3 h in the dark at 22°C, LysoTracker Redwas added to the culture medium to a final concentration of 2 mM and thesample was incubated for 30 min at 22°C. Unbound LysoTracker Red was re-moved by washing, and the root tips were transferred to fresh culture mediumcontaining 100 mM E-64d or 1% (v/v) ethanol. The samples were cultured for18 h in the dark at 22°C with shaking at 50 rpm before observing fluorescence.

Seeds were plated on MS medium containing 10 mM E-64d as described(Yamada et al., 2005) or 2 mM, 3 mM, or 5 mM tyrphostin A23 (Sigma, T7165) for6 d prior to phenotypic observation. For BFA treatment, Pro-35S:ACR4-GFP andProACR4:ACR4 (DTNFR)-GFP were grown on MS medium for 5–6 d, and theseedlings then were treated with 25 mM BFA for 1.5 h before taking pictures.

Protein Analysis

Protein analysis was performed as described (Mou et al., 2003), with minorchanges. Proteins were extracted from 10-d-old seedlings including Pro-35S:ACR4-HA-Flag, Pro-35S:ACR4 (ET)-HA-Flag, and Pro-35S:ACR4 (ID)-HA-Flag transgenic plants by homogenizing the plants in 300 mL neutral pHextractionbuffer (25mMHEPES, pH7.4, 2mMMgCl2, 1mMEDTA, pH8.0, 100mM

NaCl, 10% [v/v] glycerol, and 1% [v/v] Triton X-100) with 13 protease inhibitorcocktail (Roche, RE04693132001). The extracts were centrifuged at 12,000g for20min at 4°C. A 300-mL aliquot of supernatant was transferred into a 1.5mL tube,combined with 60 mL 63 SDS sample buffer without b-mercaptoethanol (non-reducing conditions), and thoroughly mixed. The protein sample was dividedinto equal parts, combined with DTT (Sigma, D9779) or H2O2 as indicated, andthoroughly mixed. The samples were incubated at room temperature for 30 min,during which time they were mixed twice. The protein samples were heated at60°C for 10min, centrifuged at 12,000g for 10min, and loadedonto 6%SDS-PAGEgels. The proteins were detected using anti-HA (Abmart, M20003) and anti-ACTIN (Abmart, M20009) antibodies or Rubisco.

For protein expression in protoplasts, Pro-35S:ACR4 (ET)-HA-Flag, Pro-35S:ACR4 (ID)-HA-Flag, Pro-35S:ACR4ET (12C-A)-HA-Flag [ET (12C-A)], Pro-35S:ACR4ET (11C-A)-HA-Flag [ET (11C-A)], Pro-35S:ACR4ET (DTNFR)-HA-Flag [ET (DTNFR)], and Pro-35S:ACR4Repeats-HA-Flag (Repeats) were puri-fied using a Plasmid Maxiprep Kit (Vigorous Biotechnology) and transferredinto Arabidopsis protoplasts as described by Jin et al. (2001). The proteins wereextracted by homogenizing in 200 mL neutral pH extraction buffer, followed bythe steps described above.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL datalibraries under accession numbers as follows: AT3G59420 (ACR4), AT4G11690(ABO8), At4g23100 (PAD2), AT5G47910 (ATRBOHD), AT1G64060 (ATRBOHF),AT5G05730 (ASA1), AT1G70940 (PIN3), AT3G62980 (TIR1), AT4G03190 (AFB1),AT3G26810 (AFB2), AT1G12820 (AFB3), AT5G12990 (CLE40), AT3G11260(WOX5), AT3G20840 (PLT1), AT1G51190 (PLT2), At5g53540 (APP1), AT1G75820(CLV1), AT2G42500 (PP2A-3), AT3G58500 (PP2A-4).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Disruption of redox homeostasis affects the dif-ferentiation of DSCs in the root meristem.

Supplemental Figure S2. Different effects of photoperiod on DSCdifferentiation.

Supplemental Figure S3. The asa1-1 and tir1-1 afb1,2,3mutants accumulatemore H2O2 than the wild type in root tips.

Supplemental Figure S4. The acr4-2, cle40 and cle40 acr4-2 mutants accu-mulate more superoxide than the wild type in root tips.

Supplemental Figure S5. Schematic of the cle40 and cle40 acr4-2 CRISPR/Cas9 mutants.

Supplemental Figure S6. Synthetic CLE40 peptide (CLE40p) treatmentrestores the DSC status of cle40 and weakly restores the DSC status ofcle40 acr4-2.

Supplemental Figure S7. Redox-mediated DSC differentiation is indepen-dent of CLV1.

Supplemental Figure S8. PP2A-3/4 responds to redox change.

Supplemental Figure S9. Relative GFP fluorescence in the plasma mem-brane and the cytoplasm of the root tips in ProACR4:ACR4-GFP trans-genic seedlings.

Supplemental Figure S10. Quantitative analyses of DSC layers in the rootsof complementation materials as indicated on MS medium.

Supplemental Figure S11. The different domains of ACR4 play differentroles in DSC maintenance and silique development.

Supplemental Figure S12. ACR4 with deletion of the TNFR domain didnot respond to BFA.

Supplemental Figure S13. Redox homeostasis mediates DSC differentia-tion independently of WOX5.

Supplemental Figure S14. PLTs act at or downstream of ACR4 for DSCdifferentiation.

Supplemental Table S1. Primers used in this study.

ACKNOWLEDGMENTS

We thank Dr. Chuanyou Li at the Institute of Genetics and DevelopmentalBiology, China Academy of Science, and Chunming Liu at the Institute of CropScience, China Academy of Agricultural Science, for providing relativematerials.

Received May 24, 2019; accepted August 20, 2019; published August 30, 2019.

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Redox-Mediated Endocytosis of a Receptor-Like Kinase ... · observed DSC layers using Lugol’s...

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Transcript of Redox-Mediated Endocytosis of a Receptor-Like Kinase ... · observed DSC layers using Lugol’s...