Noncanonical signaling regulates cell division pattern during lateral development

Rongfeng 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 Science Laboratory 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 be in regulating cell division during . The NPK1- orchestrated to ensure proper organogenesis. The cell division NQK1-NRK1 cascade regulates cytokinesis in Nicotiana tabacum patterns control the shape of growing organs, yet how they are (26, 27), and the activated NPK1, a MAPKKK, acts at the late precisely determined and coordinated is poorly understood. In M-phase to mediate the cell plate formation (28). MKK4/MKK5 plants, the distribution of the phytohormone auxin is tightly together with MPK3/MPK6 regulate stomatal patterning by con- linked to organogenesis, including lateral root (LR) development. trolling asymmetric cell division (29) and inflorescence architec- Nevertheless, how auxin regulates cell division pattern during ture by promoting localized cell proliferation in Arabidopsis (30). lateral root development remains elusive. Here, we report that Recently, MPK6 was proved to affect cell division orientation in auxin activates Mitogen-Activated Protein Kinase (MAPK) signal- both primary and lateral (31, 32), and the MKK4/MKK5- ing via transmembrane kinases (TMKs) to control cell division MPK3/MPK6 module was essential for LR emergence (33). pattern during lateral root development. Both TMK1/4 and MKK4/5- Notably, mutual regulation between plant MAPK signal MPK3/6 pathways are required to properly orient cell divisions, transduction modules and auxin signaling has been reported which ultimately determine lateral root development in response (34, 35). Exogenous auxin application can specifically activate to auxin. We show that TMKs directly and specifically interact with MPK3/MPK6 in root (33, 36). However, it is still unknown how and phosphorylate MKK4/5, which is required for auxin to activate MPK3/MPK6 are activated by auxin. In this study, we demon- PLANT BIOLOGY MKK4/5-MPK3/6 signaling. Our data suggest that TMK-mediated noncanonical auxin signaling is required to regulate cell division strate that the local auxin maximum at LR initiation sites trig- pattern and connect auxin signaling to MAPK signaling, which are gers TMK1/TMK4-mediated phosphorylation and activation of both essential for plant development. MKK4/MKK5, followed by the activation of MPK3/MPK6 to regulate cell division pattern during LR development. auxin | TMKs | MKK4/5 | MPK3/6 | cell division pattern Results TMK1 and TMK4 Are Required for Auxin-Regulated Cell Division oordinated cell division and cell expansion are essential for Pattern during LR Development. To investigate potential non- proper organ development in both animals and plants (1–3). C transcriptional mechanisms underlying auxin-mediated regulation Cell division orientation determines cell fate, and formative cell of LR development in Arabidopsis, we examined a transmembrane division drives organ morphogenesis and tissue differentiation. kinases (TMKs) family that was recently found to participate in Extensive research has focused on the regulation of the polarized direction of cell expansion, such as the interdigitated pavement cell expansion (4, 5), the tip growth of pollen tubes (6), and root Significance hair cells (7), but little is known about how the direction of cell division is determined. Development in multicellular organisms consists of a series of The phytohormone auxin plays a pivotal role in regulating cell divisions followed by cell expansions, which are tightly plant growth and development in a concentration-dependent controlled both spatially and temporally. Disorganized cell di- manner. Disruption of the auxin concentration gradient causes vision causes serious growth defects in both animals and severe defects in cell division patterns and aberrant organ devel- plants. Auxin has been repeatedly implicated in regulating cell opment (8–13). Lateral root (LR) initiation and development, a division patterns during organogenesis in plants; however, the underlying mechanisms remain to be further investigated. tightly coordinated process involving the strictly controlled cell Here, we demonstrate that auxin signaling controls cell di- division pattern (14), provides a model system to study the un- vision pattern by activating Mitogen-Activated Protein Kinase derlying mechanism of auxin regulation in cell division patterns (MAPK)-signaling cascade during lateral root development. Our during organogenesis (15, 16). work reveals how signal transduction pathways regulate cell Previous work demonstrated that abundant key transcrip- division pattern and also provides a connection between auxin tion factors, regulated by the TIR1/AFB-based canonical auxin signaling and MAPK signaling in plants. pathway, were essential for organogenesis, including LR de- – velopment (17 20). Recently, TMK-mediated auxin signaling Author contributions: T.X. designed research; R.H., R.Z., J.H., Z.Z., and J.W. performed was identified to regulate the ROP signaling pathways in the research; Y.X. contributed new reagents/analytic tools; R.H., R.Z., J.H., and J.W. analyzed pavement cells morphogenesis (21) and the noncanonical Aux/ data; and R.H., J.H., and T.X. wrote the paper. IAAs in the apical hook development (22). This discovery The authors declare no competing interest. provides a new perspective for understanding the action of This article is a PNAS Direct Submission. auxin in many developmental processes. Published under the PNAS license. Mitogen-Activated Protein Kinase (MAPK) cascades are highly 1R.H. and R.Z. contributed equally to this work. conserved signaling modules found in all eukaryotic cells. Plant 2To whom correspondence may be addressed. Email: [email protected] or [email protected]. MAPK pathways, which convert signals into cellular responses, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. regulate various aspects of plant growth and development (23–25). 1073/pnas.1910916116/-/DCSupplemental. Accumulating evidences illustrate that plant MAPKs are involved

www.pnas.org/cgi/doi/10.1073/pnas.1910916116 PNAS Latest Articles | 1of6 Downloaded by guest on September 27, 2021 auxin signaling (22). Of the 4 TMK members, tmk1-1 and tmk4-1 the 5-d-old noninduced mutant to the 1/2MS medium containing mutants showed slightly fewer LRs and lower LR density than wild 50 μM estradiol for another 5 d and found that the lateral roots type (SI Appendix,Fig.S1A–C), and LRs were mostly abolished in were partially abolished in the newly generated primary roots of −/− the tmk1-1tmk4-1 double-mutant (Fig. 1 A and B and SI Appendix, the estradiol treated tmk1 tmk4RNAi-est mutant (SI Appendix, Fig. S1 A and B), suggesting that TMK1 and TMK4 play an es- Fig. S5E). We further noticed that the cell division patterns were sential but redundant role in regulating LR development. Notably, disorganized at the different stages of LR development in the GUS reporter analyses driven by the native promoters of both tmk1-1tmk4-1 mutant, leading to the LR growth defects in the TMK1 and TMK4 suggested that TMK1 and TMK4 were highly mutant compared to the wild type (Fig. 1 C and D and SI Ap- expressed at the different stages of LR development (SI Appendix, pendix,Fig.S3B). Together, these data suggest that TMK1 and Fig. S2), which were confirmed by pTMK1-TMK1-GFP;tmk1-1 TMK4 are required to regulate cell division pattern and LR de- and pTMK4-TMK4-GFP;tmk4-1 transgenic lines (SI Appendix,Fig. velopment in response to auxin. S3A). These were in agreement with the strong LR phenotype in the tmk1-1tmk4-1 mutant. Exogenous auxin treatment with TMK1 and TMK4 Interact with MKK4 and MKK5. To determine how 150 nM NAA (1-Naphthaleneacetic acid) strongly promoted LR TMK1/TMK4 regulate cell division orientation and LR de- development including LR number and lateral root primordia velopment, we identified proteins that interact with TMK1 in (LRP)inwildtypebutnotinthetmk1-1tmk4-1 mutant (Fig. 1 A Arabidopsis. Briefly, we used GFP-trap agarose beads to immu- and B and SI Appendix,Fig.S4). However, the tmk1-1tmk4-1 noprecipitate TMK1-GFP and interacting proteins from pTMK1- mutant had a bit higher LRP density compared to wild type (SI TMK1-GFP transgenic plants, followed by mass spectrometry. Appendix,Fig.S4B), which may be caused by the severe growth Among the TMK1-interaction candidates, we were particularly defect, such as the much shorter primary root. Therefore, we interested in MAP-kinase kinase 4 (MAPKK4 or MKK4), a key generated an estradiol-inducible TMK4 RNAi in the tmk1 component of the MAPK-signaling pathway that was previously mutant background to further investigate the role of TMK1 reported to regulate both cell division and LR development in and TMK4 in LR development. The estradiol treatment in the plant (30, 33). −/− tmk1 tmk4RNAi-est mutant resulted in the reduced expres- Arabidopsis contains 10 MKK family members (37). To com- sion and protein level of TMK4 (SI Appendix,Fig.S5D and F). prehensively investigate these MKKs, we used the unbiased yeast This mutant showed similar phenotype as in the tmk1-1tmk4-1 2-hybrid assay to screen all MKK proteins for interactions with the double-mutant (SI Appendix,Fig.S5A–C). To avoid the effect C terminus of the 4 TMK family members. We found that the C of primary root defect in the tmk1-1tmk4-1 mutant, we transferred terminus of TMK1 and TMK4 specifically interacted with MKK4

A tmk1-1 gTMK1-flag; C Col-0 tmk4-1 tmk1-1tmk4-1 Stage I Stage II Stage III Stage IV NAA -+-+- + Col-0

B 30 DMSO NAA 25 b e 20

15 a d tmk1-1 tmk4-1

LR number 10

5 c c 0 n=25 26 25 25 26 26 Col-0 tmk1-1 gTMK1-flag; D tmk4-1 tmk1-1tmk4-1 60 Stage I 60 Stage II 60 Stage III 60 Stage IV 50 Col-0 50 Col-0 50 Col-0 50 Col-0 tmk1-1 tmk1-1 tmk1-1 tmk1-1 40 tmk4-1 40 tmk4-1 40 tmk4-1 40 tmk4-1 30 30 30 30 20 20 20 20 10 10 10 10 distribution (%) distribution (%) distribution (%) Frequency of angle Frequency of angle Frequency of angle distribution (%) Frequency of angle 0 0 0 0 20 40 60 80100120140160 20 40 60 80100120140160 20 40 60 80100120140160 20 40 60 80100120140160 Angle (degree) Angle (degree) Angle (degree) Angle (degree)

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, tmk1-1tmk4-1 and 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 NAA treatment. Values indicate mean ± SD from 3 individual biological repeats. n denotes number of independent seedlings, and different letters denote significant differences 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 individual seedlings showed similar results.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1910916116 Huang et al. Downloaded by guest on September 27, 2021 and MKK5 (Fig. 2A and SI Appendix,Fig.S6A), which belong to insensitive to auxin, but LRP density in the mutant was a bit the same subfamily (SI Appendix,Fig.S6C). The TMK2C terminus higher than in the wild type (SI Appendix,Fig.S9A and B). To interacted with MKK5 (SI Appendix, Fig. S6A); however, TMK2 avoid differences in primary root growth, we grew wild-type was not expressed in the LR (38). Transiently expressed MYC- and mkk4,5RNAi-est seedlings in 1/2 MS medium for 5 d and then tagged MKK4 and MKK5 coimmunoprecipitated with HA-tagged transferred them to the medium containing 50 μM estradiol to- TMK1 from protoplasts (Fig. 2B). Consistently, HA-tagged MKK4 gether with 150 nM NAA for another 5 d (SI Appendix,Fig.S10A). and MKK5 coimmunoprecipitated with MYC-tagged TMK1 (SI Again, we observed defective LR development in the estradiol- Appendix,Fig.S6B). These interactions were further confirmed on treated mkk4,5RNAi-est seedlings that was not rescued by auxin plasma membrane by the FLIM-FRET (Fluorescence Lifetime treatment (Fig. 3 A and B). These defects in the estradiol-treated Imaging Microscopy–Fluorescence Resonance Energy Transfer) mkk4,5RNAi-est mutant were also associated with the disorga- assay in protoplasts (Fig. 2 C and D). Similar to TMK1 and TMK4, nized cell division patterns at different stages of LR development both MKK4 and MKK5 were also highly expressed at the different (Fig. 3 E and F). Thus, MKK4 and MKK5 also regulate LR de- stages of LR development, assessed in pMKK4-MKK4-RFP and velopment in response to auxin. pMKK5-MKK5-RFP transgenic lines (SI Appendix,Fig.S7). Based on these data, we hypothesized that MKK4/5 participate with Auxin Induces TMK1/4-Dependent Phosphorylation of MKK4/5. The TMK1/4 to regulate LR development. TMK-mediated signaling pathway has been implicated in auxin signal transduction (22, 38), which led us to hypothesize that MKK4 and MKK5 Regulate Cell Division Pattern during LR Development auxin triggers the activation of MKK4 and MKK5 via TMKs. To in Response to Auxin. The mkk4mkk5 double-mutant is embryonic test this hypothesis, we treated Arabidopsis protoplasts tran- lethal; therefore, we generated an estradiol-inducible MKK4 siently expressing MKK4 or MKK5 with 100 nM NAA for 1 h. and MKK5 RNAi mutant to further investigate the role of Auxin treatment led to the mobility shift of MKK4 and MKK5 in MKK4 and MKK5 in LR development. Estradiol treatment of Phos-tag polyacrylamide gels that was abolished by Lambda the mkk4,5RNAi-estmutantresultedinthereducedexpression Protein Phosphatase (λ-PPase) treatment (Fig. 4A), suggesting of MKK4 and MKK5,butnototherMKKs (SI Appendix,Fig.S8A that auxin induces MKK4/5 phosphorylation. Using the same and C). Similar to the tmk1-1tmk4-1 mutant, the estradiol-treated approach, we found that coexpression of TMK1, but not a kinase mkk4,5RNAi-est mutant showed strong defects in LR development inactive TMK1(K616E), induced the phosphorylation of MKK4/5 which were also insensitive to auxin treatment (Fig. 3 A and B). (Fig. 4B). These data suggest that TMK1 also can phosphor-

TheLRdensityintheestradiol-treated mkk4,5RNAi-est was ylate MKK4/5. Indeed, an in vitro phosphorylation assay con- PLANT BIOLOGY firmed that both MKK4 and MKK5 were specifically and directly phosphorylated by a recombinant TMK1C terminal fragment (Fig. 4C). These observations collectively suggest that TMK- AB Prey based auxin signaling may participate in regulating the phos- AD MKK4 MKK5 Input IP phorylation of MKK4/5, which is consistent with the similar LR BD TMK1-HA +++ +++ defects in both tmk1-1tmk4-1 and mkk4,5RNAi-est mutants. -TL MKK4-MYC +-- +-- TMK1C MKK5-MYC -+- -+- Auxin Activates MPK3 and MPK6 through TMK1/4 to Regulate Cell α-HA Division Pattern in LR. MPK3/6 have been reported to function BD - downstream of MKK4/5 in multiple developmental processes TLHA α-MYC (29, 30, 39, 40). Similar to TMK1/4 and MKK4/5, both MPK3 TMK1C and MPK6 were also highly accumulated at different stages of LR development (SI Appendix, Fig. S7). To determine whether C D 2.8 **** MPK3/6 play a role in LR development, we generated an FLU Intensity FLU Lifetime MPK3 RNAi mpk6 2.6 estradiol-inducible in the mutant, since the mpk3mpk6 double-mutant was also embryonic lethal. Estradiol 4 2.4 −/− 0 treatment of the mpk6 mpk3RNAi-est mutant resulted in 2.2 the reduced expression of MPK3 in the mpk6 mutant back- ground (SI Appendix,Fig.S8B). Similar to the tmk1-1tmk4-1 Lifetime [ns] 2.0 and estradiol-inducible mkk4,5RNAi-est mutants, estradiol treat- −/− 4 Lifetime (ns) GFP 1.8 ment of mpk6 mpk3RNAi-est also led to LR developmen- 0 1.6 n=32 31 23 tal defects (SI Appendix,Figs.S9C and D and S10B)andthe TMK1-GFP +++ disorganized cell division pattern, which was insensitive to MKK4-RFP - + -

Lifetime [ns] MKK5-RFP - - + auxin (Fig. 3 C–F). To further verify whether auxin activates MPK3/6 to regulate Fig. 2. TMK1 interacts with MKK4 and MKK5. (A) Yeast 2-hybrid assay of LR development, we examined yuc1-D mutant, which produces TMK1C-terminus (as bait) and MKK4 and MKK5 protein (as prey). Three in- excessive auxin (41), and wei8-3tar2-1 mutant, which produces dependent biological repeats. AD, activation domain; BD, DNA-binding reduced auxin (42). The levels of phosphorylated MPK3/6 corre- domain; -TL, synthetic dextrose minimal medium without leucine and tryp- SI Appendix tophan; -TLHA, synthetic dextrose minimal medium without leucine, tryp- lated with auxin levels in the respective mutants ( ,Fig. tophan, adenine, and histidine. (B) α-MYC coimmunoprecipitated TMK1-HA S11 A and B). In addition, we treated wild-type seedlings with with MKK4-MYC and MKK5-MYC in protoplasts. The 35S-TMK1-HA was L-kynurenine (L-Kyn) (43) to inhibit auxin biosynthesis, followed transiently coexpressed with 35S-MKK4-MYC or 35S-MKK5-MYC in Arabi- by the exogenous auxin (NAA or IAA) treatment. We found that dopsis protoplasts. Three independent repeats showed similar results. (C) L-Kyn treatment led to slightly reduced levels of phosphorylated Images of FLIM-FRET (Fluorescence Lifetime Imaging Microscopy–Fluores- MPK3/6, which was restored by auxin treatment. Auxin treatment cence Resonance Energy Transfer) assay between TMK1 and MKK4 or MKK5. also restored MPK3/6 phosphorylation in the auxin-deficient wei8- The 35S-TMK1-GFP was transiently coexpressed with 35S-MKK4-RFP or 35S- 3tar2-1 mutant (Fig. 4 D and E and SI Appendix,Fig.S11C MKK5-RFP in Arabidopsis protoplasts, and the protoplasts were harvested D after 6 h for image analysis. (Scale bar, 10 μm.) (D) Quantification of lifetimes and ). It has been reported that MKK4/5 mediate the acti- of GFP signals (C). n denotes number of independent protoplasts. Asterisks vation of MPK3/6 by auxin (33). More importantly, we found indicate a statistically significant difference (****P value < 0.0001), accord- that the auxin-induced phosphorylation of MPK3/6 was par- −/− ing to a Student’s t test. tially abolished in the tmk1 tmk4RNAi-est mutant, but not

Huang et al. PNAS Latest Articles | 3of6 Downloaded by guest on September 27, 2021 A Col-0 mkk4,5RNAi-est B E EST -+ + -++ Stage I Stage II Stage III Stage IV NAA -- + --+ 25 DMSO EST b EST+NAA 20

15

a a Col-0 10 a LR number c c 5

0 n=21 20 20 21 19 20

Col-0 mkk4,5 -est RNAi-est C Col-0 mpk6-/-mpk3RNAi-est D EST -+ + -+ + NAA -- + - -+ 25 DMSO b EST 20 EST+NAA mkk4,5RNAi

15 a a a -est 10 LR number

5 c c mpk3RNAi 0 -/- n=25 25 25 42 42 43 mpk6-/-mpk3

Col-0 mpk6 RNAi-est F 60 60 60 60 mpk6 mkk4,5RNAi Col-0 mkk4 Col-0 mpk6 mkk4,5RNAi Col-0 mpk6 mpk6 Stage I Stage II mkk4 Col-0 Stage III Stage IV 50 50 50 50 , -/- -/- -/- , -/- 5RNAi 5RNAi mpk3RNAi mpk3RNAi mpk3RNAi 40 40 40 mpk3RNAi 40 30 30 30 30 -est -est -est -est 20 20 20 20 - -est - -est est 10 10 10 10 est Frequency of angle distribution (%) Frequency of angle distribution (%) distribution (%) distribution (%) Frequency of angle 0 0 0 Frequency of angle 0 20 40 60 80100120140160 20 40 60 80100120 160140 20 40 60 80100120 160140 20 40 60 80100120 160140 Angle (degree) Angle (degree) Angle (degree) Angle (degree)

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 in Col-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 SD of 3 individual biological repeats. n denotes number of independent seedlings. (C) Lateral root phenotype of 9-d-old seedlings in Col-0 and estradiol-inducible mutant 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 comparisons − − test 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.

in the tir1afb2afb3 mutant (Fig. 4 F and G and SI Appendix, auxin action at the cellular level. Previous work demonstrated that Fig. S11 E and F). Taken together, our observations collectively TIR1-based auxin signaling regulates lateral root development, suggestthatauxinactivatesthe MKK4/5-MPK3/6-signaling especially initiation, through the transcriptional regulation of the cascade via TMK1/4 to regulate cell division pattern during key components involved in this developmental process. That LR development. TMK1/TMK4 mediate auxin activation of the MKK4/5-MPK3/6 cascade provides a posttranscriptional regulation mechanism, Discussion which coordinates with the TIR1 pathway to control the LR de- The molecular mechanism underlying the strict control of cell velopment precisely (SI Appendix,Fig.S12). This also fills a missing division pattern, by which multicellular organisms produce new link in our understanding of both auxin and MAPK-signaling and specific tissues and organs, is a basic and central theme in events, whose modes of action are critical for plant growth and developmental biology. The formative cell divisions are required development. Given the pivotal roles of auxin in cell division for multiple developmental processes both in plants and animals. patterning, our findings shed light on how hormonal signaling leads Unlike animals, most plant organs, including lateral roots, are to changes in cell division orientation, which is important for plant primarily postembryonically initiated from continuous de novo cells (44), but distinct from animal cells. development, implying that these mechanisms establish tight The new lateral roots grow from LR initiation sites where spatiotemporal coordination of cell division and patterning (16). auxin accumulates (SI Appendix,Fig.S2)(9),thesameplaceas In this study, we show that auxin activates MKK4/5-MPK3/6 the protein abundance of TMK1/TMK4, MKK4/MKK5 and cascade through TMK1/4 to regulate cell division pattern during MPK3/MPK6 (SI Appendix,Figs.S2,S3A, and S7). Both auxin LR morphogenesis. TMKs, as the key components of auxin sig- and MKK4/5-MPK3/6 activities are required for LR develop- naling on plasma membrane, mediate noncanonical auxin-signaling ment(9,33,45).Inthemeanwhile,MKK4andMKK5,aswell transduction to control versatile developmental processes (21, 22), as MPK3 and MPK6, have redundant functions in diverse bi- which is distinct from TIR1-based canonical transcriptional auxin ological processes, including stomatal differentiation (29), in- signaling. This finding provides a mechanism to regulate cell di- florescence localized cell division (30), and zygote division vision pattern during organ morphogenesis and a perspective of (46). Although our work here focuses on the roles of this

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1910916116 Huang et al. Downloaded by guest on September 27, 2021 A MKK4-MYC MKK5-MYC C His-MBP λ-PPase --++ -- -TMK1C + - + - + NAA --+ + ++ His-MBP - + - + - Phos-tag gel GST-MKK4(PD) ++--- α-MYC GST-MKK5(PD) --++- Normal gel GST - -- -+ [32P]TMK1C - B MKK4-MYC MKK5-MYC 32 - TMK1-HA - + - + --+ + [ P]MKK4/5(PD) TMK1-HA - --+ --+ - (K616E) TMK1C - - - λ-PPase - + --- + MKK4 - (PD) - Phos-tag gel MKK5(PD) α-MYC His-MBP- Normal gel GST - α-HA D E 4 MPK6 *** MPK3 Col-0 wei8-3tar2-1 3 *** NAA --+ -+ ** L-Kyn --+ + - 2 ** -MPK6-p - MPK3-p 1 MPK3 activity -MPK6 Relative MPK6 and -MPK3 0

NAA - - + - + - - + - + PLANT BIOLOGY L-Kyn - + + -- - + + -- Col-0 wei8-3 Col-0 wei8-3 F G tar2-1 tar2-1 4 MPK6 Col-0 tmk1-/-tmk4RNAi-est MPK3 EST ++ + +++ 3 * NAA --+ --+ **** L-Kyn - + + - + + ** 2 *** -MPK6-p - MPK3-p 1 - n.s. n.s. MPK3 activity MPK6 Relative MPK6 and - MPK3 0 EST + + ++++ ++ + + + + NAA - - + - - + - - + - - + L-Kyn - + + - + + - + + - + + Col-0 tmk1-/-tmk4 Col-0 tmk1-/-tmk4 RNAi-est RNAi-est

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 and MKK5. 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; dimethyl sulfoxide (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 transiently expressed 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)InvitrokinaseassayofTMK1kinasedomainwithMKK4(PD) and MKK5(PD) protein. 32 Phosphorylation is detected by radioactive P .MKK4(PD), kinase inactive MKK4(K108M); MKK5(PD), kinase inactive MKK5(K99M). Protein loading was detected by Western 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-Kyn treatment 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 and MPK3 phosphorylation partially via TMK1 and TMK4. The 6-d-old tmk1−/−tmk4RNAi-est seedlings grown on 1/2MS were transferred to medium containing 10 μM estradiol for another 4 d, the seedlings were harvested for 100 μM L-Kyn treatment for 1 h, then added back 10 μM NAA, and the seedling roots were harvested for − − protein extraction. Three biological repeats. (G) Quantification of relative MPK6 and MPK3 kinase activity in tmk1 / tmk4RNAi-est seedlings’ treatment with auxin shown 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.

auxin-signaling mechanism in LR cells, it is likely that similar auxin- regulates cell division orientation. Moreover, MPK3 and MPK6 TMK-MAPK modules operate in other plant cells and tissues. are protein kinases and likely function with downstream substrates Plant cell division orientation relies on the PPB (preprophase or transcription factors, whose identification will be crucial to band), a specialized MT (microtubule) apparatus composed of understand how this signaling executes and exports outputs. MTs and F-actins, to organize the cell division plane (47). Recent The MKK4/5-MPK3/6 cascade has been reported as down- evidence indicates that MPK6 is associated with MT dynamics (48) stream of several RLKs [e.g., ER (30), FLS2 (49), HAE/HSL2 (50), and involved in the control of cell division plane formation (32), CLV1/CLV2 (51)] and senses various upstream signals (e.g., which provides some hints for further exploring the detailed various stress stimuli and development signals). For example, mechanism of how the auxin–TMK–MAPK pathway elaborately IDA-HAE/HLS2 ligand–receptor mediates auxin-facilitated LR

Huang et al. PNAS Latest Articles | 5of6 Downloaded by guest on September 27, 2021 formation through MKK4/5-MPK3/6. Auxin also activates MPK3 auxin accumulates (SI Appendix,Fig.S2). Thus, it is worthwhile and MPK6 in an IDA-HAE/HSL2-independent manner, suggesting to understand how these multiple signaling pathways conju- the existence of unknown signaling pathways (33). The finding of gated to regulate cellular events in response to the complex the TMK-MKK4/5-MPK3/6 cascade may fill the gap between the developmental cues. auxin and MAPK cascade. Due to the overlapped downstream components, the auxin–TMK-signaling pathway may reciprocally Materials and Methods crosstalk with IDA-HAE/HLS2 signaling, which means phyto- Plant growth conditions and materials, phenotype analyses, plasmid con- hormone and plant small peptides coordinate to regulate LR structs, GUS staining and imaging, RT-qPCR analyses, recombinant protein development. Meanwhile, it remains unclear how plants apply the purification, Western blots, Coimmunoprecipitation, FLIM-FRET assay, in vitro same signaling pathway in distinct biological processes. One pos- kinase assay, MAPK activity assay, and primers (SI Appendix, Table S1) are sibility is that the MKK4/5-MPK3/6 cascade functions as a mo- provided in SI Appendix, SI Materials and Methods. lecular switch over the course of evolution, which links the various ACKNOWLEDGMENTS. This work was supported by the National Key R&D tissue- or cell-specific activation of upstream inputs and the spa- Program of China (2016YFA0503200), the National Natural Science Founda- tiotemporal control of downstream substrates, i.e., TMK1/TMK4, tion of China (Grants 31870256 and 31422008, to T.X.), and by National Nat- are highly expressed at LR primordia where the upstream input ural Science Foundation of China (Grant 31470359, to J.H.).

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