Karrikin Signaling Acts Parallel to and Additively withStrigolactone Signaling to Regulate Rice MesocotylElongation in Darkness[OPEN]

Jianshu Zheng,a,1 Kai Hong,a,1 Longjun Zeng,a,b,1 Lei Wang,a Shujing Kang,a,c Minghao Qu,a Jiarong Dai,b,c

Linyuan Zou,a Lixin Zhu,d Zhanpeng Tang,a Xiangbing Meng,e Bing Wang,e Jiang Hu,d Dali Zeng,d Yonghui Zhao,b

Peng Cui,a Quan Wang,a Qian Qian,a,d Yonghong Wang,f,g,h Jiayang Li,e,g and Guosheng Xionga,b,1

a Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, 518120, Chinab Plant Phenomics Research Center, Nanjing Agricultural University, Nanjing, 210095, ChinacCollege of Agriculture, Nanjing Agricultural University, Nanjing, 210095, Chinad State Key Laboratory of Rice Biology, China National Rice Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou310006, Chinae State Key Laboratory of Plant Genomics, and National Center for Plant Gene Research (Beijing), Institute of Genetics andDevelopmental Biology, The Innovative Academy for Seed Design, Chinese Academy of Sciences, Beijing, 100101, Chinaf State Key Laboratory of Plant Genomics, and National Center for Plant Gene Research (Beijing), Institute of Genetics andDevelopmental Biology, The CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Beijing, 100101,ChinagUniversity of Chinese Academy of Sciences, Beijing, 100039, ChinahCollege of Life Sciences, Shandong Agricultural University, Taian, 271018, China

ORCID IDs: 0000-0001-6083-5251 (J.Z.); 0000-0002-8795-7678 (K.H.); 0000-0002-1355-4761 (L.J.Z.); 0000-0002-0356-3592 (L.W.);0000-0003-1272-018X (S.K.); 0000-0001-6158-6103 (M.Q.); 0000-0002-2232-0510 (J.D.); 0000-0002-1544-5913 (L.Y.Z.); 0000-0003-2687-6396 (L.X.Z.); 0000-0001-9102-5921 (Z.T.); 0000-0001-7040-7138 (X.M.); 0000-0002-2759-1512 (B.W.); 0000-0002-3560-4256(J.H.); 0000-0002-0126-6906 (Y.Z.); 0000-0003-2349-8633 (D.Z.); 0000-0003-2989-571X (P.C.); 0000-0003-2912-1263 (Q.W.); 0000-0002-0349-4937 (Q.Q.); 0000-0003-0721-1989 (Y.W.); 0000-0002-0487-6574 (J.L.); 0000-0002-4312-1676 (G.X.)

Seedling emergence in monocots depends mainly on mesocotyl elongation, requiring coordination between developmentalsignals and environmental stimuli. Strigolactones (SLs) and karrikins are butenolide compounds that regulate variousdevelopmental processes; both are able to negatively regulate rice (Oryza sativa) mesocotyl elongation in the dark. Here, wereport that a karrikin signaling complex, DWARF14-LIKE (D14L)-DWARF3 (D3)-O. sativa SUPPRESSOR OF MAX2 1(OsSMAX1) mediates the regulation of rice mesocotyl elongation in the dark. We demonstrate that D14L recognizes thekarrikin signal and recruits the SCFD3 ubiquitin ligase for the ubiquitination and degradation of OsSMAX1, mirroring the SL-induced and D14- and D3-dependent ubiquitination and degradation of D53. Overexpression of OsSMAX1 promotedmesocotyl elongation in the dark, whereas knockout of OsSMAX1 suppressed the elongated-mesocotyl phenotypes of d14land d3. OsSMAX1 localizes to the nucleus and interacts with TOPLESS-RELATED PROTEINs, regulating downstream geneexpression. Moreover, we showed that the GR24 enantiomers GR245DS and GR24ent-5DS specifically inhibit mesocotylelongation and regulate downstream gene expression in a D14- and D14L-dependent manner, respectively. Our workrevealed that karrikin and SL signaling play parallel and additive roles in modulating downstream gene expression andnegatively regulating mesocotyl elongation in the dark.

INTRODUCTION

Seed germination and seedling establishment require the co-ordination of developmental programs in response to variousenvironmental signals (Gommers and Monte, 2018). Epigealseedlings of dicotyledonous plants growing in soil or in the darkhave elongated hypocotyls that allow the seedlings to emerge

from the soil to reach the light (de Wit et al., 2016). By contrast,monocot seedling emergence depends onmesocotyl elongation,an important agronomic trait of grain crop species (de Wit et al.,2016). Mesocotyl elongation reflects the coordination of cellelongation andcell division processes that are regulated preciselyby phytohormones that integrate developmental signals andenvironmental stimuli (Sawers et al., 2002). The phytohormonesthat promote rice (Oryza sativa) mesocotyl elongation includeauxin (Epel et al., 1992), brassinosteroids (BRs; Yamamuro et al.,2000), cytokinins (Huet al., 2014), ethylene (Xionget al., 2017), andgibberellins (Takahashi, 1972), whereas abscisic acid (Takahashi,1972), jasmonic acid (JA; Riemann et al., 2003; Xiong et al., 2017),and strigolactones (SLs; Hu et al., 2010) inhibit mesocotyl elon-gation. The interaction of SLs with cytokinins (Hu et al., 2014) and

1Address correspondence to gsxiong@njau.edu.cn.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: gsxiong@njau.edu.cn.[OPEN]Articles can be viewed without a subscription.1These authors contributed equally to this work.www.plantcell.org/cgi/doi/10.1105/tpc.20.00123

The Plant Cell, Vol. 32: 2780–2805, September 2020, www.plantcell.org ã 2020 ASPB.

BRs in the regulation of dark-induced rice mesocotyl elongation(Sun et al., 2018) suggests that mesocotyl development is anexcellent model for investigating the crosstalk between SLs andother phytohormones.

SLs are butenolide compounds that are involved in variousdevelopmental processes and environmental responses, in-cluding parasitic weed germination stimulation, arbuscular my-corrhizal (AM) fungi hyphal branching, shoot branching inhibition,root system architecture formation, secondary growth of thecambium, leaf senescence, and drought tolerance (Al-Babili andBouwmeester, 2015). Deficiency in SL biosynthesis or signalingresults in tiller bud outgrowth in rice (Ishikawa et al., 2005), in-creased tiller angles (Sang et al., 2014), enhanced sensitivity tosenescence (Yamada et al., 2014), and mesocotyl elongation inthedark (Hu et al., 2010). SLs arederived fromb-carotene, and theactivities of the following catalytic enzymes lead to carlactoneproduction: carotene isomerase DWARF27 (D27; Lin et al., 2009;Alder et al., 2012), carotenoid cleavage dioxygenase HTD1/D17(CCD7; Zou et al., 2006), and CCD8 (Arite et al., 2007). Carlactoneis further converted by the MORE AXILLARY GROWTH1 (MAX1;Lazar and Goodman, 2006; Abe et al., 2014; Cardoso et al., 2014;de Saint Germain et al., 2016; Yoneyama et al., 2018; Zhang et al.,2018; Wakabayashi et al., 2019) and the LATERAL BRANCHINGOXIDOREDUCTASE (LBO) enzymes (Brewer et al., 2016). The SLsignaling cascade is initiated when the receptor DWARF14 (D14),an a/b hydrolase fold protein (Arite et al., 2009; de Saint Germainet al., 2016; Yao et al., 2016; Shabek et al., 2018; Seto et al., 2019),covalently binds and hydrolyzesSL (deSaint Germain et al., 2016;Yao et al., 2016), enhancing the interactions of D14with the F-boxprotein DWARF3 (D3; Yan et al., 2007; Hamiaux et al., 2012; Jianget al., 2013; Zhou et al., 2013) and the Clp ATPase proteinDWARF53 (D53; Jiang et al., 2013; Zhou et al., 2013). In the

presence of SL, the D53 protein is ubiquitinated by SCFD3 fordegradation via the 26S proteasome pathway, relieving theTOPLESS-RELATED PROTEIN (TPR) transcriptional cor-epressors that mediate repression of D53 downstream targets(Jiang et al., 2013; Zhou et al., 2013).SL signalingwas revealed to bemore complexwhenMAX2was

discovered to participate in both SL and karrikin pathways.Karrikins are butenolide chemical regulators present in the smokeof burned plant material, and their molecular structure is similar tothat of the D-ring of SLs (Scaffidi et al., 2013). Karrikins canstimulate seed germination (Flematti et al., 2004) and are involvedin regulating root skewing (Swarbreck et al., 2019) and root hairdevelopment (Villaécija-Aguilar et al., 2019) in Arabidopsis. Ge-netic screening of Arabidopsis karrikin-insensitive (kai) mutantsrevealed that loss of function of MAX2/KAI1 (Nelson et al., 2011)led to an elongated-hypocotyl phenotype in the light. HTL/KAI2was initially identified by screening light-responsive mutants (Sunand Ni, 2011; Waters et al., 2012) and later was found to be in-sensitive to karrikin (Nelson et al., 2011; Waters et al., 2012).Compared with the wild type, both max2 and kai2 are hyper-sensitive to drought stress (Bu et al., 2014; Ha et al., 2014; Li et al.,2017), indicating that karrikin plays roles in drought adaptation.The phenotypes of Atd14 and kai2 single-mutant seedlingssomewhat resemble the phenotype of max2 seedlings (Waterset al., 2012;Scaffidi et al., 2013), and theAtd14kai2doublemutantphenotypically mimics max2 (Waters et al., 2012). Neither thehypocotyl-elongation phenotype nor the shoot-branching phe-notype of Atd14 kai2 and max2 respond to karrikin or rac-GR24(Waters et al., 2012; Scaffidi et al., 2013). Loss of function ofSMAX1 and SMXL2 can restore most aspects of the max2seedling phenotype, but not shoot-branching defects of max2(Stanga et al., 2013, 2016; Soundappan et al., 2015), while the

Karrikin and Strigolactone Repress Mesocotyl Elongation 2781

max2 shoot-branching phenotype is rescued by the smxl6 smxl7smxl 8 triplemutation (Soundappan et al., 2015;Wang et al., 2015;Liang et al., 2016). Recently, SMAX1 and SMXL2 were shown tosuppress the root-skewing and root-hair phenotypes of max2(Swarbreck et al., 2019; Villaécija-Aguilar et al., 2019). The karrikinsignaling and SL signaling pathways act in parallel to regulateMAX2 activity in a ligand-dependent manner (Soundappan et al.,2015;Wang et al., 2015). The commonly used synthetic SL analograc-GR24 is amixture of two enantiomers, GR245DS andGR24ent-5DS (Scaffidi et al., 2014), and the effects of GR245DS andGR24ent-5DS on the inhibition of hypocotyl elongation and the regulation ofgene expression seem to be mediated by AtD14 and KAI2, re-spectively (Scaffidi et al., 2014). Collectively, these genetic datasuggest that theSLand karrikin signaling pathways act in a similarmanner: they recruit different members of the same receptorfamily (a/b fold hydrolase) that recognize different ligands totrigger the SCFMAX2 complex to target different members of thesame protein family for degradation (Morffy et al., 2016).

Under phosphorus-limiting conditions, increased SL bio-synthesis and exudation from roots promote AM symbiosis withplants (Akiyamaet al., 2005). In rice,D3, but notD14, is required forroot colonization by AM fungi (Yoshida et al., 2012). The karrikinsignaling component DWARF14-LIKE (D14L) is required for col-onization of rice roots by AM fungi (Gutjahr et al., 2015b). TheD14L-mediated pathway may act in parallel with the CERK1-dependent pathway (Gutjahr et al., 2015b; Chiu et al., 2018).Moreover, the mesocotyls of the d3mutant are longer than thoseof other SL pathway mutants, including d10 and d14 (Hu et al.,2010), and loss of function ofD14L increases mesocotyl length inthe dark (Gutjahr et al., 2015b; Kameoka and Kyozuka, 2015).These findings suggest the existence of a D14L-D3–dependent(but D14-independent) karrikin signal cascade in rice.

Here, we report that mesocotyl elongation in the dark is regu-lated by the D14L-D3-O. sativa SUPPRESSOR OF MAX2 1(OsSMAX1) module in rice. OsSMAX1 interacted with TPR tran-scriptional corepressors in an Ethylene-responsive elementbinding factor-associated amphiphilic repression (EAR) motif-dependent manner and regulated the expression of down-stream target genes. D14L and D3 were required for the karrikinsignal-induced degradation of OsSMAX1, which is necessary forinhibition of mesocotyl elongation in the dark, but not the regu-lation of shoot branching. We further revealed that D14L and D14are required for the recognition of the stereospecific enantiomersof GR24 and for the recruitment of SCFD3 for ubiquitination anddegradation of substrate proteins, respectively. Our work dem-onstrates that the parallel and additive actions of SL and karrikinsignaling in the regulation of mesocotyl elongation in the darklargely depend on their convergence in the regulation of the ex-pression of common downstream genes.

RESULTS

D14L Acts Parallel to and Additively with D14 to RegulateRice Mesocotyl Elongation in the Dark

Both the karrikin and SL signaling pathways are involved in theregulation of mesocotyl elongation in the dark, which requires the

function of D14L and D14, respectively (Hu et al., 2010; Gutjahret al., 2015b; Kameoka and Kyozuka, 2015). A deficiency in SLbiosynthesis or signaling leads to the accumulation of D53 andresults in tiller bud outgrowth (Jiang et al., 2013; Zhou et al., 2013).As a gain-of-function SL-insensitive mutant, the shoot-branchingphenotype of d53 is similar to that of the loss-of-function SLbiosynthesis mutants d27, d17, and d10 and the loss-of-functionSL signaling mutants d14 and d3 (Jiang et al., 2013; Zhou et al.,2013).Comparedwith thewild type,both theSL-deficientmutants(d27, d17, and d10) and the SL-insensitive mutants (d14 and d3)have longermesocotyls (Hu et al., 2010). To investigate the role ofD53 in the inhibition of mesocotyl elongation in the dark, wemeasured the mesocotyl length of d53, which was as long as thatofd14 (Figures1Aand1B).ACT:D53m-GFP transgenic seedlings,which constitutively express d53 and GFP fusion proteins, ex-hibited a shoot-branching phenotype similar to that of d53(Supplemental Figures 1A and 1B; Jiang et al., 2013). The mes-ocotyl length of the ACT:D53m-GFP transgenic seedlings wasalso similar to that of d14 (Supplemental Figures 1C and 1D).These resultssuggested thatD53accumulation in theseseedlingspromotes mesocotyl elongation in the dark and confirmed thatD14- and D3-dependent D53 degradation is involved in the in-hibition of rice mesocotyl elongation in the dark. However, the d3mesocotyl length was longer than that of other SL mutants, in-cluding d14 and d53, strongly suggesting that dark-inducedmesocotyl elongation in rice is also regulated by an SL-independent pathway. D14L, D14L2, D14L3, and D14L4 arehomologs of D14 (Supplemental Figure 2; Supplemental Data Set1; Arite et al., 2009; Walker et al., 2019). Moreover, D14L is ho-mologous toHTL/KAI2and is involved in regulating ricemesocotylelongation in the dark (Gutjahr et al., 2015b; Kameoka and Kyo-zuka, 2015). D14L2 and D14L3 are similar to DLK2, which can beinduced by karrikin treatment (Nelson et al., 2011), and enhancehypocotyl elongation (Végh et al., 2017).To investigate how karrikin regulates rice mesocotyl de-

velopment in the dark, we generated D14L knockout plants usingclustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated 9 (Cas9)–mediated genome edit-ing as previously described (Song et al., 2017). The d14l-1mutantcontained a 1-bp deletion at site 493 of D14L that shifted thereading frame, the d14l-2 mutant contained a 3-bp in-frame de-letion at site 493 to 495, and the d14l-3 mutant had a G-to-Tsubstitution at site 495 and a 7-bp deletion at site 496 to 502,causing a reading frameshift inD14L (Supplemental Figure 3). Thed14l-1 line was used for further analysis and is referred to as d14lhenceforth (Supplemental Figure 3). Under light conditions,mesocotyl elongationwasnotobserved ind14,d14l,d3,d14d14l,or wild-type seedlings (Supplemental Figure 4). In the dark, themesocotyl length of d14l was similar to that of d14, which waslonger than that of thewild typebut shorter than that ofd3 (Figures1A and 1B). The d14l mesocotyl phenotype in this study is con-sistent with the phenotypes reported previously (Gutjahr et al.,2015b; Kameoka and Kyozuka, 2015). However, unlike the D14LRNAi d14 seedlings in a previous study (Kameoka and Kyozuka,2015), themesocotyl length of the d14 d14l doublemutant was aslongas that ofd3 (Figures1Aand1B). In addition, themesocotyl ofthed53d14ldoublemutantwas longer than thatofd14landsimilarto that of d3 (Supplemental Figures 1E and 1F). These results

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Figure 1. D14L Acts in Parallel and Additively with D14 in Mesocotyl Inhibition in the Dark.

(A) Mesocotyls of seedlings grown for 7 d in darkness; the arrowheads indicate the boundaries of the mesocotyl. Bar 5 5 mm. WT, wild type.(B) Lengths of mesocotyls of dark-grown seedlings. Data are presented as means6 SDs, and the numbers above the columns indicate the sample sizes.Statistically significant differenceswere determined byone-wayANOVA; the different letters indicate significant differences between samples according toDuncan’s test (P < 0.05; Supplemental Data Set 5). WT, wild type.(C) and (D)Relative expression levels of themesocotyl-related genesOsTCP5 (C) andGY1 (D) detected by RT-qPCR in seedlings of mutants compared tothe wild type (WT).(E) Venn diagram of genes for which expression was upregulated in the mutants compared to the wild type.

Karrikin and Strigolactone Repress Mesocotyl Elongation 2783

suggested that the D14L-mediated pathway acts additively withthe D14-mediated pathway in the inhibition of rice mesocotylelongation in the dark. SL negatively regulates the expression ofOsTCP5 during mesocotyl development (Hu et al., 2014). De-ficiency in either D14 or D14L resulted in the downregulation ofOsTCP5 (Figure 1C). GY1 is involved in JA biosynthesis andnegatively regulates mesocotyl elongation (Xiong et al., 2017).Deficiency in either D14 or D14L resulted in significant down-regulation of GY1 expression (Figure 1D). The downregulation ofOsTCP5andGY1expression ind14d14landd3wassimilar to thatin d14 and d14l (Figures 1C and 1D), indicating that the D14- andD14L-mediated pathways potentially regulate common targetgenes to modulate mesocotyl elongation in the dark.

Next, we compared the gene expression profiles of wild-type,d14,d14l,d3, andd14d14l seedlingsgrown in thedark.More thantwo-thirds of the differentially expressed genes (DEGs) betweenthe wild type and d3 overlapped with the DEGs between the wildtype and d14 d14l. Interestingly, more than one-third of the DEGsbetween thewild typeandd14overlappedwith theDEGsbetweenthewild typeandd14l (Figures1Eand1F).GeneOntologyanalysisof these DEGs revealed a significant enrichment of genes asso-ciated with the responses to abiotic stress and light stimuli, whichare likely subject to regulation by both the D14- and D14L-mediated pathways (Figure 1G). Loss of function of either D14or D14L resulted in a decrease in the expression of D14L (Fig-ure 1H). The expression of D14L2 has been suggested to bedependent on D14L (Gutjahr et al., 2015b). Indeed, disruptingeither D14 or D14L function led to a dramatic decrease in D14L2expression (Figure 1I). Moreover, the expression of D14L3 alsodependedon the functionof bothD14andD14L (Figure 1J). Takentogether, these results suggested that, in termsof the regulationofrice mesocotyl elongation in the dark, the D14L- and D14-mediated pathways act additively and in parallel, and both de-pend on the function of D3.

D14L Interacts with OsSMAX1 and D3

The D53-like/SMAXL protein family has nine members in rice(Jiang et al., 2013). In the presence of SL, D14 recruits D3 todegrade D53 (Jiang et al., 2013; Zhou et al., 2013). We hypoth-esized that D14L may be similar to D14 with respect to the re-cruitment of D3 for the ubiquitination of aD53-like/SMAXL proteinin the regulation of mesocotyl elongation in the dark. First, wetested the interaction of individual D53-like/SMAXL proteins withD14, D14L, D14L2, andD14L3proteins (Supplemental Figure 2) ina yeast two-hybrid assay. Among the rice D53-like/SMAXL pro-teins, D53L, which is encoded by LOC_Os12g01360, was mostsimilar to D53 (Supplemental Figure 5; Supplemental Data Set 2)

and could interact with D14 in the presence of rac-GR24(Supplemental Figure 6). The protein encoded by LOC_Os08g15230 was most similar to Arabidopsis (Arabidopsisthaliana) SMAX1 and could interact with D14L (Figure 2A; Jiangetal., 2013;Stangaetal., 2013), butnotwithD14,D14L2,orD14L3(Supplemental Figure 7). We therefore refer to LOC_Os08g15230as OsSMAX1 hereafter. Pull-down assays confirmed that OsS-MAX1 interactswithD14L, but notD14 (Figure 2B).Moreover, HA-OsSMAX1was coprecipitated byGFP-D14L, but not byGFP-D14when tagged proteins were transiently expressed in rice proto-plasts, further confirming the interaction between OsSMAX1 andD14L (Figure 2C).We found that D14L interactswithOsSMAX1, but notwith other

D53-like/SMAXL proteins in the yeast two-hybrid assay(Supplemental Figure 6). To identify the OsSMAX1 domain re-sponsible for its interaction with D14L, various OsSMAX1fragments were tested via yeast two-hybrid assays and pull-down assays. The putative interaction domain was narrowed toOsSMAX1 (191 to 444; Figure 2D; Supplemental Figure 8A),which contained a region similar to theD14-interacting fragmentof D53 (181 to 404; Supplemental Figure 8B; Zhou et al., 2013).D14 displays hydrolase activity on rac-GR24 (Zhao et al., 2013a),enhancing the interaction between D14 and D53 (Jiang et al.,2013). However, rac-GR24 showed little effect on the interactionbetween D14L and OsSMAX1 (Supplemental Figure 7). Wemutated the conserved sites of the hydrolase catalytic triad inD14L (Ser-96, Asp-218, andHis-247) and found that themutatedD14L proteins still interacted with OsSMAX1 (SupplementalFigure 9). These findings are consistent with previous findingsthat rice D14L has little hydrolase activity on GR24 (Zhao et al.,2013a).To detect the interaction between D14L and D3, we performed

a pull-down assay. Nus-D14L was pulled down by glutathioneS-transferase (GST)-D3, but not by GST, suggesting that D14Lcan interact with D3 (Figure 2E). Both HA-D3 and GFP-D14Lwere transiently expressed in rice protoplasts. HA-D3 could becoimmunoprecipitated by GFP-D14L, but not by GFP(Figure 2F). These results suggested that D14L may forma complex with D3 and OsSMAX1, as D14 forms a complex withD3 and D53.

Accumulation of OsSMAX1 Determines Rice MesocotylElongation in the Dark

SL-induced D53 degradation requires the function of D14 and D3(Jiang et al., 2013; Zhou et al., 2013). Mutants deficient in SLbiosynthesis or signaling accumulate D53 and showoutgrowth oftiller buds (Jiang et al., 2013; Zhou et al., 2013). To investigate

Figure 1. (continued).

(F) Venn diagram of genes for which expression was downregulated in the mutants compared to the wild type.(G) Pathways identified by enrichment analysis of genes with upregulated expression in all indicated mutants compared with the wild type.(H) to (J) Relative expression of D14L (H), D14L2 (I), and D14L3 (J) in seedlings of mutants compared to the wild type (WT). In (C), (D), and (H–J), theexpression of each indicated gene is relative to that of ACTIN as the internal reference; data shown are from one of three replicate experiments. Data arepresented as means 6 SEs (n 5 3). Statistically significant differences were determined by one-way ANOVA; the different letters indicate significantdifferences between samples according to Tukey’s test (P < 0.05; Supplemental Data Set 5).

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whether the protein stability of OsSMAX1 requires the function ofD14L and D3 in vivo, we used OsSMAX1 antibodies to measureOsSMAX1 protein levels in the wild-type, d14, d14l, d3, and d14d14l seedlings (Supplemental Figure 10). OsSMAX1 proteinaccumulated in d14l, d3, and d14 d14l, but not in the wild type ord14 (Figure 3A). These results indicated that the regulation ofOsSMAX1 protein stability depends on the function of D14L andD3, but not on that of D14. By contrast, we found that the ac-cumulation of D53 in d14l was similar to that in the wild type(Figure 3B), suggesting that D14L has little effect on the abun-dance of D53.

In d53 plant, the lack of the Gly-Lys-Thr (GKT) motif preventsd53 from being subjected to SL-induced degradation. When theGKT motif-mutated D53 (D53m) was overexpressed in the wildtype, theshoot-branchingphenotypesweresimilar to thoseofd53(Supplemental Figures 1A and 1B; Jiang et al., 2013). Since the

GKT motif region of OsSMAX1 is highly similar to that of D53, wegenerated a mutated OsSMAX1 (OsSMAX1m) by an in-framedeletion of the GKT motif at 744 to 747 (Gly-Lys-Thr-Ala;Figure 3C), which is similar to that in D53 at 813 to 817 (Gly-Lys-Thr-Gly-Ile). Since d53 interacts with D14 as well as D53, yeasttwo-hybrid assays (Figure 3D) and pull-down assays (Figure 3E)wereconducted toverify the interactionbetweenOsSMAX1mandD14L. The results showed thatOsSMAX1m interactswithD14Laswell as OsSMAX1.To further evaluate the stability of D53/SMAXL family proteins,

we performed a StrigoQuant-like reporter transient expressionassay in rice protoplasts (Supplemental Figure 11A; Samodelovet al., 2016). To verify the viability of this assay, we first tested theeffectsof rac-GR24on thestabilityofD53andD53mreporters thatwere transiently expressed in thewild-type,d3,d14,d14l, andd14d14l protoplasts (Supplemental Figure 11B). The ratio of firefly

Figure 2. D14L Forms a Complex with OsSMAX1 and D3.

(A) D14L interacts with OsSMAX1 in a yeast two-hybrid assay.(B) In vitro pull-down assay with amylose resin. His-MBP-OsSMAX1was detected by anti-His antibodies. His-Nus-D14L and His-Nus-D14 were detectedby anti-Nus antibodies. MBP was detected by anti-MBP antibodies.(C) Co-IP of transiently expressed proteins in rice protoplasts by GFP-Trapcoupled agarose beads. HA-OsSMAX1 was detected by anti-HA antibodies.GFP-D14L and GFP-D14 were detected by anti-GFP antibodies.(D) In vitro pull-down assay by amylose resin. His-MBP-OsSMAX1 (191 to 444) was detected by anti-His antibodies. His-Nus-D14L andHis-Nus-D14weredetected by anti-Nus antibodies.(E)Pull-downassayusingglutathionemagnetic agarosebeads.GST-D3-Hiswasdetectedbyanti-His antibodies.His-Nus-D14Lweredetectedbyanti-Hisantibodies. GST was detected by anti-GST antibodies.(F) Co-IP of transiently expressed proteins in rice protoplasts using GFP-Trapcoupled agarose beads. HA-D3 was detected by anti-HA antibodies. GFP-D14L and GFP were detected by anti-GFP antibodies. The proteins indicated by red arrows were used as bait, and the proteins indicated by black arrowswere used as prey.

Karrikin and Strigolactone Repress Mesocotyl Elongation 2785

Figure 3. Accumulation of OsSMAX1 Leads to Rice Mesocotyl Elongation in the Dark.

(A) OsSMAX1 protein levels in the wild-type (WT) and mutant seedlings detected by immunoblotting with anti-OsSMAX1 polyclonal antibodies.(B) D53 protein levels in wild-type (WT) and mutant seedlings detected by immunoblotting with anti-D53 polyclonal antibodies.(C) Mutation sites of OsSMAX1m.(D) Yeast two-hybrid analysis showing that D14L interacts with both OsSMAX1and OsSMAX1m equally well.(E) In vitro pull-down assay using amylose resin. His-MBP-OsSMAX1 andHis-MBP-OsSMAX1mwere detected by anti-His antibodies. His-Nus-D14L andHis-Nus-D14weredetectedbyanti-Nusantibodies. Theprotein indicatedby the redarrowwasusedasbait, and theproteins indicatedbyblackarrowswereused as prey.(F)35S:REN-2A-OsSMAX1-FFand35S:REN-2A-OsSMAX1m-FF transiently expressed in riceprotoplasts of thewild type (WT) andmutants. TheFF:REN isthe average ratio of the bioluminescence of firefly luciferase to that of Renilla luciferase. The lowercase letters indicate samples expressing 35S:REN-2A-OsSMAX1-FF, and the uppercase letters indicate samples expressing 35S:REN-2A-OsSMAX1m-FF.(G)Seedlingsofwild-type (WT)and transgenicUbi:OsSMAX1-GFP-3XFlagandUbi:OsSMAX1m-GFP-3XFlagseedlingsgrown in thedark for7d.Thecenterseedling is shown enlarged in the top right corner, and the mesocotyl is indicated by the arrow. Bar 5 2 cm.

2786 The Plant Cell

luciferase activity toRenilla luciferase activity (FF:REN) of the D53reporter in d14, d3, and d14 d14l treated with 1mM rac-GR24washigher than that in the wild type and d14l. By contrast, there wereno obvious differences in the FF:REN of the D53m reporter inresponse to rac-GR24 treatment among the wild type, d3, d14,d14l, and d14 d14l (Supplemental Figure 11B). To evaluate thestability ofOsSMAX1andOsSMAX1m,StrigoQuant-like reportersof OsSMAX1 andOsSMAX1mwere transiently expressed in wild-type, d14, d14l, d3, and d14 d14l protoplasts. The stability ofOsSMAX1, indicated by the FF:REN, was higher in d14l, d3, andd14 d14l than in the wild type, while the stability of OsSMAX1 ind14 was similar to that in the wild type (Figure 3F). However, thestability of OsSMAX1mdid not obviously differ in d14, d14l, d3, ord14 d14l compared to that in the wild type (Figure 3F). Theseresults suggested that the stability of OsSMAX1 is regulated byD14L and D3, but not by D14, and that the mutation of the GKTmotif of OsSMAX1 enables OsSMAX1m to resist D14L- and D3-dependent degradation.

To test whether the elongation of the mesocotyls of d14l, d3,andd14d14l resulted fromtheaccumulationofOsSMAX1 in thesemutants, we constructed vectors to express OsSMAX1 andOsSMAX1m driven by the maize UBIQUITIN promoter(Ubi:OsSMAX1-GFP-3XFlag and Ubi:OsSMAX1m-GFP-3XFlag,respectively) and transformed them into rice cv Nipponbare(Figures 3G to 3I). OsSMAX1 and OsSMAX1m overexpressionpromotedmesocotyl elongation in the dark (Figures 3G and 3H).The length of the mesocotyls in the transgenic seedlings wasconsistent with the protein levels of the OsSMAX1 or OsS-MAX1m fusion proteins (Figure 3I). We further generated con-structs overexpressingOsSMAX1andOsSMAX1mdrivenby therice ACTIN promoter (ACT:OsSMAX1-Flag and ACT:OsS-MAX1m-Flag, respectively), and transformed them into Nip-ponbare. In addition, we generated an OsSMAX1 promoter-driven OsSMAX1m expression vector (OsSMAX1:-OsSMAX1m-GFP-3XFlag) and subsequently obtained trans-genic seedlings (Supplemental Figure 12A). The length of themesocotyl of the ACT:OsSMAX1-Flag seedlings was similar tothat of wild-type seedlings. The length of the mesocotyl of theACT:OsSMAX1m-Flag and OsSMAX1:OsSMAX1m-GFP-3XFlag transgenic seedlingswassignificantlygreater than thatofwild-type seedlings (Supplemental Figure 12B), which is con-sistent with the abundance of OsSMAX1m-Flag fusion protein inthe ACT:OsSMAX1m-Flag transgenic seedlings and the OsS-MAX1m-GFP-Flag fusion protein in theOsSMAX1:OsSMAX1m-GFP-3XFlag transgenic seedlings (Supplemental Figure 12C).These results indicated that OsSMAX1m is more likely resistantto ligand-induced degradation than OsSMAX1 and that the

accumulation of OsSMAX1 determines the rice mesocotyl-elongation phenotype in the dark.

Karrikin-Like Signals (KLs) Induce Ubiquitination andDegradation of OsSMAX1

To determine the role of karrikins in the inhibition of rice mes-ocotyl elongation in the dark, we added either KAR1 or KAR2 tothe medium used for seedling growth in the dark for 7 d (Figures4A to 4C). Treatment with either 20 mM KAR1 or KAR2 inhibitedthe elongation of the mesocotyls of the wild-type and d14seedlings but did not inhibit the elongation of the mesocotyls ofeither thed14lor thed3 seedlings (Figures 4Aand4B). Treatmentwith karrikins consistently resulted in decreased abundance ofOsSMAX1 in the wild type and d14 but had little effect on theabundanceofOsSMAX1 ind14landd3 (Figure4C). These resultsindicated that karrikins could induce the degradation of OsS-MAX1, which is dependent on the function of D14L and D3. Toconfirm whether karrikins induce the degradation of OsSMAX1,which depends on the function of the GKTmotif, we treated calliderived from Ubi:OsSMAX1-GFP-Flag and Ubi:OsSMAX1m-GFP-Flag transgenic seedlings with 10 mMKAR1 and found thatKAR1 had no obvious effects on the induction of OsSMAX1degradation within 2 h (Figure 4D).It has been suggested that different GR24 stereoisomers have

different effects and that the nonnatural enantiomer GR24ent-5DS

can mimic the inhibitory effects of karrikin on hypocotylelongation in Arabidopsis (Scaffidi et al., 2014). We thereforemeasured the abundance of the OsSMAX1-GFP-Flag fusionprotein in response to GR24 stereoisomer treatment. OsS-MAX1-GFP-Flag degraded upon treatment with 10 mMGR24ent-5DS, but not with 10 mM GR245DS (Figure 4D). How-ever, the abundance of OsSMAX1m was not affected bytreatment with either GR24ent-5DS or GR245DS (Figure 4D).Moreover, the results showed that GR24ent-5DS treatmentcould induce the ubiquitination of OsSMAX1, but not ofOsSMAX1m (Figure 4E). Taken together, these results in-dicated that different GR24 stereoisomers have distinct ef-fects on the induction of OsSMAX1 degradation and that themutation of theGKTmotif of OsSMAX1 is resistant to GR24ent-5DS-induced ubiquitination and degradation.To further determine whether karrikins could enhance the in-

teraction betweenD14L andOsSMAX1 in amanner similar to thatof SLs with respect to the interaction between D14 and D53, weperformed a yeast two-hybrid assay with different small mole-cules. The results showed that the addition of 20 mM rac-GR24 orGR245DScouldenhance the interactionbetweenD14andD53,but

Figure 3. (continued).

(H) Length of the mesocotyl of wild-type (WT) and transgenic Ubi:OsSMAX1-GFP-3XFlag and Ubi:OsSMAX1m-GFP-3XFlag seedlings.(I) OsSMAX1 protein levels in seedlings of the wild-type (WT) and transgenic Ubi:OsSMAX1-GFP-3XFlag and Ubi:OsSMAX1m-GFP-3XFlag seedlings asrevealed by immunoblottingwith anti-Flagmonoclonal antibodies. In (F), data are presented asmeans6 SEs (n5 3). In (H), the numbers above the columnsindicate the sample sizes. Data are presented asmeans6 SDs. Statistically significant differenceswere determinedby one-wayANOVA; thedifferent lettersindicate significant differences between samples according to Duncan’s test (P < 0.05; Supplemental Data Set 5).

Karrikin and Strigolactone Repress Mesocotyl Elongation 2787

neither rac-GR24 and GR245DS nor KAR1 and GR24ent-5DS couldenhance the interaction between D14L andOsSMAX1 (SupplementalFigure 13). The pull-down assay also revealed that addition of 20 mMrac-GR24,KAR1,GR24

5DS,orGR24ent-5DShasnoobviouseffectontheinteraction between D14L and OsSMAX1. Even when the

concentration of these chemicals was increased to 50mM, KAR1 andGR24ent-5DSstillhadnoobviouseffectonthe interactionbetweenD14Land OsSMAX1 (Supplemental Figure 14). These results indicated thattheremaybedifferentrecognitionmechanismsbetweenKLsandD14Land between SLs and D14.

Figure 4. KL Signal-Induced Degradation of OsSMAX1 Depends on the Function of D14L and D3.

(A)Dark-grown 7-d-old wild-type (WT), d14, d14l, and d3 seedlings under karrikin treatment. KAR1 and KAR2 (20 mM) were added to themedia. The centerseedling is shown enlarged in the top right corner, and the mesocotyl is indicated by the arrows. Bars 5 2 cm.(B)Lengthof themesocotyl of the indicatedseedlingsgrown in thedark for7dunderkarrikin treatment.Dataarepresentedasmeans6SDs, and thenumbersabove the columns indicate the sample sizes. Statistically significant differences were determined by one-way ANOVA; the different letters indicatesignificant differences between samples according to Duncan’s test (P < 0.05; Supplemental Data Set 5).(C)OsSMAX1protein levels in the dark-grown 7-d-oldwild-type (WT),d14,d14l, andd3 seedlings under karrikin treatment. Anti-OsSMAX1 andanti-ACTINantibodies were used for immunoblotting.(D)OsSMAX1andOsSMAX1mprotein levels in calli ofUbi:OsSMAX1-GFP-Flag andUbi:OsSMAX1m-GFP-Flag transgenic plants after chemical treatment(10 mM KAR1, GR245DS, and GR24ent-5DS) at the indicated time points. The anti-Flag and anti-ACTIN antibodies were used for immunoblotting.(E) Ubiquitination assay of OsSMAX1-GFP-Flag and OsSMAX1m-GFP-Flag in response to 10 mM GR24ent-5DS. Polyubiquitinated proteins detected bymonoclonal anti-ubiquitin antibodies. OsSMAX1-GFP-Flag, OsSMAX1m-GFP-Flag, and GFP proteins detected by anti-GFP antibodies. IP, immuno-precipitation; Ub, ubiqitin.

2788 The Plant Cell

OsSMAX1 Acts Downstream of D14L and D3 in RegulatingMesocotyl Elongation

To further dissect the role of OsSMAX1 in the regulation ofmesocotyl elongation in the dark, we generated OsSMAX1knockout plants by CRISPR/Cas9-mediated genome editing(Supplemental Figure 15) and found that loss of function ofOsSMAX1 reduced rice mesocotyl elongation in the dark com-pared to that of thewild type (Supplemental Figure 15).Ossmax1-1, which has a 4-bp deletion at site 70 to 73, was used for furtheranalysis and ishenceforth referred to asOssmax1. Theexpressionlevelsof theOsTCP5,GY1, andOsGSK2genesareconsistentwiththe mesocotyl phenotypes of Ossmax1 and OsSMAX1:OsS-MAX1m-GFP-3XFlag, referred to OsSMAX1m-overexpression(OE) hereafter (Supplemental Figures 16A to 16C). These re-sults indicated thatOsSMAX1may regulatemesocotyl elongationin the dark by regulating the expression of these genes duringmesocotyl development. Moreover, the expression of the D14L,D14L2, andD14L3 genes increased inOssmax1 but decreased inOsSMAX1m-OE (Supplemental Figures 16D to 16F). Taken to-gether, these results indicated that the D14L-D3-OsSMAX1complex might act additively in conjunction with the D14-D3-D53complex to control the expression of downstream genes, such asthe D14L, D14L2, and D14L3.

In Arabidopsis, loss of function of SMAX1 and SMXL2 sup-presses the hypocotyl-elongation phenotype of max2 (Stangaet al., 2013, 2016; Soundappan et al., 2015). To test whetherOsSMAX1 acts downstream of D14L and D3 in the regulation ofmesocotyl elongation, we obtained Ossmax1 d14l and Ossmax1d3 double mutant plants. The length of the mesocotyl of both theOssmax1 d14l and Ossmax1 d3 double mutants in the dark wassimilar to that of Ossmax1 (Figures 5A and 5B), indicating thatOsSMAX1 acts downstream of D14L and D3. Moreover, wemeasured theprotein levels ofOsSMAX1 in thewild type,d3,d14l,Ossmax1,Ossmax1d3, andOssmax1d14land found that the lossof function ofOsSMAX1 resulted in no accumulation ofOsSMAX1in d3 and d14l (Figure 5C). The transcript levels of both OsTCP5and GY1 in these mutants compared with the wild type wereconsistent with the abundance of OsSMAX1 and with themesocotyl-elongation phenotypes in these mutants (Figure 5D).These results strongly suggested that OsSMAX1 acts down-streamofD14L andD3 to inhibit mesocotyl elongation in the dark.

D3 can be recruited by D14 to degrade D53 (Jiang et al., 2013;Zhou et al., 2013) and can be recruited by D14L to degradeOsSMAX1 (Figures 2 and 4D). Since the mesocotyl phenotype ofd14 d14l is similar to that of d3, it is reasonable to expect that lossof function of OsSMAX1 may rescue the elongated-mesocotylphenotype of d14l and that the mesocotyl length of Ossmax1 d3would be similar to that of d14. Surprisingly, the loss of function ofOsSMAX1 fully rescued the elongated-mesocotyl phenotype ofd3 instead of partially rescuing the mesocotyl phenotype of d3 inthe dark (Figures 5A to 5D). Thus, wegeneratedOssmax1 d10 andOssmax1 d14 double mutants. The length of the mesocotyls ofboth Ossmax1 d10 and Ossmax1 d14 was similar to that ofOssmax1 (Figures 5E and 5F). Furthermore, the transcript levels ofboth OsTCP5 and GY1 in Ossmax1 d10 and Ossmax1 d14 weresimilar to those in Ossmax1 (Figures 5G and 5H), and the ex-pression levels of OsTCP5 and GY1 were consistent with the

observed mesocotyl-elongation phenotypes of these mutants.Together, these findings indicated that OsSMAX1 might actdownstream of D14-D3-D53 signaling in the regulation of ricemesocotyl elongation in the dark.OsSMAX1 transcript levelsweredownregulated ind14l,d3,d14

d14l, andOsSMAX1m-OE compared to those of the wild type butwere upregulated in Ossmax1 and Ossmax1 d3 (SupplementalFigure 16G), indicating thatOsSMAX1 expression was negativelyregulated by the accumulation of the OsSMAX1 protein. Sur-prisingly, OsSMAX1 transcript levels were also downregulated ind14 and D53m-OE but upregulated in Ossmax1 d14(Supplemental Figure 16G). D53 transcription is subject to feed-back regulation fromSLsignaling (Jianget al., 2013). Interestingly,we found that D53 expression levels decreased in d14l andOsSMAX1m-OE, similar to that in d14 and D53m-OE, but in-creased in Ossmax1 (Supplemental Figure 16G). Collectively,these results indicated that the D14-D3-D53 and D14L-D3-OsSMAX1 signaling complexes act interdependently to maintainthe expression levels of D53 and OsSMAX1.

OsSMAX1 Is Required for SL-Mediated Regulation ofMesocotyl Elongation, but Not for SL-Mediated Inhibition ofShoot Branching

To determine whether D14L-D3-OsSMAX1 regulates shootbranching like D14-D3-D53 does, we measured the tiller numberof thewild-type,d3,d14,d14l, andd14d14lplants (Figures6Aand6B). No significant differences in tiller number were detectedbetween the wild type and d14l, and the tiller number of d14 d14lwas similar to that of d14 and d3 (Figures 6A and 6B). Moreover,the tiller number of the Ubi:OsSMAX1-GFP-3XFlag andUbi:OsSMAX1m-GFP-3XFlag transgenic plants did not signifi-cantly differ from that of wild-type plants (Supplemental Figures17A and 17B). Consistent with these results, the tiller numbers ofthe ACT:OsSMAX1-Flag, ACT:OsSMAX1m-Flag, andOsSMAX1:OsSMAX1m-GFP-3XFlag transgenic plants were also similar tothat of the wild type (Supplemental Figures 17C and 17D). Al-though the loss of function of OsSMAX1 inhibits mesocotylelongation in the dark, comparedwith that of wild-type plants, thetiller number of OsSMAX1 knockout plants differed little(Supplemental Figures 17Eand17F). Although the loss of functionof OsSMAX1 suppresses the elongated-mesocotyl phenotype ofd3 (Figures5Aand5B), the tiller numberofOssmax1d3wassimilarto that of d3 (Figures 6A and 6B), which indicated that OsSMAX1has little effect on rice shoot branching. Furthermore, the tillernumbers ofOssmax1 d14 andOssmax1 d10were similar to thosein d14 and d10 (Figures 6A and 6B), respectively. These resultssuggested that the D14L-D3-OsSMAX1 complex, unlike the D14-D3-D53 complex, is not required to regulate shoot branchingin rice.We observed that the plant height was reduced both in the

OsSMAX1 andOsSMAX1moverexpression plants (SupplementalFigure 17G) and in the Ossmax1 plants (Figure 6C). Similarly, theplant height ofOssmax1d3,Ossmax1d14, andOssmax1d10wasalso reduced compared to that of d3, d14, and d10, respectively(Figure 6C). These results suggested that theD14L-D3-OsSMAX1complex may be involved in regulating plant height in rice.

Karrikin and Strigolactone Repress Mesocotyl Elongation 2789

Figure 5. Loss of Function of OsSMAX1 Suppresses the Elongated-Mesocotyl Phenotype of d14l and d3.

(A)Dark-grown7-d-oldseedlingsof thewild type (WT),d14l,d3,Ossmax1,Ossmax1d14l, andOssmax1d3. Thecenter seedling is shownenlarged in the topright corner, and the mesocotyl is indicated by the arrows. Bar 5 2 cm.(B) Length of the mesocotyl of dark-grown 7-d-old seedlings in (A).(C) Abundance of OsSMAX1 proteins in dark-grown 7-d-old seedlings in (A).(D) Relative expression levels of OsTCP5 and GY1 in dark-grown 7-d-old seedlings of the wild type (WT), d14l, d3, Ossmax1, Ossmax1 d14l, andOssmax1 d3.(E) Dark-grown 7-d-old wild-type (WT), d10, d14,Ossmax1,Ossmax1 d10, andOssmax1 d14 seedlings. The center seedling is shown enlarged in the topright corner, and the mesocotyl is indicated by the arrows. Bar 5 2 cm.(F) Length of the mesocotyl of dark-grown 7-d-old seedlings in (E).(G) Relative expression levels of OsTCP5 and GY1 in dark-grown 7-d-old seedlings of the wild type (WT), d10, Ossmax1, and Ossmax1 d10.(H) Relative expression levels ofOsTCP5 andGY1 in dark-grown 7-d-old seedlings of the wild type (WT), d14,Ossmax1, andOssmax1 d14. In (B) and (F),data are presented asmeans6 SDs, and the numbers above the columns indicate the sample sizes. Statistically significant differenceswere determined byone-way ANOVA; the different letters indicate significant differences between samples according to Duncan’s test (P < 0.05; Supplemental Data Set 5). In(D), (G), and (H), theexpressionofeach indicatedgene is relative to thatofACTINas the internal referenceand is replicated three times.Dataarepresentedasmeans6 SEs (n5 3). Statistically significant differences were determined by one-way ANOVA; the different letters indicate significant differences betweensamples according to Tukey’s test (P < 0.05; Supplemental Data Set 5).

2790 The Plant Cell

OsSMAX1 Interacts with TPRs to Regulate DownstreamGene Expression

D53/SMAXL family proteins are localized in the nucleus and areable to recruit TPR transcriptional corepressors through their EAR

motifs to regulate the expression of downstream genes (Causieret al., 2012; Jiang et al., 2013; Liang et al., 2016; Ma et al., 2017).Similar to D53, OsSMAX1 has an EAR motif (SupplementalFigure 18A), indicating that OsSMAX1might interact with TPRs toregulatedownstreamgeneexpression, asobserved forD53 (Jianget al., 2013; Ma et al., 2017). Interactions between OsSMAX1 andrice TPRs were detected via two-hybrid assays (Figure 7A), andthe interaction between OsSMAX1 and the N terminus of TPR2was further verified via pull-down assays (Figure 7B). ThemutatedEAR motif of OsSMAX1 disrupted the interaction between OsS-MAX1 and OsTPR2 (Supplemental Figure 18B). In addition,OsSMAX1-GFP-Flag and OsSMAX1m-GFP-Flag fusion proteinslocalized mainly to the nucleus (Figure 7C). These observationsindicated that OsSMAX1 may recruit TPR transcriptional cor-epressors to regulate the expression of downstream genes.To identify the downstream genes regulated by OsSMAX1, we

compared the gene expression profiles of thewild type,Ossmax1,andOsSMAX1m-OE and identified 84 genes potentially regulatedby OsSMAX1. Among these genes, the expression of 18 geneswas upregulated inOssmax1 and downregulated inOsSMAX1m-OE, and the expression of 66 genes was downregulated in Oss-max1 and upregulated in OsSMAX1m-OE (Figure 7D). The ex-pression profiles of these OsSMAX1 negatively regulated genesand positively regulated genes are shown, respectively, ina heatmap (Figure 7E; Supplemental Figure 19). We selectedseveral genes for RT-qPCR analysis and confirmed their ex-pression levels. The expression of LOC_Os06g49750, LOC_Os02g40240, and LOC_Os05g11414 was downregulated inOsSMAX1m-OE and upregulated in Ossmax1 (Figures 7F to 7H).LOC_Os06g49750 encodes the rice homolog of the karrikin-inducible marker gene KARRIKIN UPREGULATED F-BOX1(KUF1) in Arabidopsis (Figure 7F; Nelson et al., 2011). LOC_Os02g40240 (Figure 7G) is a light-inducible gene that encodesaplasmamembrane receptor-likekinaseofLEAFPANICLE2 (LP2;Thilmonyet al., 2009).LOC_Os05g11414encodes theMADS-boxfamily transcription factor OsMADS58, which regulates the ex-pression of photosynthesis-related genes (Figure 7I; Chen et al.,2015). By contrast, the expression of LOC_Os04g15840, LOC_Os06g32355, and LOC_Os03g64330, which encode members ofthe Expansin, Thionin, and Aquaporin family proteins, re-spectively, was upregulated in OsSMAX1m-OE and down-regulated in Ossmax1 (Figures 7I and 7J). These results providedfurther evidence that OsSMAX1 influences rice mesocotyl elon-gation in the dark through the regulation of the expression ofdownstream genes.

KL and SL Signaling Complexes Perceive Different GR24Stereoisomers to Inhibit Rice Mesocotyl Elongation inthe Dark

To determine the role of karrikin and SL in the inhibition of mes-ocotyl elongation in the dark, we added chemicals to the mediaused for seedling growth and observed the seedlings underdarkness for 7d (Supplemental Figure20).We found that1mM rac-GR24 or GR24ent-5DS is sufficient to inhibit the elongation of themesocotyl of d10, but not of the wild type, and that 10 mM rac-GR24 or GR24ent-5DS is able to significantly inhibit the elongationof themesocotyls of the wild type and d10. Treatment with 20 mM

Figure 6. D14L-D3-OsSMAX1–Mediated Signaling Pathway Does NotRegulate Shoot Branching.

(A) Morphology of wild-type (WT), d10, d14, d3, d53, d14l, Ossmax1,Ossmax1d10,Ossmax1d14,Ossmax1d3,d53d14l, andd14d14lplantsatthe heading stage.(B) Tiller number of the indicated plants at the heading stage.(C) Height of the indicated plants at the heading stage. Bars5 5 cm. Dataare presented asmeans6 SDs (n5 10). Statistically significant differenceswere determined by one-way ANOVA; the different letters indicate sig-nificant differences betweensamples according toDuncan’s test (P<0.05;Supplemental Data Set 5).

Karrikin and Strigolactone Repress Mesocotyl Elongation 2791

Figure 7. OsSMAX1 Interacts with TPRs and Regulates Downstream Gene Expression.

(A) Yeast two-hybrid assay showing that OsSMAX1 interacts with the N terminus (1 to 300) of TPRs.

2792 The Plant Cell

rac-GR24 caused more obvious inhibitory effects than didtreatment with 10 mM rac-GR24 for the wild type and d10, whiletreatment with 20 mM GR24ent-5DS caused a more obvious in-hibitory effect than did 10 mMGR24ent-5DS in thewild type, but notin d10 (Supplemental Figure 20). Treatment with 20mM rac-GR24dramatically inhibited theelongationof themesocotyls of thewild-type and d17 seedlings, slightly inhibited the elongation of themesocotyls of d14 and d14l, and had little effect on the elongationof the mesocotyls of the d3 and d14 d14l mutants. However,20mMKAR1 inhibited theelongation of themesocotyls of thewild-type, d17, and d14 seedlings but had little effect on those of thed14l, d3, and d14 d14l mutants (Figures 8A and 8B). rac-GR24treatment induced D53 expression in a D14-dependent manner,while KAR1 treatment induced OsSMAX1 expression in a D14L-dependent manner (Figures 8C and 8D). The expression of Os-KUF1 responded more specifically to KAR1 than to rac-GR24 inthe wild type and responded to KAR1 only in d17 and d14(Figure 8E). Expression of the OsSMAX1-downregulated geneLP2 was induced in response to both KAR1 and rac-GR24 in thewild type and d17 and induced in response to KAR1 in d14(Figure8F).Expressionof theOsSMAX1-upregulatedgenesLOC_Os04g15840 andLOC_Os06g32355wasnot induced in responseto KAR1 or rac-GR24 in the wild type (Figures 8G and 8H). De-ficiency of the SL or karrikin pathway led to increased expressionof these two genes, such that their expression was inhibited inresponse to both KAR1 and rac-GR24 in the d17mutants (Figures8G and 8H). The expression of LOC_Os04g15840 and LOC_Os06g32355 in d14, d14l, and d14 d14l was responsive to bothKAR1 and rac-GR24. However, rac-GR24 did not inhibit theirexpression in d14l aswell as KAR1 did in d14 (Figures 8G and 8H),which was consistent with the inhibition effect of rac-GR24 andKAR1 on the mesocotyl elongation in d14l and d14 (Figure 8B).Given that rac-GR24 is a racemic mixture of two enantiomers(GR245DS and GR24ent-5DS) and that GR24ent-5DS triggers theubiquitination and degradation of OsSMAX1, we expected rac-GR24 to be capable of activating both D14- and D14L-mediateddownstream responses in thewild typeandactivating thekarrikin-induced response in d14 and the SL-induced response in d14l,respectively. Surprisingly, seedlings treatedwith 20mM rac-GR24showedaslight inhibition in theelongationof themesocotyls in thed14 and d14l seedlings (Figures 8A to 8C; Supplemental

Figure 20). Treatment of seedlings with 40mM rac-GR24 revealeda significant inhibitory effect on the elongation of the mesocotylsof thewild typeandd14butno inhibitoryeffecton theelongationofthe mesocotyl of d14l (Supplemental Figure 20). However, whenthe wild-type, d17, d14, d14l, d3, and d14 d14l seedlings weregrown inmediaonlywith20mMGR245DSorGR24ent-5DS,GR245DS

inhibited the elongation of the mesocotyls of the wild-type, d17,and d14l seedlings, but not of the d14 seedlings (Figures 9A and9B). By contrast, GR24ent-5DS inhibited the elongation of themesocotyls of thewild-type,d17, andd14seedlings, but notof thed14l seedlings (Figures 9A and 9B). NeitherGR245DS norGR24ent-5DShadasignificant inhibitoryeffecton the lengthof themesocotylof d3 or d14 d14l (Figures 9A and 9B). Consistent with its ability toinhibit mesocotyl elongation, GR24ent-5DS treatment reduced theaccumulation of OsSMAX1 in the wild-type, d17, and d14seedlingsbuthad little effecton theabundanceofOsSMAX1 in thed14l and d14 d14l seedlings (Figures 9C and 9D). Taken together,these results showed that D14L responds specifically to GR24ent-5DS in the regulation of mesocotyl development.GR245DS induced D53 expression in the wild-type, d17, and

d14l seedlings, but not in d14, d14 d14l, and d3 seedlings(Figure 9E). By contrast, GR24ent-5DS induced OsSMAX1 ex-pression in the d17 and d14 seedlings, but not in the wild-type,d14l, d14 d14l, and d3 seedlings (Figure 9F). RT-qPCR analysisshowed that LP2 is responsive to GR24ent-5DS or GR245DS ina D14- or D14L-dependent manner, respectively (Figure 9G). TheOsKUF1 seems responsive specifically to GR24ent-5DS in a D14L-andD3-dependentmanner. However,OsKUF1 is also responsiveGR245DS in d17, but not in d14l (Figure 9H). In addition, bothLOC_Os04g15840 and LOC_Os06g32355 are responsive toGR245DS or GR24ent-5DS in a D14- or D14L-dependent manner,respectively (SupplementalFigure21).Our resultsshowedthat theOsSMAX1-regulated genes either respond specifically toGR24ent-5DS or respond to both GR245DS and GR24ent-5DS. Theresponse to GR245DS depends on the function of D14 and D3,while the response to GR24ent-5DS depends on the function ofD14L and D3. These results demonstrated that the nonnaturalenantiomer GR24ent-5DSmaymimic the inhibitory effect of karrikinon mesocotyl elongation in the dark, as it did with the inhibition ofArabidopsis hypocotyl elongation under light conditions. Thesefindings suggested that D14-D3-D53 and D14L-D3-OsSMAX1

Figure 7. (continued).

(B) In vitro pull-down assay by amylose resin. His-MBP-OsSMAX1 was detected by anti-His antibodies. TPR2(1-600)-His was detected by anti-Hisantibodies.MBPwasdetectedbyanti-MBPantibodies. Theproteins usedasbait are indicatedby redarrows, and theproteins usedaspreyare indicatedbyblack arrows.(C)Subcellular localization ofOsSMAX1-GFP-FlagandOsSMAX1m-GFP-Flag fusionproteins. The images show the roots ofUbi:OsSMAX1-GFP-Flag andUbi:OsSMAX1m-GFP-Flag transgenic plants. Bars 5 100 mm. DIC, differential interference contrast; PI, propidium iodide.(D)Venn diagram of differentially regulated genes inOssmax1 andOsSMAX1:OsSMAX1m-GFP-Flag transgenic plants compared towild-type (WT) plants.(E)Heatmapof the expression fold change of upregulated genes inOssmax1mutant plants anddownregulated genes inOsSMAX1:OsSMAX1m-GFP-Flag(OsSMAX1m-OE) transgenic plants compared to wild-type (WT) plants.(F) to (H)Relativeexpression levelsof the indicatedOsSMAX1repressedgenes,LOC_Os06g49750 (F),LOC_Os02g40240 (G), andLOC_Os05g11414 (H), indifferent mutant and transgenic plants.(I) to (K)Relative expression levels of the indicatedOsSMAX1-activated genes, LOC_Os04g15840 (I), LOC_Os06g4032355 (J), and LOC_Os03g64330 (K),in differentmutant and transgenic plants compared towild-type (WT) plants. In (F) to (K), the expression of each indicatedgene is relative to that ofACTIN asthe internal reference. Data are presented as means6 SEs (n5 3). Statistically significant differences were determined by one-way ANOVA; the differentletters indicate significant differences between samples according to Tukey’s test (P < 0.05; Supplemental Data Set 5).

Karrikin and Strigolactone Repress Mesocotyl Elongation 2793

Figure 8. Mesocotyl Elongation in the Dark in Response to Both SL and Karrikin Signals.

(A) Dark-grown7-d-oldwild-type (WT),d17,d14,d14l,d14d14l, andd3seedlingswith orwithout treatmentwith the indicated chemical at 20mM.Thecenterseedling is shown enlarged in the top right corner, and the mesocotyl is indicated by the arrows. Bars 5 2 cm.(B) Length of the mesocotyls of seedlings shown in (A). WT, wild type.(C) Relative expression levels of D53 in response to chemical treatment. WT, wild type.(D) Relative expression levels of OsSMAX1 in response to chemical treatment. WT, wild type.(E) Relative expression levels of the OsSMAX1-repressed gene KUF1 (LOC_Os06g49750) in response to chemical treatment. WT, wild type.(F) Relative expression levels of the OsSMAX1-repressed gene LP2 (LOC_Os02g40240) in response to chemical treatment. WT, wild type.(G) Relative expression levels of the OsSMAX1-enhanced gene LOC_Os04g15840 in response to chemical treatment. WT, wild type.(H)Relativeexpression levelsof theOsSMAX1-enhancedgeneLOC_Os06g32355 in response tochemical treatment. In (B), dataarepresentedasmeans6SDs, and the numbers above the columns indicate the sample sizes. Statistically significant differences were determined by one-way ANOVA; the differentletters indicate significant differencesbetween samples according toDuncan’s test (P <0.05; Supplemental DataSet 5). In (C) to (H), the expressionof eachindicated gene is relative to that ofACTIN as the internal reference. The expression values are scaled to the expression levels in themock-treated wild type(WT). Data are presented as means 6 SEs (n 5 3). Statistically significant differences were determined by one-way ANOVA; the different letters indicatesignificant differences between samples according to Tukey’s test (P < 0.05; Supplemental Data Set 5).

2794 The Plant Cell

Figure 9. SL and Karrikin Signaling Complexes Selectively Perceive GR24 Stereoisomers in the Inhibition of Mesocotyl Elongation in the Dark.

(A)Dark-grown 7-d-old wild-type (WT), d17, d14, d14l, d14 d14l, and d3 seedlings with or without treatment of the indicated chemical at 20 mM. The centerseedling is shown enlarged in the top right corner, and the mesocotyl is indicated by the arrows. Bars 5 2 cm.(B) Length of themesocotyls of seedlings shown in (A). Data are presented asmeans6 SDs, and the numbers above the columns indicate the sample sizes.Statistically significant differenceswere determined byone-wayANOVA; the different letters indicate significant differences between samples according toDuncan’s test (P < 0.05; Supplemental Data Set 5). WT, wild type.(C) OsSMAX1 protein levels in the dark-grown 7-d-old wild-type (WT), d14, and d14l seedlings treated with GR24 stereoisomers.

Karrikin and Strigolactone Repress Mesocotyl Elongation 2795

specifically recognize different GR24 stereoisomers and regulatespecific and common downstream target genes during ricemesocotyl elongation in the dark.

DISCUSSION

Comparative approaches to the study of karrikin signaling and SLsignaling have provided crucial information about both pathways,with progress in one helping to inform the other (Waters et al.,2017). KAR2 and rac-GR24 inhibit hypocotyl elongation in the lightinArabidopsis (Nelsonet al., 2011;Waters et al., 2012). In addition,in Arabidopsis, a previous promoter-swapping analysis of KAI2and D14 revealed that their distinct roles in plant growth are likelydue to the formation of distinct signaling complexes (Waters et al.,2015b). It has been reported that SMAX1 and SMXL6/SMXL7/SMXL8 proteins can suppress distinct or overlapping phenotypesof max2 (Soundappan et al., 2015). AtD14, MAX2, and SMXL6/SMXL7/SMXL8 are responsible for SL responses in the regulationof shoot branching (Soundappan et al., 2015; Wang et al., 2015).Genetic analysis revealed that SMAX1 and SMXL2 act down-streamofMAX2 andKAI2 in karrikin signaling (Stanga et al., 2013,2016; Villaécija-Aguilar et al., 2019). Both SMAX1 and SMXL2 canbedegraded in response tokarrikinsignaling inaKAI2-andMAX2-dependentmanner inArabidopsis (Khosla et al., 2020;Wanget al.,2020). The specificity of the karrikin or SL signaling pathway isdetermined by the perception of ligands by the karrikin or SLreceptor (KAI2 or AtD14, respectively), each of which can recruitSCFMAX2, leading to a decrease in the stability of different sub-groups of D53/SMAXL proteins (Soundappan et al., 2015; Wanget al., 2015, 2020; Liang et al., 2016; Khosla et al., 2020). Previouswork in rice revealed thatD14 andD14Lplay distinct roles in shootbranching andAMsymbiosis, bothofwhich require the function ofD3 (Arite et al., 2009; Yoshida et al., 2012; Jiang et al., 2013; Zhouet al., 2013; Gutjahr et al., 2015b). D53 has been revealed to actdownstream of D14 and D3 and regulate the shoot branching ofrice (Jiang et al., 2013; Zhou et al., 2013). By contrast, OsSMAX1acts downstream of D14L and D3 to regulate root colonization byAM fungi and the elongation of the mesocotyl in rice (Choi et al.,2020). In this study, we show that OsSMAX1 acts downstream ofD14L and D3 and parallel to D14-D3-D53 in regulation the elon-gation of the mesocotyl. We further demonstrate that D14 andD14L specifically recognize different GR24 stereoisomers, butboth are required for the function of D3 to regulate the accumu-lation of specificmembers of the D53/SMAXL family protein in theregulation of downstreamevents. In addition, we determined that,similar to AtKUF1 in Arabidopsis, OsKUF1 in rice is regulated

specifically by KL signals (Figures 8C and 9E). These results in-dicate that the karrikin signaling pathway is relatively conserved indicotyledonous and monocotyledonous plants.Here, we proposed a working model for the interaction of

karrikin signaling in rice (Figure 10). In the absence of a KL ligand,the activity of unidentified OsSMAX1-interacting transcriptionfactors is repressed by the OsSMAX1-TPR complex. In thepresenceof aKLsignal,D14Lperceives the ligandand recruits theSCFD3 complex to polyubiquitinate OsSMAX1, which is sub-sequently degraded by the 26S proteasome. The degradation ofOsSMAX1 leads to the release of OsSMAX1-interacting tran-scription factors from the TPR transcriptional corepressor andalters the expression of its downstream target genes. Our resultssuggested that the specificity of karrikin andSL signaling could bedetermined by the degradation of distinct D53/SMAXL proteins inresponse to the recognition of specific ligands by their receptors.Thus, SL and KL signals act independently to trigger the ex-pression of specific downstream genes that distinguish D14L-dependent processes (AM symbiosis) and D14-dependent pro-cesses (shoot branching) mediated by transcription factors thatinteract specifically with D53 and OsSMAX1, respectively. Dis-ruption of either theSLor karrikin pathway alters the expression ofcommondownstream target genes, such asD14L2 andD14L3. Insome cases, the disruption of both the SL and karrikin pathwaysresulted in additive changes in the expression levels of down-streamgenes, such as LOC_Os01g41310 and LOC_Os03g52080(SupplementalDataSet3). TheGR24stereoisomersGR245DSandGR24ent-5DS could mimic SL-specific and KL-specific signals andact through D14 and D14L to degrade D53 and OsSMAX1, re-spectively, leading to the release of activity of common tran-scription factors from repressionmediated by TPR transcriptionalcorepressors, which resulted in changes in the expression ofcommon target genes and inhibited rice mesocotyl elongation inthe dark.Our work on OsSMAX1 and the work on SMXL2 revealed that

downstream genes may be subject to regulation by both the SLsignaling pathway and the karrikin pathway (Wang et al., 2020).These findings may help to explain why it is difficult to investigatethe expression of downstream gene in response to rac-GR24treatment. We showed that the karrikin pathway is not involved inthe regulation of the branching of rice shoots (Figure 6). Bycontrast, the SL-mediated regulation of mesocotyl elongationrequires the function ofOsSMAX1 (Figure 5). The transcript levelsof D53 andOsSMAX1 were revealed to be controlled by negativefeedback regulation (Supplemental Figure 16G). In addition, theexpression of D53 and OsSMAX1 could respond to both SL and

Figure 9. (continued).

(D) OsSMAX1 protein levels in the dark-grown 7-d-old (WT), d17, and d14 d14l seedlings treated with GR24 stereoisomers. The anti-OsSMAX1 and anti-ACTIN antibodies were used for immunoblotting in (C) and (D).(E) Relative expression levels of D53 in response to GR24 stereoisomer treatment. WT, wild type.(F) Relative expression levels of OsSMAX1 in response to GR24 stereoisomer treatment. WT, wild type.(G) Relative expression levels of LP2 (LOC_Os02g40240) in response to GR24 stereoisomer treatment. WT, wild type.(H)Relative expression levels ofKUF1 (LOC_Os06g49750) in response toGR24stereoisomer treatment. In (E) to (H), the expressionof each indicated geneis relative to that ofACTIN as the internal reference. The expression values are scaled to the expression level in themock-treatedwild type (WT). All data arepresented as means 6 SEs (n 5 3). Statistically significant differences were determined by one-way ANOVA; the different letters indicate significantdifferences between samples according to Tukey’s test (P < 0.05; Supplemental Data Set 5).

2796 The Plant Cell

KLsignals in aD14- andD14L-dependentmanner (Figures 9Eand9F). These results indicated a possible mechanism of crosstalkbetween theSLpathwayandKLpathway.Notably, theexpressionof aMAX1homolog in rice (Os01g0700900),which is referred to asboth LOC_Os01g50520 and LOC01g50530 (Cardoso et al., 2014;Zhang et al., 2014), was downregulated in the d14, d14l, d14 d14l,and d3 mutants as well as in D53m-OE and OsSMAX1m-OEtransgenic seedlings but was upregulated in the Ossmax1, Oss-max1 d14, and Ossmax1 d3 seedlings (Figure 7E). Therefore,OsSMAX1 negatively regulates SL biosynthesis, suggesting analternative mechanism of the crosstalk between the SL pathwayand KL pathway (Choi et al., 2020).

The stereospecificity of SL analogs is crucial for their recog-nition and for the inductionof downstreamactivities (Scaffidi et al.,2014). Although the protein structures of both D14 andD14L havebeen resolved, it remains unclear how their stereospecific

recognition of substrates is achieved. In SL signaling, ligandbinding or hydrolysis-induced conformational changes of re-ceptor is essential for the interactions between receptor and itsdownstream signaling partners (Yao et al., 2016; Shabek et al.,2018; Burger et al., 2019). Previous reports have indicated thatD14/AtD14 require an intact catalytic triad for signal perceptionand interaction with D3/MAX2 (Hamiaux et al., 2012; Jiang et al.,2013; Kagiyama et al., 2013; Zhao et al., 2013a; Waters et al.,2015b; de Saint Germain et al., 2016; Yao et al., 2016; Shabeket al., 2018; Xu et al., 2018). However, when the catalytic triad ofD14L was mutated, there was no significant change in the in-teraction of D14L with OsSMAX1 (Supplemental Figure 9). InArabidopsis, bothAtD14andKAI2hydrolyze rac-GR24 (Zhaoetal., 2013a; Toh et al., 2014). However, rice D14, but not D14L, hy-drolyzes rac-GR24 (Zhao et al., 2013a). In addition, AtD14 isdegraded in response to SL in a MAX2-dependent manner

Figure 10. KL Signaling Pathway Mirrors the SL Signaling Pathway.

A working model of karrikin signaling mediated by the D14L-D3-OsSMAX1 complex. Karrikin signaling mirrors the SL signaling complex in rice. In theabsence of ligands, bothOsSMAX1 andD53 are able to interact with TPR transcriptional corepressors and repress the expression of downstreamgenes. Inthe presence of ligands, D14L and D14 perceive specific ligands (such as GR24ent-5DS and GR245DS) and recruit the SCFD3 complex to ubiquitinateOsSMAX1andD53 for degradation by the 26Sproteasome. In turn, this releasesOsSMAX1- andD53-mediated repression of the activity of their interactingtranscription factors, thus regulating the expression of downstream target genes. SL signals specifically regulate shoot branching, and KL signals mightspecifically regulate root colonization byAM fungi. The specificity of the output of SL signals andKL signals is likely determined by transcription factors thatinteract specifically with D53 or OsSMAX1. It is possible that some common transcription factors both interact with D53 and are responsible for theexpression of a subset of common genes, which could respond to both KL signaling and SL signaling. During skotomorphogenesis, KL and SL signals actthrough the D14L-D3-OsSMAX1 complex and D14-D3-D53, respectively, and act in parallel and/or additively to trigger the expression of their specific orcommonly regulated downstream genes, which leads to the inhibition of rice mesocotyl elongation.

Karrikin and Strigolactone Repress Mesocotyl Elongation 2797

(Chevalier et al., 2014), whereas KAI2 is degrade in response tokarrikin in a MAX2-independent manner (Waters et al., 2015a).Surprisingly, recent work indicated that the turnover of SMAX1could be mediated by KAI2 and MAX2 and may be subjected toa MAX2-independent regulation (Khosla et al., 2020). These re-sults suggest that the recognition of ligands might differ betweenD14andD14L. It is possible thatKAR1orGR24ent-5DSmight not bethe direct ligands perceived by D14L, which is stimulated by anunidentified endogenous KL signal. In this scenario, KAR1 orGR24ent-5DS would exhibit activity in the in planta assay, but not inthe in vitro assays.

Given that GR24ent-5DS is a nonnatural compound that canmimic karrikin activity and that carlactone requires MAX1-dependent activity for repressing shoot branching and limitedactivity for inhibiting hypocotyl elongation (Scaffidi et al., 2013), ithas been proposed that endogenous KL molecules from a car-lactone-independent pathway may be responsible for the regu-lation of seedling development (Conn and Nelson, 2016; Burgeret al., 2019). KAI2 and AtD14 homologs are present throughoutseed plants (Bythell-Douglas et al., 2017), and SMXL proteins canbe traced back tomoss (Bennett and Leyser, 2014). The evolutionof distinct ligand recognition by KAI2 and AtD14 homologs in-dicated that, compared with the AtD14 signaling pathway, theKAI2-mediated signaling pathway is more ancient (Bythell-Douglas et al., 2017). KAR1 and GR24ent-5DS were found to berecognized by different groups of KAI2-like proteins in moss(Burger et al., 2019). Diversification of KAI2 proteins in Brassicatournefortii conferred differential responses to karrikins (Sunet al.,2020). SL perception through KAI2 homologs occurs in parasiticplants of theOrobanchaceae family,which seems tohaveevolvedrelatively more recently (Conn et al., 2015; Toh et al., 2015;Tsuchiya et al., 2015; Xu et al., 2018). When a KAI2 homolog fromSelaginella moellendorffii was expressed in Arabidopsis kai2, itcouldcomplement theseedlingand leafdevelopmentphenotypesof kai2, but the plants were not responsive to SL or karrikintreatment (Waters et al., 2015b). SMXL2 can be degraded in re-sponse to both karrikin signaling and SL signaling (Wang et al.,2020). When ShHTL receptors from Striga were expressed inArabidopsis, the seeds could germinate without the requirementof gibberellin in aMAX2- andSMAX1-dependentmanner (Bunsicket al., 2020). These results support that coevolution ofreceptor–target pairswas involved in the establishment of distinctsignaling pathways for SL and karrikin, which have been selectedin adaptive evolution of land plants (Waters et al., 2017).

Identifying the endogenous KL signal that is recognized byD14L, resolving the structure of the D14-D3-D53 complex,identifying the transcriptional factors that interact with D53/SMXLproteins, and characterizing the D14L-D3-OsSMAX1 complex ofdifferent specieswill provide insight into themolecularmechanismunderlying how these signals are perceived and passed todownstream signaling components and into how stereospecificrecognition of substrates is achieved and evolves. It has beensuggested that D14L acts in parallel with the OsCERK1-mediatedcommon symbiosis signaling pathway (CSSP) in the regulation ofearly colonization events of AM fungus–plant symbiosis (Gutjahret al., 2015b; Chiu et al., 2018). However, the genes involved inCSSP downstream ofOsCERK1 were found to be upregulated inOssmax1 (Choi et al., 2020). It seems that the karrikin signaling

pathway plays an essential role in coordinating with CSSP and SLsignaling during AM symbiosis. To reveal how karrikin signalingmediates the communication between plants and AM fungi re-quires intensive further investigation. The signaling paradigm ofkarrikin strongly suggests that some unidentified chemicals playroles in AM symbiosis (Choi et al., 2020). The recent discovery ofzaxinonesynthase (Wangetal., 2019) andb-cyclocitral (Dickinsonet al., 2019) and their roles in rice root development and stressadaptation indicated that apocarotenoid-derived smallmoleculesmight be candidates for the KLmolecule that regulatesmesocotyldevelopment and/or symbiosis between plant and AM fungi.In rice, coleoptile and mesocotyl elongation protects the shoot

apical meristem during seedling emergence from the soil. Themesocotyl immediately stops expanding upon exposure to light,while the coleoptile still grows even when mesocotyl elongationceases (Takahashi, 1972). The relative lengthof themesocotyl andcoleoptile are affected by developmental and environmentalfactors (Huet al., 2010), whichcould beusedas amodel system toinvestigate the molecular mechanism underlying crosstalk be-tween developmental and environmental factors in monocots.Elongation of the mesocotyl and coleoptile has been observed inthe light-signaling-deficient mutants phyA and cpm1 (Takanoet al., 2001;Biswas et al., 2003;Hagaet al., 2005) and JA-deficientmutants hebiba and cpm2 (Riemann et al., 2003, 2013). SL andkarrikin negatively regulate rice mesocotyl elongation in the dark.In Arabidopsis, SL inhibition of hypocotyl elongation depends onboth cryptochrome and phytochrome signaling (Jia et al., 2014).Notably, KAI2 is required for normal seedling photomorphogen-esis but does not affect seedling morphology in darkness, sug-gesting that the regulation of seedling development mediated bykarrikin signaling may act downstream of light signaling.The cell length of the lower parts of the mesocotyls of d10 and

d14 is similar to that of the wild type, while the cells of the lowerparts of themesocotyl ofd3are shorter than thoseof thewild type.However, there are more cells in the mesocotyls of d10 and d14than in those of the wild type, and there are more cells in themesocotyl ofd3 than ind10andd14 (Huet al., 2010). These resultsindicated that the SL-mediated inhibition ofmesocotyl elongationoccurs via negative regulation of cell division. SL has been sug-gested to be involved in crosstalk with cytokinin and BRs toregulate cell division in the mesocotyl (Sun et al., 2018). Since themesocotyl length in d14 d14l is similar to that in d3, disruption ofboth the SL and karrikin pathways resulted in downregulation ofthe expression of OsTCP5 and GY1, and loss of OsSMAX1function reversed the downregulated expression of OsTCP5 andGY1 as well as the elongated-mesocotyl phenotypes of d10, d14,d14l, and d3 (Figure 5). It is possible that karrikin may controlmesocotyl development by negatively controlling the action ofcytokinin aswell as JA to regulate cell division and cell elongation.It hasbeen reported that JAbiosynthesis in seedlings is induced

by red light (Haga and Iino, 2004) and that exogenous applicationof JA can suppress the elongation of the mesocotyls of dark-grown dmutants (Hu et al., 2010). The facts that karrikin signalingregulates the expression of light-responsive genes (such as LP2)and that JAsynthesis genes canbe inducedby red light (Haga andIino, 2004) in rice seedlingsmayhelp explainwhy the elongationofthe mesocotyls of the SL- and KL-deficient mutants was notobservedunder lightconditions.Previousworksuggested that the

2798 The Plant Cell

elongation of the mesocotyls of hebiba was enhanced underdarkness as well as under red light because of deficiency in JAbiosynthesis (Riemann et al., 2003). However, the large fragmentdeletion found in hebiba included not only a JA biosynthesis genebut also D14L (Riemann et al., 2013; Gutjahr et al., 2015b). Thedefective colonization by AM fungi in hebiba is determined by thefunction of D14L and not by the deficiency in JA biosynthesis(Gutjahr et al., 2015a, 2015b). Thus, further investigation of theinteraction between karrikin signaling and light signaling aswell asthe role of JA in mesocotyl development may help elucidate themolecular mechanism underlying crosstalk between karrikinsignaling and light signaling as well as other phytohormone sig-naling pathways in plants.

Because of limited water resources and labor costs, there hasbeen a trend toward rice cultivation by direct seeding instead oftransplanting in rice-growing areas in recent years (Feng et al.,2017). Breeding elite rice varieties suitable for direct seeding re-quires the improvement of various early developmental traits,including fast and uniform seed germination, high seedling vigor,early tillering capability, strong root growth, and lodging re-sistance. Recent identification of natural variation in GY1 (Xionget al., 2017) andOsGSK2 (Sun et al., 2018) highlights the practicalapplication of beneficial alleles of phytohormone-related genesthat regulate mesocotyl growth. Our work has indicated thatkarrikin signaling andSLsignalingmayact upstreamof cytokinin-,JA-, andBR-mediated pathways in the regulation of belowgrounddevelopment of rice seedlings (Supplemental Figures16A to16C).Improving our understanding of the molecular mechanismthrough which karrikin and SL regulate mesocotyl developmentand mining the natural variation in karrikin signaling and SL sig-naling components as well as OsSMAX1-regulated downstreamgeneswill contribute to the development of new elite rice varietiessuitable for direct seeding.

METHODS

Plant Materials and Growth Conditions

Plants were grown in paddy fields at the experimental stations of theChinaNational Rice Research Institute at Fuyang, Zhejiang Province, in thesummer or at Lingshui, Hainan Province in the winter. Oryza sativa cvNipponbare, mutants (d10, d17, d14, d3, and d53), and the pACT:D53m-GFP transgenic plants in the Nipponbare genetic background were thesame as previously described by Jiang et al. (2013). d14l and Ossmax1mutants in the Nipponbare genetic background were generated viaCRISPR/Cas9genomeediting (Song et al., 2017). Themutation sites of thed14l and Ossmax1mutants are shown in Supplemental Figures 3 and 15,respectively. d14 d14l, d53 d14l, Ossmax1 d14, Ossmax1 d14l, Ossmax1d3, and Ossmax1 d10 double mutants were generated by crossing. Plantheight and tiller number were recorded at the heading stage.

Mesocotyl Length Measurements

For phenotype determination, unshelled rice seeds were sterilized withsodium hypochlorite for 1 h and then germinated for 20 h at 30°C. Uniformseedswere selected, placedonsolidmediumconsistingof 0.9% (w/v) agar(Sigma-Aldrich), and then grown in the dark at 30°C for 7 d. For seedlingsgrown in the light, the growth conditions consisted of 30°C with a 16-h-light/8-h-dark cycle viafluorescent lampswitha light intensity of 75mEm22

s21. For chemical treatment, seedlings were grown on 0.9% (w/v) agar

plates with the indicated concentrations of chemicals (KAR1, rac-GR24,GR245DS, and GR24ent-5DS). Except where specified, at least 20 seedlingswere used for mesocotyl length measurements that were performedmanually or via ImageJ.

Plasmid Construction

For yeast two-hybrid analyses, the coding DNA sequences (CDSs) ofOsSMAX1,D14, andD53were amplifiedandcloned intobothpGBKT7andpGADT7betweenEcoRI andBamHI. To construct themutatedOsSMAX1-binding domain with the GKT motif deletion (Figure 3C) or EAR mutation(Supplemental Figure 18A), two fragments of each indicated OsSMAX1mutationwere individually amplifiedand thencloned together intopGBKT7via an In-Fusion HD cloning kit (Clontech). The CDSs of D53L, DL1, DL2,DL3, DL4, DL5, and DL6 were subsequently amplified and cloned intopGBKT7. The different truncated CDSs of OsSMAX1 and D53 were am-plified using the indicated primers and then cloned into pGBKT7, and thecoding regions ofD14L,D14L2, andD14L3were amplified and cloned intopGADT7. To construct different site-specificmutants at potential catalytictriad sites of D14L, two fragments of a given mutated D14L were in-dividually amplifiedand thencloned together into pGADT7via an In-FusionHD cloning kit. To construct TPRs-activation domain (AD), TPR1(1-300)-AD, TPR2(1-300)-AD, and TPR3(1-300)-AD, theDNA sequences encodingthe full-length CDS of TPR2 and the 1 to 300 N-terminal amino acid res-idues of eachTPRwere amplifiedandcloned into pGADT7via an In-FusionHD cloning kit.

Toconstructvectors forexpressing recombinantproteins inEscherichiacoli, the full-length, truncated, or mutated CDSs of the genes of interestwere amplified using the primers listed in Supplemental Table 1 and thencloned into pDONR221 via BP reactions (Invitrogen) to generate entryvectors, followed by LR reactions (Invitrogen) with pET-55-DEST, pET-57-DEST, pET-60-DEST, and pDEST-His-Maltose binding protein (MBP)destination vectors. TPR2(1-600) was cloned into a pET-55-DEST desti-nation vector, D14 and D14L were cloned into a pET-57-DEST destinationvector,D3wascloned intoapET-60-DESTdestinationvector, andD53andOsSMAX1 were cloned into a pDEST-His-MBP destination vector. E. colistrain BL21 was used for expression vectors and for purification of taggedproteins. For coimmunoprecipitation (Co-IP) transient expression vectors,pBeacon-EGFP and pBeacon-HA were used to transiently express pro-teins with the respective recombinant tags in rice protoplasts (Wang et al.,2015). The genes were cloned into pDONR221 by BP reactions to createentry plasmids, followed by LR reactions with appropriate transient ex-pression vectors.

Togenerate aPUC57-StrigoQuant-like backbone vector containing thecauliflower mosaic virus-35S promoter, Renilla luciferase-2A-firefly lucif-erase coding sequences, and the nopaline synthase terminator, the cor-responding elements were synthesized by GENEWIZ. Multiple cloningsiteswere located between the 2A and firefly luciferase sequences. For theproteins of interest, the full-length CDSs of the corresponding genes wereamplified and cloned into PUC57-StrigoQuant using an In-Fusion HDcloning kit through the SpeI and HpaI double digestion sites. All primersused for vector construction are listed in Supplemental Table 1.

Plant Transformation

The oligos corresponding to the guide RNA sequences of D14L andOsSMAX1 were synthesized and cloned into the CRISPR/Cas9 genomeediting vector as previously described by Song et al. (2017). A pCAM-BIA1300-pUbi-GFP-3XFlag binary vector was derived from pCAM-BIA1300 andwas generated by inserting themaize Ubi promoter, nopalinesynthase terminator, andGFP-33FlagCDSusing an In-FusionHD cloningkit between KpnI and HindIII. The BamHI and PmlI sites were introducedbetween Ubi promoter and GFP-33Flag CDS. To construct

Karrikin and Strigolactone Repress Mesocotyl Elongation 2799

pUbi:OsSMAX1-GFP-3XFlag and pUbi:OsSMAX1m-GFP-3XFlag over-expression vectors, the OsSMAX1 and OsSMAX1m coding regions wereamplified and then cloned into pCAMBIA1300-pUbi-GFP-3XFlag betweenBamHI and PmlI using an In-Fusion HD cloning kit. For pOsSMAX1:-OsSMAX1m-GFP-Flag, theOsSMAX1promoterwasamplifiedandused toreplace the pUbi promoter betweenKpnI andBamHI. For pAct:OsSMAX1-Flag and pAct:OsSMAX1m-Flag, the tag-fused OsSMAX1 and OsS-MAX1mcoding regionswereamplifiedanddigestedbyApaIandSpeI, afterwhich they were ligated into an AHLG vector (Actin promoter: HA-linker-GFP-Nos terminator expression cassette in pCAMBIA 1300). All over-expressionplasmidswereconfirmedbysequencing (SangonBiotech). Thebinary vectors for D14L and OsSMAX1 knockout or for overexpression ofOsSMAX1 and OsSMAX1m were transformed into Nipponbare by theAgrobacterium-mediated transgenic method (Hiei et al., 1994). All primersused for vector construction are listed in Supplemental Table 1.

Yeast Two-Hybrid Assays

To detect protein interactions, plasmids were transformed into Y2H-GoldYeast (Clontech) cells using a lithium acetate and polyethylene glycol–mediated protocol. After incubation for 2 d at 28°C in synthetic defined(SD)/-Leu-Trp medium (Clontech), the yeast clones were transferred toSD/-Leu-Trp-His-Ade medium (Clontech) with or without chemical treat-ment. To assay the effects of rac-GR24, GR245DS, GR24ent-5DS, and KAR1

on the interaction of OsSMAX1 and D14L, the yeasts cotransformed withOsSMAX1-binding domain and D14L-AD were diluted to different con-centrations and grown for 60 h on SD/-Leu-Trp-His-Ade medium with theindicated chemicals at 20 mM. rac-GR24 and KAR1 were obtained fromChiralix (Nijmegen), and the GR24 stereoisomers GR245DS and GR24ent-5DS were prepared as described by Wang et al. (2020).

Protein Expression and Purification

E. coli strain BL21 (CWBIO) containing the fusion protein expressionplasmids was grown in Luria-Bertani broth with the corresponding anti-biotics to an OD600 of 0.6 to 0.8 and then continuously cultured at 28°Covernight together with 0.4mM isopropyl-b-D-thiogalactopyranoside. Thecollected cells were lysed using a JN-02C low-temperature high-pressurebiomixer (JNBIO) and then centrifuged at 8000g for 15 min. The super-natants were subsequently collected for recombinant protein purification.For GST-tagged recombinant protein purification, the supernatants andglutathione magnetic agarose beads (BeaverBeads GSH; Beaver) wereincubated together for 3 h. After washing three times with 13 PBS, therecombinant protein eluent was removed from the beads by competitivebinding with 20 mM reduced glutathione. For MBP-tagged recombinantprotein purification, the supernatants were incubated with amylose resin(BioLabs) for 3hand thenwashed three timeswithwashbuffer (20mMTris-HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA, and 10 mM 2-mercaptoethanol),after which the recombinant protein eluent from amylose resin was ob-tained by competitive binding with 10 mM maltose. The His-MBP-OsS-MAX1 protein was concentrated using Amicon Ultra-4 centrifugal filters(Ultracel-100K; Millipore). For His-tagged protein purification, the super-natantswere incubatedwithNiSepharose6FastFlow (GEHealthcare) for 3h. After washing three timeswith PBS, the recombinant protein eluent fromSepharose waswashedwith a gradient of different concentrations (10, 20,50, and 250 mM) of imidazole in elution buffer consisting of 50 mM NaH2

PO4, pH 7.8, 300 mM NaCl, and 0.2% (v/v) Tween 20. All steps wereperformed at 4°C or on ice.

Pull-Down Assays

To detect protein interactions, His-tagged proteins and GST-taggedproteins (3 mg) or GST (6 mg) were incubated together at 4°C in a 700-

mL incubation buffer (13 protease inhibitor cocktail [Roche], MG132[Millipore], and 0.5% [v/v] Triton X-100 [Sigma-Aldrich] in PBS). After in-cubation for 1 h, glutathionemagnetic agarose beads (BeaverBeads GSH;50 mL) were added, followed by incubation at 4°C for 2 h. The beads werethen washed three times with wash buffer (0.1% Triton X-100 in PBS) andthen eluted with 30 mL of SDS-PAGE sample buffer for SDS-PAGE andimmunoblotting; Nus protein (6 mg) was used as a negative control. Todetect the interaction between His-MBP–tagged proteins and His-Nus–tagged proteins, 2 mg of His-MBP–tagged recombinant proteinswas bound to amylose resin, after which it and 4 mg of His-Nus–taggedproteinswere incubated together at 4°C in 700mL of binding buffer (50mMTris-HCl, pH 8.0, 150 mM NaCl, 10 mM EDTA, 13 protease inhibitorcocktail, 50 mM MG132, and 1% [v/v] Triton X-100) for 2 h. The amyloseresin was washed three times with wash buffer (50 mM Tris-HCl, pH 8.0,150 mM NaCl, and 0.1% [v/v] Triton X-100); Nus (6 mg) and MBP (6 mg)proteinswere used as negative controls. The proteinswere detected usingthe following monoclonal antibodies: mouse anti-His (Transgen Biotech,cat. no.HT501-02) ata1:5000dilution;mouseanti-GST (TransgenBiotech,cat. no. HT601-02) at a 1:10,000 dilution; anti-NusA (Abnova, cat. no.MAB0049-M01) at a 1:10,000 dilution; and mouse anti-MBP (TransgenBiotech, cat. no. HT701-02) at a 1:10,000 dilution. To assay the effects ofrac-GR24,GR245DS,GR24ent-5DSandKAR1on the interactionofOsSMAX1andD14L, the indicatedconcentrations (20or50mM)of thechemicalswereadded to the incubation buffer.

Co-IP Assays

Protoplasts generated from the young stems of 3-week-old rice seedlingsgrown under light were transformedwith transient expression plasmids aspreviously described by Bart et al. (2006). After incubation of the proto-plasts at 28°C for 12 h, protein extraction buffer (50 mM Tris-HCl, pH 8.0,150mMNaCl, 10mMEDTA, 13 protease inhibitor cocktail, 50mMMG132,and1%[v/v] TritonX-100)wasadded, afterwhich themixturewasvortexed(IKA Vortex). The lysate was centrifuged at 14,000g for 15min at 4°C, afterwhich the supernatantwascollected forCo-IP experiments. In accordancewith the manufacturer’s instructions, 25 mL of GFP-Trap coupled toagarose (ChromoTek) was added to 1 mL of extracted protein and in-cubated for 3 h at 4°C. After incubation, the agarose was washed threetimeswithwashbuffer (50mMTris-HCl, pH8.0, 150mMNaCl, and0.1%[v/v] TritonX-100), followedby elutionwith 30mLof SDS-PAGEsample bufferfor SDS-PAGE electrophoresis and immunoblot analysis. GFPprotein wasused as a negative control in each set of experiments. The proteins weredetected by mouse anti-GFP monoclonal antibodies (Roche, cat. no.11814460001) at a 1:5000dilution or by anti-HAantibodies (Roche, cat.no.11867423001) at a 1:5000 dilution.

Dual Luciferase Reporter Assays in Protoplasts

To determine the stability of D53, the respective PUC57-StrigoQuant-likereporter vectors of D53 and D53m were transformed into rice protoplastsfrom the wild-type, d3, d14, d14l, and d14 d14l plants as previously de-scribed by Bart et al. (2006). After incubation at 28°C for 11 h, 1 mM rac-GR24 was added, followed by incubation for another 1 h. The Renilla andfirefly luciferase activities were then measured using a Dual LuciferaseReporter Assay System (Promega). To test the stability of OsSMAX1, therespective PUC57-StrigoQuant-like reporter vectors of OsSMAX1 andOsSMAX1mwere transformed into rice protoplasts from the wild type, d3,d14, d14l and d14 d14l as described. After incubation at 28°C for 12 h, theRenilla and firefly luciferase activities were measured using a Dual Lucif-erase Reporter Assay System. FF:REN was used as a metric of proteinstability.

2800 The Plant Cell

Antibody Preparation

TodetectendogenousOsSMAX1, thecDNAfragmentencodingOsSMAX1(571 to 951 amino acids) was cloned into pDONR221 via BP reactions(Invitrogen) and then cloned into a pDEST-His-MBP destination vector byLR reactions (Invitrogen) to express the MBP-OsSMAX1 (571- to 951–aminoacid) fusionprotein. The fusionproteinwassubsequently expressedin E. coli BL21 and then purified with amylose resin. The MBP-OsSMAX1(571 to951aminoacids) purifiedproteinwasusedasanantigen toproducepolyclonal anti-OsSMAX1 antibodies in rabbits. The specificity of the anti-OsSMAX1antibodieswasconfirmedby immunoblot analysis ofOsSMAX1knockout plants (Figure 5C) and OsSMAX1m-OE transgenic plants(Supplemental Figure 10).

Endogenous and Transgenic Protein Detection

The aerial parts of 7-d-old light-grown rice seedlings were ground in liquidnitrogen and incubated in extraction buffer (50 mM Tris-HCl, pH 7.4,150 mM NaCl, 10 mM EDTA, 1% [w/v] SDS, and 13 protease inhibitorcocktail) for 20min at 4°C. After centrifugation for 10min at 14,000g, SDS-PAGE sample buffer was added to the supernatant for SDS-PAGE elec-trophoresis and immunoblot analysis. EndogenousOsSMAX1proteinwasdetected by anti-OsSMAX1 antibodies at a 1:4000 dilution. For endoge-nousD53, the shoot bases of 3-week-old plantswere used, after which thedetectionwasperformedusinganti-D53polyclonal antibodies (Jianget al.,2013).Todetermine theeffectsof thechemicalsonendogenousOsSMAX1degradation, the aerial parts of seedlings treated with 20 mMKAR1, KAR2,GR245DS, or GR24ent-5DS and grown in the light for 7 d were collected forimmunoblotting. rac-GR24 and KAR1 were obtained from Chiralix, andGR245DS andGR24ent-5DSwere prepared as described (Wang et al., 2020).KAR2was obtained fromDaqin. Except where specified, the abundance ofOsSMAX1 or OsSMAX1m proteins from light-grown 7-d-old seedlings ofdifferentOsSMAX1andOsSMAX1m transgenic seedlingswasmeasurebyanti-Flag antibodies (DDDDK monoclonal antibody; MBL, cat. no. M185-3L) at a 1:10,000 dilution. To determine the stability of OsSMAX1 in re-sponse to rac-GR24, GR245DS, GR24ent-5DS, and KAR1 treatment, calli ofOsSMAX1-GFP-Flag and OsSMAX1m-GFP-Flag transgenic plants werecultured on solid medium for 6 d at 28°C and then transferred to liquidmedia containing a specific chemical compound at a specific concen-tration (Jiang et al., 2013). After treatment with 10mM rac-GR24, GR245DS,GR24ent-5DS,andKAR1, thecalliwerecollectedatspecifiedtimepoints.TheOsSMAX1-GFP-Flag and OsSMAX1m-GFP-Flag proteins collected fromthe calli were detected by anti-Flag antibody. In the ubiquitin assay, theOsSMAX1-GFP-Flag and OsSMAX1m-GFP-Flag proteins were detectedby anti-GFP monoclonal antibody (Roche) at a 1:5000 dilution andmonoclonal anti-ubiquitin antibody (Zhao et al., 2013b). The rice ACTINprotein was detected by anti-ACTIN antibodies (Abmart, cat. no. M20009)at a 1:4000 dilution, as an internal control.

Subcellular Localization

To observe the subcellular localization of OsSMAX1 in plants, the primaryroots of 5-d-old seedlings of Ubi:OsSMAX1-GFP-Flag and Ubi:OsS-MAX1m-GFP-Flag transgenic plants were used for analysis. The GFPsignal was detected using a confocal microscope (SP8; Leica) at an ex-citation wavelength of 488 nm. Propidium iodide was used for cell wallstaining, and the excitation wavelength was 540 nm.

RNA Sequencing and Data Analysis

Dark-grown 7-d-old seedlings were collected for total RNA extraction.Eight seedlings were pooled as one sample for each indicated genotype.Three biological replicates were used for RNA extraction and RNA se-quencing. Total RNAwasextracted usingTRI-Reagent (MRC)according to

the manufacturer’s protocol. RNA-seq was performed on an IlluminaNovaSeq platform. The reads were aligned to the reference genome MSUversion_7.0 and the gene model annotation file (http://rice.plantbiology.msu.edu/) using TopHat2. Gene expression profiling and the DEGsidentification were performed with cufflinks. The genes whose expressionwas upregulated in themutants and transgenic plants of all three biologicalreplicates are provided inSupplemental DataSets 3 and4. AVenndiagramand heatmap were constructed by R using the VennDiagram package(https://cran.r-project.org/web/packages/VennDiagram) and the pheat-mappackage (https://cran.r-project.org/web/packages/pheatmap), re-spectively. Gene ontology and Kyoto Encyclopedia of Genes andGenomes analyses were performed by the online tool KOBAS (http://kobas.cbi.pku.edu.cn).

RT-qPCR Analysis

All the aerial parts of dark-grown 7-d-old seedlings were sampled for totalRNAextraction. The totalRNAwasextractedusingTRI-Reagent accordingto the manufacturer’s instructions. The total RNA (0.5 to 0.7 mg) was usedfor first-strand cDNA synthesis using ReverTra Ace qPCR Master Mix inconjunction with gDNA Remover (Toyobo). RT-qPCR analysis was per-formed using gene-specific primers (Supplemental Table 2) on a CFXConnect Real-Time System (Bio-Rad) according to the manufacturer’sinstructions. The 10-mL reaction volumeconsistedof 1mLof diluted cDNA,primers at 0.3 mM, and 5 mL of ChamQ Universal SYBR qPCRMaster Mix(Vazyme). The rice ACTIN gene was used as an internal control.

Accession Numbers

The RNA-seq information has been deposited in the BioProject IDPRJNA553596. Sequence data from this article can be found in theGenBank/EMBL libraries under the following accession numbers: D10(LOC_Os01g54270); D17 (LOC_Os04g46470); D14 (LOC_Os03g10620);D14L (LOC_Os03g32270); D14L2 (LOC_Os05g51240); D14L3 (LO-C_Os01g41240); D3 (LOC_Os06g06050); D53 (LOC_Os11g01330); D53L(LOC_Os12g01360); OsSMAX1 (LOC_Os08g15230); DL1 (LO-C_Os02g26600); DL2 (LOC_Os02g33460); DL3 (LOC_Os02g54720); DL4(LOC_Os04g23220); DL5 (LOC_Os04g33980); DL6 (LOC_Os11g05820);TPR1 (LOC_Os01g15020); TPR2 (LOC_Os08g06480); TPR3 (LO-C_Os03g14980); OsTCP5 (LOC_Os02g51280); OsGSK2 (LO-C_Os05g11730); GY1 (LOC_Os01g67430); LP2 (LOC_Os02g40240).

Supplemental Data

Supplemental Figure 1. Accumulation of D53 promotes mesocotylelongation in the dark.

Supplemental Figure 2. D14 subfamily proteins in rice.

Supplemental Figure 3. D14L knockout plants generated by CRISPR/CAS9.

Supplemental Figure 4. Seedlings of different mutants in the light andin the dark.

Supplemental Figure 5. Phylogenetic tree of D53/SMAX1 familyproteins.

Supplemental Figure 6. Interaction of D14 homologs with D53 familyproteins in yeast two-hybrid assay.

Supplemental Figure 7. Interaction of OsSMAX1 with a D14 homologin yeast two-hybrid assay.

Supplemental Figure 8. Yeast two-hybrid analysis of OsSMAX1domain interaction with D14L.

Karrikin and Strigolactone Repress Mesocotyl Elongation 2801

Supplemental Figure 9.Mutation of the catalytic triad of D14L did notaffect its interaction with OsSMAX1.

Supplemental Figure 10. Validation of the anti-OsSMAX1 antibody.

Supplemental Figure 11. StrigoQuant-like reporters transiently ex-pressed in protoplasts.

Supplemental Figure 12. OsSMAX1 accumulation promotes meso-cotyl elongation in the dark.

Supplemental Figure 13. Interaction between D14L and OsSMAX1 isnot enhanced by 20 mM Karrikin treatment.

Supplemental Figure 14. Interaction between D14L and OsSMAX1 isnot enhanced by 50 mM Karrikin treatment.

Supplemental Figure 15. OsSMAX1 knockout plants generated byCRISPR/CAS9.

Supplemental Figure 15. Relative expression levels of OsSMAX1-regulated genes.

Supplemental Figure 17. OsSMAX1 is not involved in regulation ofshoot branching.

Supplemental Figure 18. The EAR motif of OsSMAX1 is essential forthe interaction between OsSMAX1 and TPR2.

Supplemental Figure 19. Heatmap of OsSMAX1 positively regulatedgenes in wild-type, mutant, and transgenic plants.

Supplemental Figure 20. Mesocotyl elongation in response todifferent concentration of chemicals in the dark.

Supplemental Figure 21. Relative expression levels of OsSMAX1-regulated genes in response to GR24 stereoisomers.

Supplemental Table 1. Primers used for vector construction.

Supplemental Table 2. Primers used for RT-qPCR.

Supplemental Data Set 1. Text file of the alignment used for thephylogenetic analysis of D14 proteins shown in SupplementalFigure 2.

Supplemental Data Set 2. Text file of the alignment used for thephylogenetic analysis of D53/SMAX1 proteins shown in SupplementalFigure 5.

Supplemental Data Set 3. Downregulated genes in mutants andoverexpression plants compared to the wild type.

Supplemental Data Set 4. Upregulated genes in mutants and over-expression plants compared to the wild type.

Supplemental Data Set 5. Statistical Analyses.

ACKNOWLEDGMENTS

We thank Shengben Li (Nanjing Agricultural University) and Zhixi Tian(Institute of Genetics and Developmental Biology, Chinese academy ofSciences) for critical reading. We thank Qi Xie (Institute of Genetics andDevelopmental Biology, Chinese Academy of Sciences) for providing theantibody of ubiquitin. We thank High-Performance Computing Centers atAgricultural Genomics Institute at Shenzhen, Chinese Academy of Agri-cultural Sciences, for bioinformatics support and also thank JiangsuCollaborative InnovationCenter forModernCropProduction for support.This work was supported by founding from National Key Research andDevelopment Program of China (grant 2016YFD0101801); NationalNatural Science Foundation of China (31501384 and 31201004); and theScience, Technology and Innovation Commission of Shenzhen Munic-ipality (grants JCYJ20170303154319837, JCYJ20170412155447658,and KQJSCX2018323140312935).

AUTHOR CONTRIBUTIONS

J.L. and G.X. conceived the project. J.Z., K.H., L.J.Z., G.X., and J.L.designed the experiments. J.Z., K.H., L.J.Z., L.W., S.K., J.D., L.Y.Z.,L.X.Z., Z.T., X.M., J.H., Y.Z., and Q.W. performed experiments. J.Z.,K.H., L.J.Z., M.Q., D.Z., B.W., C.P., Q.W., Q.Q., Y.W., G.X., and J.L.analyzed data. J.Z., G.X., and J.L. wrote the article.

Received February 24, 2020; revised June 2, 2020; accepted July 9, 2020;published July 14, 2020.

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Karrikin and Strigolactone Repress Mesocotyl Elongation 2805

DOI 10.1105/tpc.20.00123; originally published online July 14, 2020; 2020;32;2780-2805Plant Cell

Peng Cui, Quan Wang, Qian Qian, Yonghong Wang, Jiayang Li and Guosheng XiongZou, Lixin Zhu, Zhanpeng Tang, Xiangbing Meng, Bing Wang, Jiang Hu, Dali Zeng, Yonghui Zhao,

Jianshu Zheng, Kai Hong, Longjun Zeng, Lei Wang, Shujing Kang, Minghao Qu, Jiarong Dai, LinyuanMesocotyl Elongation in Darkness

Karrikin Signaling Acts Parallel to and Additively with Strigolactone Signaling to Regulate Rice

 This information is current as of May 17, 2021

 

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