Noncanonical auxin signaling regulates cell divisionpattern during lateral root developmentRongfeng Huanga,1, Rui Zhengb,c,1, Jun Hea,2, Zimin Zhoud, Jiacheng Wanga, Yan Xionga, and Tongda Xua,b,2

aHorticulture Biology and Metabolomics Center, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University, 350002 Fuzhou,Fujian, China; bShanghai Center for Plant Stress Biology, Center for Excellence in Molecular Plant Sciences, Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences, 201602 Shanghai, China; cUniversity of Chinese Academy of Sciences, 100049 Beijing, China; and dTemasek Life ScienceLaboratory Ltd., National University of Singapore, 117604 Singapore

Edited by Natasha V. Raikhel, Center for Plant Cell Biology, Riverside, CA, and approved September 12, 2019 (received for review June 25, 2019)

In both plants and animals, multiple cellular processes must beorchestrated to ensure proper organogenesis. The cell divisionpatterns control the shape of growing organs, yet how they areprecisely determined and coordinated is poorly understood. Inplants, the distribution of the phytohormone auxin is tightlylinked to organogenesis, including lateral root (LR) development.Nevertheless, how auxin regulates cell division pattern duringlateral root development remains elusive. Here, we report thatauxin activates Mitogen-Activated Protein Kinase (MAPK) signal-ing via transmembrane kinases (TMKs) to control cell divisionpattern during lateral root development. Both TMK1/4 andMKK4/5-MPK3/6 pathways are required to properly orient cell divisions,which ultimately determine lateral root development in responseto auxin. We show that TMKs directly and specifically interact withand phosphorylate MKK4/5, which is required for auxin to activateMKK4/5-MPK3/6 signaling. Our data suggest that TMK-mediatednoncanonical auxin signaling is required to regulate cell divisionpattern and connect auxin signaling to MAPK signaling, which areboth essential for plant development.

auxin | TMKs | MKK4/5 | MPK3/6 | cell division pattern

Coordinated cell division and cell expansion are essential forproper organ development in both animals and plants (1–3).

Cell division orientation determines cell fate, and formative celldivision drives organ morphogenesis and tissue differentiation.Extensive research has focused on the regulation of the polarizeddirection of cell expansion, such as the interdigitated pavementcell expansion (4, 5), the tip growth of pollen tubes (6), and roothair cells (7), but little is known about how the direction of celldivision is determined.The phytohormone auxin plays a pivotal role in regulating

plant growth and development in a concentration-dependentmanner. Disruption of the auxin concentration gradient causessevere defects in cell division patterns and aberrant organ devel-opment (8–13). Lateral root (LR) initiation and development, atightly coordinated process involving the strictly controlled celldivision pattern (14), provides a model system to study the un-derlying mechanism of auxin regulation in cell division patternsduring organogenesis (15, 16).Previous work demonstrated that abundant key transcrip-

tion factors, regulated by the TIR1/AFB-based canonical auxinpathway, were essential for organogenesis, including LR de-velopment (17–20). Recently, TMK-mediated auxin signalingwas identified to regulate the ROP signaling pathways in thepavement cells morphogenesis (21) and the noncanonical Aux/IAAs in the apical hook development (22). This discoveryprovides a new perspective for understanding the action ofauxin in many developmental processes.Mitogen-Activated Protein Kinase (MAPK) cascades are highly

conserved signaling modules found in all eukaryotic cells. PlantMAPK pathways, which convert signals into cellular responses,regulate various aspects of plant growth and development (23–25).Accumulating evidences illustrate that plant MAPKs are involved

in regulating cell division during organogenesis. The NPK1-NQK1-NRK1 cascade regulates cytokinesis in Nicotiana tabacum(26, 27), and the activated NPK1, a MAPKKK, acts at the lateM-phase to mediate the cell plate formation (28). MKK4/MKK5together with MPK3/MPK6 regulate stomatal patterning by con-trolling asymmetric cell division (29) and inflorescence architec-ture by promoting localized cell proliferation in Arabidopsis (30).Recently, MPK6 was proved to affect cell division orientation inboth primary and lateral roots (31, 32), and the MKK4/MKK5-MPK3/MPK6 module was essential for LR emergence (33).Notably, mutual regulation between plant MAPK signal

transduction modules and auxin signaling has been reported(34, 35). Exogenous auxin application can specifically activateMPK3/MPK6 in root (33, 36). However, it is still unknown howMPK3/MPK6 are activated by auxin. In this study, we demon-strate that the local auxin maximum at LR initiation sites trig-gers TMK1/TMK4-mediated phosphorylation and activation ofMKK4/MKK5, followed by the activation of MPK3/MPK6 toregulate cell division pattern during LR development.

ResultsTMK1 and TMK4 Are Required for Auxin-Regulated Cell DivisionPattern during LR Development. To investigate potential non-transcriptional mechanisms underlying auxin-mediated regulationof LR development in Arabidopsis, we examined a transmembranekinases (TMKs) family that was recently found to participate in

Significance

Development in multicellular organisms consists of a series ofcell divisions followed by cell expansions, which are tightlycontrolled both spatially and temporally. Disorganized cell di-vision causes serious growth defects in both animals andplants. Auxin has been repeatedly implicated in regulating celldivision patterns during organogenesis in plants; however, theunderlying mechanisms remain to be further investigated.Here, we demonstrate that auxin signaling controls cell di-vision pattern by activating Mitogen-Activated Protein Kinase(MAPK)-signaling cascade during lateral root development. Ourwork reveals how signal transduction pathways regulate celldivision pattern and also provides a connection between auxinsignaling and MAPK signaling in plants.

Author contributions: T.X. designed research; R.H., R.Z., J.H., Z.Z., and J.W. performedresearch; Y.X. contributed new reagents/analytic tools; R.H., R.Z., J.H., and J.W. analyzeddata; and R.H., J.H., and T.X. wrote the paper.

The authors declare no competing interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1R.H. and R.Z. contributed equally to this work.2To whom correspondence may be addressed. Email: hejun@sibs.ac.cn or tdxu@sibs.ac.cn.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1910916116/-/DCSupplemental.

First published September 30, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1910916116 PNAS | October 15, 2019 | vol. 116 | no. 42 | 21285–21290

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auxin signaling (22). Of the 4 TMK members, tmk1-1 and tmk4-1mutants showed slightly fewer LRs and lower LR density than wildtype (SI Appendix, Fig. S1 A–C), and LRs were mostly abolished inthe tmk1-1tmk4-1 double-mutant (Fig. 1 A and B and SI Appendix,Fig. S1 A and B), suggesting that TMK1 and TMK4 play an es-sential but redundant role in regulating LR development. Notably,GUS reporter analyses driven by the native promoters of bothTMK1 and TMK4 suggested that TMK1 and TMK4 were highlyexpressed at the different stages of LR development (SI Appendix,Fig. S2), which were confirmed by pTMK1-TMK1-GFP;tmk1-1and pTMK4-TMK4-GFP;tmk4-1 transgenic lines (SI Appendix, Fig.S3A). These were in agreement with the strong LR phenotypein the tmk1-1tmk4-1 mutant. Exogenous auxin treatment with150 nM NAA (1-Naphthaleneacetic acid) strongly promoted LRdevelopment including LR number and lateral root primordia(LRP) in wild type but not in the tmk1-1tmk4-1 mutant (Fig. 1 Aand B and SI Appendix, Fig. S4). However, the tmk1-1tmk4-1mutant had a bit higher LRP density compared to wild type (SIAppendix, Fig. S4B), which may be caused by the severe growthdefect, such as the much shorter primary root. Therefore, wegenerated an estradiol-inducible TMK4 RNAi in the tmk1mutant background to further investigate the role of TMK1and TMK4 in LR development. The estradiol treatment in thetmk1−/−tmk4RNAi-est mutant resulted in the reduced expres-sion and protein level of TMK4 (SI Appendix, Fig. S5 D and F).This mutant showed similar phenotype as in the tmk1-1tmk4-1double-mutant (SI Appendix, Fig. S5 A–C). To avoid the effectof primary root defect in the tmk1-1tmk4-1 mutant, we transferred

the 5-d-old noninduced mutant to the 1/2MS medium containing50 μM estradiol for another 5 d and found that the lateral rootswere partially abolished in the newly generated primary roots ofthe estradiol treated tmk1−/−tmk4RNAi-est mutant (SI Appendix,Fig. S5E). We further noticed that the cell division patterns weredisorganized at the different stages of LR development in thetmk1-1tmk4-1 mutant, leading to the LR growth defects in themutant compared to the wild type (Fig. 1 C and D and SI Ap-pendix, Fig. S3B). Together, these data suggest that TMK1 andTMK4 are required to regulate cell division pattern and LR de-velopment in response to auxin.

TMK1 and TMK4 Interact with MKK4 and MKK5. To determine howTMK1/TMK4 regulate cell division orientation and LR de-velopment, we identified proteins that interact with TMK1 inArabidopsis. Briefly, we used GFP-trap agarose beads to immu-noprecipitate TMK1-GFP and interacting proteins from pTMK1-TMK1-GFP transgenic plants, followed by mass spectrometry.Among the TMK1-interaction candidates, we were particularlyinterested in MAP-kinase kinase 4 (MAPKK4 or MKK4), a keycomponent of the MAPK-signaling pathway that was previouslyreported to regulate both cell division and LR development inplant (30, 33).Arabidopsis contains 10 MKK family members (37). To com-

prehensively investigate these MKKs, we used the unbiased yeast2-hybrid assay to screen all MKK proteins for interactions with theC terminus of the 4 TMK family members. We found that the Cterminus of TMK1 and TMK4 specifically interacted with MKK4

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Fig. 1. TMK1 and TMK4 regulate auxin-mediated cell division pattern during lateral root development. (A) Lateral root from 9-d-old seedlings in Col-0, tmk1-1tmk4-1and complementing gTMK1-flag;tmk1-1tmk4-1 lines. Seedlings grown on medium with dimethyl sulfoxide (DMSO) or 150 nM NAA. (Scale bar, 1 cm.) (B)Number of LRs per seedling of 9-d-old Col-0, tmk1-1tmk4-1 mutant and gTMK1-flag in tmk1-1tmk4-1 transgenic line seedlings with or without NAAtreatment. Values indicate mean ± SD from 3 individual biological repeats. n denotes number of independent seedlings, and different letters denote significantdifferences from 1-way ANOVA with Tukey multiple comparisons test. (C) Cell division pattern in lateral root primordia at stage I to IV in Col-0, tmk1-1tmk4-1.(Scale bar, 20 μm.) (D) Frequency of angle distribution in LRP cells (From stage I to IV) in Col-0, tmk1-1tmk4-1. Ten primordia of each stage from 10 individualseedlings showed similar results.

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and MKK5 (Fig. 2A and SI Appendix, Fig. S6A), which belong tothe same subfamily (SI Appendix, Fig. S6C). The TMK2C terminusinteracted with MKK5 (SI Appendix, Fig. S6A); however, TMK2was not expressed in the LR (38). Transiently expressed MYC-tagged MKK4 and MKK5 coimmunoprecipitated with HA-taggedTMK1 from protoplasts (Fig. 2B). Consistently, HA-tagged MKK4and MKK5 coimmunoprecipitated with MYC-tagged TMK1 (SIAppendix, Fig. S6B). These interactions were further confirmed onplasma membrane by the FLIM-FRET (Fluorescence LifetimeImaging Microscopy–Fluorescence Resonance Energy Transfer)assay in protoplasts (Fig. 2 C andD). Similar to TMK1 and TMK4,both MKK4 and MKK5 were also highly expressed at the differentstages of LR development, assessed in pMKK4-MKK4-RFP andpMKK5-MKK5-RFP transgenic lines (SI Appendix, Fig. S7). Basedon these data, we hypothesized that MKK4/5 participate withTMK1/4 to regulate LR development.

MKK4 and MKK5 Regulate Cell Division Pattern during LR Developmentin Response to Auxin. The mkk4mkk5 double-mutant is embryoniclethal; therefore, we generated an estradiol-inducible MKK4and MKK5 RNAi mutant to further investigate the role ofMKK4 and MKK5 in LR development. Estradiol treatment ofthe mkk4,5RNAi-est mutant resulted in the reduced expressionof MKK4 and MKK5, but not other MKKs (SI Appendix, Fig. S8 Aand C). Similar to the tmk1-1tmk4-1 mutant, the estradiol-treatedmkk4,5RNAi-est mutant showed strong defects in LR developmentwhich were also insensitive to auxin treatment (Fig. 3 A and B).The LR density in the estradiol-treated mkk4,5RNAi-est was

insensitive to auxin, but LRP density in the mutant was a bithigher than in the wild type (SI Appendix, Fig. S9 A and B). Toavoid differences in primary root growth, we grew wild-typeand mkk4,5RNAi-est seedlings in 1/2 MS medium for 5 d and thentransferred them to the medium containing 50 μM estradiol to-gether with 150 nMNAA for another 5 d (SI Appendix, Fig. S10A).Again, we observed defective LR development in the estradiol-treated mkk4,5RNAi-est seedlings that was not rescued by auxintreatment (Fig. 3 A and B). These defects in the estradiol-treatedmkk4,5RNAi-est mutant were also associated with the disorga-nized cell division patterns at different stages of LR development(Fig. 3 E and F). Thus, MKK4 and MKK5 also regulate LR de-velopment in response to auxin.

Auxin Induces TMK1/4-Dependent Phosphorylation of MKK4/5. TheTMK-mediated signaling pathway has been implicated in auxinsignal transduction (22, 38), which led us to hypothesize thatauxin triggers the activation of MKK4 and MKK5 via TMKs. Totest this hypothesis, we treated Arabidopsis protoplasts tran-siently expressing MKK4 or MKK5 with 100 nM NAA for 1 h.Auxin treatment led to the mobility shift of MKK4 and MKK5 inPhos-tag polyacrylamide gels that was abolished by LambdaProtein Phosphatase (λ-PPase) treatment (Fig. 4A), suggestingthat auxin induces MKK4/5 phosphorylation. Using the sameapproach, we found that coexpression of TMK1, but not a kinaseinactive TMK1(K616E), induced the phosphorylation of MKK4/5(Fig. 4B). These data suggest that TMK1 also can phosphor-ylate MKK4/5. Indeed, an in vitro phosphorylation assay con-firmed that both MKK4 and MKK5 were specifically and directlyphosphorylated by a recombinant TMK1C terminal fragment(Fig. 4C). These observations collectively suggest that TMK-based auxin signaling may participate in regulating the phos-phorylation of MKK4/5, which is consistent with the similar LRdefects in both tmk1-1tmk4-1 and mkk4,5RNAi-est mutants.

Auxin Activates MPK3 and MPK6 through TMK1/4 to Regulate CellDivision Pattern in LR. MPK3/6 have been reported to functiondownstream of MKK4/5 in multiple developmental processes(29, 30, 39, 40). Similar to TMK1/4 and MKK4/5, both MPK3and MPK6 were also highly accumulated at different stages ofLR development (SI Appendix, Fig. S7). To determine whetherMPK3/6 play a role in LR development, we generated anestradiol-inducible MPK3 RNAi in the mpk6 mutant, since thempk3mpk6 double-mutant was also embryonic lethal. Estradioltreatment of the mpk6−/−mpk3RNAi-est mutant resulted inthe reduced expression of MPK3 in the mpk6 mutant back-ground (SI Appendix, Fig. S8B). Similar to the tmk1-1tmk4-1and estradiol-inducible mkk4,5RNAi-est mutants, estradiol treat-ment of mpk6−/−mpk3RNAi-est also led to LR developmen-tal defects (SI Appendix, Figs. S9 C and D and S10B) and thedisorganized cell division pattern, which was insensitive toauxin (Fig. 3 C–F).To further verify whether auxin activates MPK3/6 to regulate

LR development, we examined yuc1-D mutant, which producesexcessive auxin (41), and wei8-3tar2-1 mutant, which producesreduced auxin (42). The levels of phosphorylated MPK3/6 corre-lated with auxin levels in the respective mutants (SI Appendix, Fig.S11 A and B). In addition, we treated wild-type seedlings withL-kynurenine (L-Kyn) (43) to inhibit auxin biosynthesis, followedby the exogenous auxin (NAA or IAA) treatment. We found thatL-Kyn treatment led to slightly reduced levels of phosphorylatedMPK3/6, which was restored by auxin treatment. Auxin treatmentalso restored MPK3/6 phosphorylation in the auxin-deficient wei8-3tar2-1 mutant (Fig. 4 D and E and SI Appendix, Fig. S11 Cand D). It has been reported that MKK4/5 mediate the acti-vation of MPK3/6 by auxin (33). More importantly, we foundthat the auxin-induced phosphorylation of MPK3/6 was par-tially abolished in the tmk1−/−tmk4RNAi-est mutant, but not

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in the tir1afb2afb3 mutant (Fig. 4 F and G and SI Appendix,Fig. S11 E and F). Taken together, our observations collectivelysuggest that auxin activates the MKK4/5-MPK3/6-signalingcascade via TMK1/4 to regulate cell division pattern duringLR development.

DiscussionThe molecular mechanism underlying the strict control of celldivision pattern, by which multicellular organisms produce newand specific tissues and organs, is a basic and central theme indevelopmental biology. The formative cell divisions are requiredfor multiple developmental processes both in plants and animals.Unlike animals, most plant organs, including lateral roots, areprimarily postembryonically initiated from continuous de novodevelopment, implying that these mechanisms establish tightspatiotemporal coordination of cell division and patterning (16).In this study, we show that auxin activates MKK4/5-MPK3/6

cascade through TMK1/4 to regulate cell division pattern duringLR morphogenesis. TMKs, as the key components of auxin sig-naling on plasma membrane, mediate noncanonical auxin-signalingtransduction to control versatile developmental processes (21, 22),which is distinct from TIR1-based canonical transcriptional auxinsignaling. This finding provides a mechanism to regulate cell di-vision pattern during organ morphogenesis and a perspective of

auxin action at the cellular level. Previous work demonstrated thatTIR1-based auxin signaling regulates lateral root development,especially initiation, through the transcriptional regulation of thekey components involved in this developmental process. ThatTMK1/TMK4 mediate auxin activation of the MKK4/5-MPK3/6cascade provides a posttranscriptional regulation mechanism,which coordinates with the TIR1 pathway to control the LR de-velopment precisely (SI Appendix, Fig. S12). This also fills a missinglink in our understanding of both auxin and MAPK-signalingevents, whose modes of action are critical for plant growth anddevelopment. Given the pivotal roles of auxin in cell divisionpatterning, our findings shed light on how hormonal signaling leadsto changes in cell division orientation, which is important for plantcells (44), but distinct from animal cells.The new lateral roots grow from LR initiation sites where

auxin accumulates (SI Appendix, Fig. S2) (9), the same place asthe protein abundance of TMK1/TMK4, MKK4/MKK5 andMPK3/MPK6 (SI Appendix, Figs. S2, S3A, and S7). Both auxinand MKK4/5-MPK3/6 activities are required for LR develop-ment (9, 33, 45). In the meanwhile, MKK4 and MKK5, as wellas MPK3 and MPK6, have redundant functions in diverse bi-ological processes, including stomatal differentiation (29), in-florescence localized cell division (30), and zygote division(46). Although our work here focuses on the roles of this

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Fig. 3. MKK4/5-MPK3/6 regulates auxin-dependent cell division pattern during lateral root development. (A) Lateral root phenotype of 9-d-old seedlings inCol-0 and estradiol-inducible mutant mkk4,5RNAi-est. Seedlings grown on 1/2MS medium with dimethyl sulfoxide (DMSO) or 50 μM estradiol or 50 μM es-tradiol plus 150 nM NAA. (Scale bar, 1 cm.) (B) Number of LRs per seedling of Col-0 and estradiol-inducible mutant mkk4,5RNAi-est (A). Error bars indicate SDof 3 individual biological repeats. n denotes number of independent seedlings. (C) Lateral root phenotype of 9-d-old seedlings in Col-0 and estradiol-induciblemutant mpk6−/−mpk3RNAi-est. Seedlings grown on 1/2MS medium with DMSO or 20 nM estradiol or 20 nM estradiol plus 150 nM NAA. (Scale bar, 1 cm.) (D)Number of LRs per seedling of 9-d-old Col-0 and estradiol-inducible mutant mpk6−/−mpk3RNAi-est (C). Error bars indicate SD of 3 individual biological re-peats. n denotes number of independent seedlings, and different letters denote significant differences from 1-way ANOVA with Tukey multiple comparisonstest in B and D. (E) Cell division pattern in LRP at stage I to IV in Col-0, estradiol-inducible mkk4,5RNAi-est, and mpk6−/−mpk3RNAi-est mutant. (Scale bar,20 μm.) (F) Frequency of angles distribution in LRP cells (from stage I to IV) in Col-0, estradiol-inducible mkk4,5RNAi-est, and mpk6−/−mpk3RNAi-est mutant.Ten primordia of each stage from 10 individual seedlings showed similar results.

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auxin-signaling mechanism in LR cells, it is likely that similar auxin-TMK-MAPK modules operate in other plant cells and tissues.Plant cell division orientation relies on the PPB (preprophase

band), a specialized MT (microtubule) apparatus composed ofMTs and F-actins, to organize the cell division plane (47). Recentevidence indicates that MPK6 is associated with MT dynamics (48)and involved in the control of cell division plane formation (32),which provides some hints for further exploring the detailedmechanism of how the auxin–TMK–MAPK pathway elaborately

regulates cell division orientation. Moreover, MPK3 and MPK6are protein kinases and likely function with downstream substratesor transcription factors, whose identification will be crucial tounderstand how this signaling executes and exports outputs.The MKK4/5-MPK3/6 cascade has been reported as down-

stream of several RLKs [e.g., ER (30), FLS2 (49), HAE/HSL2 (50),CLV1/CLV2 (51)] and senses various upstream signals (e.g.,various stress stimuli and development signals). For example,IDA-HAE/HLS2 ligand–receptor mediates auxin-facilitated LR

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GST-MKK4(PD)- -

- -+ +- + - ++ - + - +

+ +GST +- - - -

-

---

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L-KynNAA -

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Col-0 wei8-3 tar2-1

Col-0 wei8-3 tar2-1

***

***

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MPK6MPK3

NAA L-Kyn

wei8-3tar2-1 Col-0

+- - -+- - - ++

--MPK6-p MPK3-p

--MPK6 MPK3

NAAL-Kyn

Col-0

+- +- - +

tmk1-/-tmk4RNAi-est

+- +- - +

EST + + + + + +

--MPK6-p MPK3-p --MPK6 MPK3

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- +- +

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- ++

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Col-0 tmk1-/-tmk4 RNAi-est

Col-0 tmk1-/-tmk4 RNAi-est

A

B

C

D E

F G

-

Fig. 4. TMKs mediate the phosphorylation of MKK4/5-MPK3/6 by auxin. (A) Phos-tag gel shift assay shows that auxin triggers phosphorylation of MKK4 andMKK5. The 35S-MKK4-MYC or 35S-MKK5-MYC (5 h) were transiently expressed in Arabidopsis protoplasts and then treated with 100 nM NAA for 1 h; dimethylsulfoxide (DMSO) was used as a control. MKK4/5 proteins were detected with an anti-MYC antibody. Similar results were obtained with 3 biological repeats. (B)Phos-tag gel shift assay shows that TMK1, but not kinase inactive TMK1(K616E), triggers phosphorylation of MKK4 and MKK5. Arabidopsis protoplasts transientlyexpressed 35S-MKK4-MYC or 35S-MKK5-MYC with 35S-TMK1-HA or 35S-TMK1(K616E)-HA for 6 h. The 35S-HA transformed with 35S-MKK4-MYC or 35S-MKK5-MYC were used as control. Two independent repeats showed similar results. (C) In vitro kinase assay of TMK1 kinase domain with MKK4(PD) and MKK5(PD) protein.Phosphorylation is detected by radioactive P32. MKK4(PD), kinase inactive MKK4(K108M); MKK5(PD), kinase inactive MKK5(K99M). Protein loading was detected byWestern blotting. (D) Auxin levels correlated with levels of phosphorylated MPK6 and MPK3 in seedlings root. The 6-d-old seedlings were used for 100 μM L-Kyntreatment for 1 h, then added back 10 μM NAA; the seedlings roots were harvested for protein extraction. Three biological repeats showed similar results. (E)Quantification of relative MPK6 and MPK3 kinase activity in auxin treatment shown in D. n = 3 biological repeats; data are mean ± SD. (F) Auxin induces MPK6 andMPK3 phosphorylation partially via TMK1 and TMK4. The 6-d-old tmk1−/−tmk4RNAi-est seedlings grown on 1/2MS were transferred to medium containing 10 μMestradiol for another 4 d, the seedlings were harvested for 100 μM L-Kyn treatment for 1 h, then added back 10 μMNAA, and the seedling roots were harvested forprotein extraction. Three biological repeats. (G) Quantification of relative MPK6 and MPK3 kinase activity in tmk1−/−tmk4RNAi-est seedlings’ treatment with auxinshown in F. n = 3 biological repeats; data are mean ± SD. Asterisks indicate a statistically significant difference (*P value < 0.05; **P value < 0.01; ***P value < 0.001;****P < 0.0001), according to a Student’s t test; n.s., not significant.

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formation through MKK4/5-MPK3/6. Auxin also activates MPK3andMPK6 in an IDA-HAE/HSL2-independent manner, suggestingthe existence of unknown signaling pathways (33). The finding ofthe TMK-MKK4/5-MPK3/6 cascade may fill the gap between theauxin and MAPK cascade. Due to the overlapped downstreamcomponents, the auxin–TMK-signaling pathway may reciprocallycrosstalk with IDA-HAE/HLS2 signaling, which means phyto-hormone and plant small peptides coordinate to regulate LRdevelopment. Meanwhile, it remains unclear how plants apply thesame signaling pathway in distinct biological processes. One pos-sibility is that the MKK4/5-MPK3/6 cascade functions as a mo-lecular switch over the course of evolution, which links the varioustissue- or cell-specific activation of upstream inputs and the spa-tiotemporal control of downstream substrates, i.e., TMK1/TMK4,are highly expressed at LR primordia where the upstream input

auxin accumulates (SI Appendix, Fig. S2). Thus, it is worthwhileto understand how these multiple signaling pathways conju-gated to regulate cellular events in response to the complexdevelopmental cues.

Materials and MethodsPlant growth conditions and materials, phenotype analyses, plasmid con-structs, GUS staining and imaging, RT-qPCR analyses, recombinant proteinpurification,Western blots, Coimmunoprecipitation, FLIM-FRET assay, in vitrokinase assay, MAPK activity assay, and primers (SI Appendix, Table S1) areprovided in SI Appendix, SI Materials and Methods.

ACKNOWLEDGMENTS. This work was supported by the National Key R&DProgram of China (2016YFA0503200), the National Natural Science Founda-tion of China (Grants 31870256 and 31422008, to T.X.), and by National Nat-ural Science Foundation of China (Grant 31470359, to J.H.).

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