ERECTA1 Acts Upstream of the Osmkkk10-Osmkk4-Osmpk6

Plant Cell Advance Publication. Published on July 2, 2020, doi:10.1105/tpc.20.00351

RESEARCH ARTICLE

ERECTA1 Acts Upstream of the OsMKKK10-OsMKK4-OsMPK6

Cascade to Control Spikelet Number by Regulating Cytokinin

Metabolism in Rice

Tao Guoa,1, Zi-Qi Lua,b,1, Jun-Xiang Shana, Wang-Wei Yea, Nai-Qian Donga, Hong-Xuan Lina,b,c* aNational Key Laboratory of Plant Molecular Genetics, CAS Centre for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China. bSchool of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China. cUniversity of the Chinese Academy of Sciences, Beijing 100049, China. 1These authors contributed equally to this work. *Corresponding Author: [email protected] or [email protected].

Short title: OsER1 regulates spikelet number in rice

One-sentence summary: The output of an RLK-MAPK signaling pathway maintains cytokinin homeostasis, thereby determining spikelet number per panicle in rice.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Hong-Xuan Lin ([email protected] or [email protected]).

ABSTRACT Grain number is a flexible trait that strongly contributes to grain yield. In rice (Oryza sativa), the OsMKKK10-OsMKK4-OsMPK6 cascade, which is negatively regulated by the dual-specificity phosphatase GSN1, coordinates the trade-off between grain number and grain size. However, the specific components upstream and downstream of the GSN1-MAPK module that regulate spikelet number per panicle remain obscure. Here, we report that ERECTA1 (OsER1), a negative regulator of spikelet number per panicle, acts upstream of the OsMKKK10-OsMKK4-OsMPK6 cascade and that the OsER1-OsMKKK10-OsMKK4-OsMPK6 pathway is required to maintain cytokinin homeostasis. OsMPK6 directly interacts with and phosphorylates the zinc finger transcription factor DST to enhance its transcriptional activation of CYTOKININ

©2020 American Society of Plant Biologists. All Rights Reserved OXIDASE2 (OsCKX2), indicating that the OsER1-OsMKKK10-OsMKK4-OsMPK6 pathway shapes panicle morphology by regulating cytokinin metabolism. Furthermore, overexpression of either DST or OsCKX2 rescued the spikelet number phenotype of the oser1, osmkkk10, osmkk4, and osmpk6 mutants, suggesting that the DST-OsCKX2 module genetically functions downstream of the OsER1-OsMKKK10-OsMKK4-OsMPK6 pathway. These findings reveal specific crosstalk between a MAPK signaling pathway and cytokinin metabolism, shedding light on how developmental signals modulate phytohormone homeostasis to shape the inflorescence.

1 INTRODUCTION

2 Rice (Oryza sativa), one of the most important staple cereal crops worldwide, feeds

3 more than half the world’s population. Increasing grain yield is a continual focus of

4 rice breeding and improvement. Rice yield is a complicated trait determined by tiller

5 number, grain weight, and grain number per panicle. Of these, grain number per

6 panicle is a more flexible component, which depends on the number of primary and

7 secondary branches, and thus plays a major role in determining grain yield (Xing and

8 Zhang, 2010). Rice has a determinate inflorescence in which meristems differentiate

9 into primary branch meristems attached to a central rachis, which then form several

10 secondary branch meristems (Zhang and Yuan, 2014). During panicle morphogenesis,

11 the multiple inﬂorescence meristems that form and give rise to spikelets, flowers, and

12 glumes are pivotal determinants of grain number and size. These spatiotemporally

13 programmed cellular processes contribute interactively to specify rice panicle

14 architecture. To date, the genetic basis underlying the determination of spikelet

15 number per panicle has been studied by mapping quantitative trait loci (QTLs) and

16 identifying mutants; these studies have found that the maintenance of meristem size

17 and activity is closely associated with panicle branching and the number of spikelets

18 (Komatsu et al., 2003; Ashikari et al., 2005; Kurakawa et al., 2007; Huang et al.,

19 2009a; Tabuchi et al., 2011; Li et al., 2013; Wu et al., 2016; Huo et al., 2017), thereby

20 supporting the hypothesis that the activity of the reproductive meristem plays an

21 essential role in determining rice grain production (Li et al., 2013).

23 Emerging compelling evidence has suggested that the phytohormone cytokinin plays

2 24 a crucial role in maintaining shoot apical meristem (SAM) activity (Ashikari et al.,

25 2005; Kurakawa et al., 2007; Zhao et al., 2010; Perales and Reddy, 2012). Either

26 reduced levels of endogenous cytokinin or the suppression of cytokinin signaling

27 inhibits SAM activity (Werner et al., 2001; Werner et al., 2003; Higuchi et al., 2004;

28 Riefler et al., 2006; Kurakawa et al., 2007), whereas increasing cytokinin levels

29 enhances SAM activity (Rupp et al., 1999; Ashikari et al., 2005). In rice, the

30 important QTL Grain number 1a encodes the cytokinin oxidase/dehydrogenase

31 CYTOKININ OXIDASE2 (OsCKX2), which catalyzes the degradation of active

32 cytokinin. This QTL negatively regulates grain number per panicle (Ashikari et al.,

33 2005). Accordingly, a decrease in OsCKX2 expression results in the overaccumulation

34 of cytokinin in inflorescence meristems, leading to increased grain production

35 (Ashikari et al., 2005). Molecular evidence indicates that the transcription factor

36 DROUGHT AND SALT TOLERANCE (DST) (Huang et al., 2009b) controls the

37 activity of the reproductive SAM by directly binding to the promoter of OsCKX2 and

38 positively regulating its expression; therefore, disrupting DST significantly increases

39 cytokinin levels and grain production (Li et al., 2013). Nevertheless, the signaling

40 pathway that regulates cytokinin metabolism during inflorescence development

41 remains elusive.

43 Mitogen-activated protein kinase (MAPK) cascades are highly conserved, ubiquitous

44 signaling pathways in eukaryotes. The sequential phosphorylation of proteins in these

45 cascades leads to altered substrate activities and regulates cell proliferation and

46 differentiation and the coordination of responses to environmental inputs (Widmann

47 et al., 1999; Xu and Zhang, 2015). MAPK cascades play essential roles in multiple

48 processes in plants, including defense, stress responses, and developmental programs

49 (Meng and Zhang, 2013; Komis et al., 2018; Zhang et al., 2018). In rice, the

50 OsMKKK10-OsMKK4-OsMPK6 cascade is negatively regulated by the

51 dual-specificity phosphatase GRAIN SIZE AND NUMBER1 (GSN1), which directly

52 dephosphorylates OsMPK6, thereby coordinating the trade-off between grain number

53 per panicle and grain size (Guo et al., 2018; Xu et al., 2018b; Xu et al., 2018a; Wang

3 54 et al., 2019). Moreover, we previously identified a potential association between the

55 GSN1-MAPK module and phytohormone signaling in determining the plasticity of

56 panicle architecture in rice (Guo et al., 2018). However, the precise genetic and

57 molecular mechanism underlying how the GSN1-MAPK module and cytokinin

58 metabolism control spikelet number per panicle is currently unclear. Identifying the

59 components upstream and downstream of the GSN1-MAPK module could reveal

60 unrecognized molecular mechanisms.

62 Here, we characterized the rice mutant oser1, which displays a markedly increased

63 number of spikelets per panicle compared with the wild type, indicating that OsER1,

64 encoding an ERECTA family protein, is a negative regulator of spikelet number per

65 panicle. We demonstrate that OsER1 acts upstream of the

66 OsMKKK10-OsMKK4-OsMPK6 cascade to control the number of spikelets produced

67 per panicle. Moreover, we show that OsMPK6 interacts with and phosphorylates DST

68 to enhance its transcriptional activation of OsCKX2, indicating that the

69 OsMKKK10-OsMKK4-OsMPK6 cascade controls the number of spikelets per

70 panicle by regulating cytokinin metabolism. These findings advance our

71 understanding of how perceived developmental signals control phytohormone

72 metabolism to shape panicle morphology. In addition, they provide a framework for

73 understanding the role of receptor and signal conversion in inflorescence development,

74 offering a potential means to improve crop yields.

76 RESULTS

77 OsER1 is responsible for rice panicle morphogenesis and plays a negative role in

78 determining spikelet number per panicle

79 The ER gene, which encodes a receptor-like protein kinase (RLK), has been

80 extensively studied in Arabidopsis thaliana, where it regulates numerous

81 developmental processes including stomatal formation and patterning, inﬂorescence

82 architecture, and ovule development (Torii et al., 1996; Shpak et al., 2003; Meng et al.,

4 83 2012; Pillitteri and Torii, 2012; Shpak, 2013). Although emerging evidence indicates

84 that the overexpression of ER confers thermotolerance to rice (Shen et al., 2015), the

85 explicit function of ER in controlling inflorescence development in rice remains

86 unclear. We therefore investigated the function of OsER1 (LOC_Os06g10230) using

87 CRISPR/Cas9 gene editing (Ma et al., 2015). Strikingly, the oser1 mutant displayed

88 increased spikelet number per panicle and reduced grain size without altered plant

89 architecture (Figure 1A to 1C). Moreover, the average spikelet number per panicle of

90 the oser1 mutant was markedly increased, with reduced grain length but enhanced

91 grain width compared to the wild type (Figure 1F to 1H). However, the average grain

92 yield per plant was comparable to that of the wild-type Fengaizhan-1 (FAZ1) (O.

93 indica) due to a reduced setting percentage in oser1 (Figure 1I and 1J). Consistent

94 with these findings, the oser1 mutant in the O. japonica variety Zhonghua-11 (ZH11)

95 background, which carries the same mutant allele of OsER1 as that in FAZ1, also

96 showed increased spikelet number per panicle (Supplemental Figures 1A to 1C and

97 2A). These results suggest that OsER1 is responsible for panicle morphogenesis in

98 rice.

100 To further confirm the phenotype of oser1, we performed a genetic complementation

101 test in which the full-length OsER1 gene from FAZ1 was introduced into the oser1

102 mutant via Agrobacterium tumefaciens-mediated transformation. A positive

103 transgenic line harboring OsER1 displayed completely wild-type phenotypes with

104 respect to spikelet number per panicle and grain size (Figure 1D and 1E and 1K to

105 1M), but overexpression of OsER1 in the wild-type FAZ1 background had no effect

106 on spikelet number per panicle (Supplemental Figure 3). These results indicate that

107 OsER1 is not only required for rice panicle development, but it is also sufficient for

108 regulating spikelet number per panicle.

109

110 In Arabidopsis, MAPK cascades function downstream of RLK signaling (Meng and

111 Zhang, 2013; Xu and Zhang, 2015). Therefore, we assayed the level of MAPK

112 phosphorylation in young panicles of the oser1 mutant and found that the

5 113 loss-of-function of OsER1 suppressed the phosphorylation of both OsMPK6 and

114 OsMPK3 without altering their abundance (Figure 1N). Moreover, we analyzed the

115 cell division rate in younger panicles using flow cytometry and found that the

116 percentage of G2/M phase cells with 4c DNA content was markedly increased in the

117 oser1 mutant but that the percentage of G1 phase cells with 2c DNA content was

118 reduced compared to the wild type (Supplemental Figure 4A to 4C). Accordingly,

119 the expression levels of cell cycle-related genes were significantly elevated in young

120 panicles of oser1 compared to wild-type FAZ1 (Supplemental Figure 4D),

121 suggesting that localized cell proliferation occurs more rapidly in oser1 during panicle

122 morphogenesis. Overall, these results suggest that OsER1 controls localized cell

123 proliferation to negatively regulate spikelet number by activating a MAPK cascade in

124 rice.

125

126 OsER1 acts upstream of the OsMKKK10-OsMKK4-OsMPK6 cascade to

127 negatively regulate spikelet number per panicle

128 We previously proposed that the GSN1-MAPK module coordinates the trade-off

129 between grain number and grain size by integrating localized cell differentiation and

130 proliferation (Guo et al., 2018). Further investigation confirmed that the gsn1 osmpk6

131 double mutant produces significantly more spikelets per panicle than gsn1 but fewer

132 than osmpk6 in the FAZ1 genetic background (Supplemental Figure 5), which

133 carries a mutant OsMPK6 allele from the dsg1 mutant (Liu et al., 2015). These results

134 strongly indicate that the GSN1-MAPK module plays a crucial role in spikelet

135 formation, with GSN1 functioning as a positive regulator of this process; however, the

136 OsMKKK10-OsMKK4-OsMPK6 cascade acts as a negative regulator of spikelet

137 formation (Guo et al., 2018). To further investigate the relationship between OsER1

138 and the OsMKKK10-OsMKK4-OsMPK6 cascade in determining panicle morphology,

139 we compared the number of spikelets per panicle and observed that the oser1,

140 osmkkk10, osmkk4, and osmpk6 mutants showed similar panicle architectures and

141 produced many more spikelets than wild-type FAZ1 (Supplemental Figure 6). These

142 results imply that both OsER1 and the OsMKKK10-OsMKK4-OsMPK6 cascade play

6 143 negative roles in determining the number of spikelets per panicle.

144

145 Moreover, crossing with oser1 rescued the decreased spikelet number phenotype of

146 the gsn1 mutant (Figure 2A, 2E), suggesting that the phenotypes observed in the gsn1

147 mutant are genetically dependent on OsER1. In addition, overexpression of GSN1 in

148 the wild-type background resulted in the production of more spikelets per panicle,

149 similar to the phenotype of the oser1 mutant (Figure 2B, 2F). Constitutively active

150 OsMKK4 and OsMKKK10 both suppressed the increased number of spikelets when

151 expressed in the oser1 mutant (Figure 2C and 2D and 2G and 2H). These results

152 indicate that OsER1 acts upstream of the GSN1-MAPK module to regulate spikelet

153 formation in rice.

154

155 Next, we assayed the phosphorylation levels of OsMPK6 in young panicles of

156 different transgenic plants and found that the loss-of-function of OsER1 in the gsn1

157 oser1 double mutant reduced the phosphorylation level of OsMPK6 compared to gsn1

158 (Figure 2I). The same decrease in phosphorylated OsMPK6 levels was observed in

159 plants overexpressing GSN1 (Figure 2J). Consistent with this finding, constitutively

160 activated OsMKK4 and OsMKKK10 significantly enhanced the phosphorylation

161 levels of OsMPK6 in oser1 (Figure 2K and 2L), implying that OsER1 associates

162 with OsMKKK10 and OsMKK4 to modulate the phosphorylation status of OsMPK6.

163 Overall, these results suggest that OsER1 acts upstream of the

164 OsMKKK10-OsMKK4-OsMPK6 cascade to negatively regulate the number of

165 spikelets per panicle.

166

167 The OsER1-OsMKKK10-OsMKK4-OsMPK6 pathway is required to modulate

168 cytokinin metabolism

169 Although the above evidence supports the hypothesis that OsER1 and the

170 OsMKKK10-OsMKK4-OsMPK6 cascade act in a common pathway to control

171 spikelet number per panicle, the downstream components responsible for spikelet

172 formation remains unknown. Notably, we previously reported that OsCKX2 is

7 173 expressed at significantly higher levels in gsn1 compared with FAZ1 during young

174 panicle developmental, but the cytokinin-activating gene LONELY GUY is expressed

175 at dramatically lower levels in this mutant (Guo et al., 2018). In agreement with this

176 finding, the levels of several cytokinins were significantly lower in the gsn1 mutant

177 than in FAZ1, thereby supporting the hypothesis that cytokinin activity is attenuated

178 in the panicle meristem of the gsn1 mutant, which contributes to the reduction in

179 spikelet number per panicle (Guo et al., 2018). Therefore, we hypothesized that the

180 OsER1-OsMKKK10-OsMKK4-OsMPK6 pathway is closely associated with the role

181 of cytokinin homeostasis in determining the number of spikelets in rice.

182

183 Interestingly, OsCKX2 was significantly downregulated in the oser1 mutant compared

184 to wild-type FAZ1 during young panicle development (Figure 3A), and OsCKX2

185 protein levels were also lower in the mutant (Figure 3B), thereby suggesting that

186 loss-of-function of OsER1 perturbs the regulation of cytokinin metabolism

187 responsible for panicle morphogenesis. We thus assayed the endogenous cytokinin

188 levels in young panicles and found that the levels of multiple cytokinins were

189 markedly elevated in the oser1 mutant (Figure 3C), confirming the notion that

190 OsER1 is required for regulating cytokinin metabolism. In addition, the oser1 mutant

191 exhibited an increased spikelet number per panicle resembling that of the osckx2

192 mutant (Supplemental Figures 1A to 1C and 2B). Overall, these results indicate that

193 loss-of-function of OsER1 results in the production of more spikelets due to the

194 disruption of cytokinin metabolism.

195

196 Furthermore, the transcript and protein levels of OsCKX2 in the osmkkk10, osmkk4,

197 and osmpk6 mutants were also lower than those in FAZ1 (Figure 3D and 3E). The

198 endogenous cytokinin levels were also higher, as expected (Figure 3F), indicating

199 that the OsER1-OsMKKK10-OsMKK4-OsMPK6 pathway is required for the

200 regulation of cytokinin metabolism. Additionally, the expression levels of

201 RESPONSE REGULATOR (OsRR) genes, which serve as markers for the cytokinin

202 response, were significantly elevated to varying degrees in the oser1 mutant (Figure

8 203 3G), indicating that loss-of-function of OsER1 enhances cytokinin signaling,

204 ultimately leading to the formation of more spikelets. Taken together, these findings

205 suggest that the OsER1-OsMKKK10-OsMKK4-OsMPK6 pathway is required to

206 modulate cytokinin metabolism, which is responsible for panicle development in rice.

207

208 OsMPK6 interacts with and phosphorylates DST

209 To further explore the molecular mechanism by which the GSN1-MAPK module and

210 cytokinin metabolism regulate spikelet number, we performed a yeast two-hybrid

211 assay to screen for proteins that physically interact with OsMPK6, which interacts

212 with and phosphorylates its substrates to modulate multiple biological processes in

213 rice (Hu et al., 2015; Ueno et al., 2015; Tian et al., 2017). Intriguingly, one of the

214 OsMPK6 interactors, the zinc finger transcription factor DST, was previously shown

215 to regulate drought and salt tolerance (Huang et al., 2009b) and to modulate grain

216 production by controlling the expression of OsCKX2 in rice (Li et al., 2013). We then

217 performed a yeast two-hybrid assay to confirm the interaction of DST with OsMPK6

218 (Figure 4A). Moreover, we performed a pull-down assay to confirm the interaction

219 between OsMPK6 and DST in vitro. His-tagged DST was pulled down by

220 MBP-OsMPK6, but not by MBP alone (Figure 4B), indicating that DST directly

221 binds to OsMPK6 in vitro.

222

223 Next, to investigate the direct physical interaction between OsMPK6 and DST in

224 planta, we performed a BiFC (biomolecular fluorescence complementation) assay in

225 which OsMPK6 and DST were fused with the N-terminal or C-terminal parts of split

226 yellow fluorescent protein (YFP); reconstitution of YFP fluorescence indicates

227 interaction between OsMPK6 and DST. The nYFP-OsMPK6 fusion protein interacted

228 with cYFP-DST, and cYFP-OsMPK6 interacted with nYFP-DST, as expected, but no

229 signal was detected between nYFP-OsMPK6 and the negative control,

230 cYFP-OsHAL3 (homolog of the halotolerance yeast protein HAL3) (Su et al., 2016),

231 suggesting that OsMPK6 associates with DST in vivo (Figure 4C). Furthermore, we

232 confirmed the interaction between OsMPK6 and DST in a Co-IP

9 233 (co-immunoprecipitation) experiment in which the DST-Flag and OsMPK6-Myc

234 fusion proteins were co-expressed in Nicotiana benthamiana leaves and the proteins

235 were immunoprecipitated with anti-Flag beads and detected by immunoblot analysis

236 with anti-Flag and anti-Myc antibodies. A band with the expected mobility of

237 OsMPK6-Myc was successfully detected in the anti-Flag immunoprecipitates from

238 leaves expressing DST-Flag and OsMPK6-Myc; by contrast, no OsMPK6-Myc was

239 detected in the absence of DST-Flag expression (Figure 4D). Taken together, these

240 results suggest that OsMPK6 interacts with DST in vitro and in vivo.

241

242 Because OsMPK6 interacts with DST, we hypothesized that DST might be a substrate

243 of OsMPK6. Therefore, we performed phosphorylation assays with fusion proteins to

244 determine whether OsMPK6 can phosphorylate DST in vitro. The recombinant

245 MBP-MPK6 strongly phosphorylated His-DST after being activated by a

CA 246 constitutively activated version of OsMKK4 known as OsMKK4 that carries the

247 Thr238Asp and Ser244Asp mutations (Yang et al., 2001); however, OsMPK6 failed

CA 248 to phosphorylate DST in the absence of constitutively activated OsMKK4 (Figure

249 4E). ERK MAP kinases speciﬁcally phosphorylate substrates with serine or threonine

250 residues followed by a proline (S/TP) (Jacobs et al., 1999). We analyzed the amino

251 acid sequence of DST and found four potential MAPK phosphorylation sites (Thr88,

252 Thr103, Thr112, Thr274) (Huang et al., 2005). After mutating all four threonine

4A 253 residues to alanine (DST ), DST was no longer phosphorylated by OsMPK6 (Figure

254 4E), suggesting that these residues are required for the phosphorylation of DST by

255 OsMPK6. Overall, these findings suggest that OsMPK6 interacts with and

256 phosphorylates DST.

257

258 OsMPK6-mediated phosphorylation enhances the transcriptional activity of DST

259 and positively regulates OsCKX2 expression

260 To determine how OsMPK6-mediated phosphorylation affects DST function and to

261 understand the biological signiﬁcance of this phosphorylation, we investigated

262 whether OsMPK6-mediated phosphorylation increases the transcriptional activation

10 263 of OsCKX2 by DST. We performed transient transactivation assays using the

264 OsCKX2 promoter fused to a luciferase (LUC) reporter gene (Figure 5A). The DST,

265 OsMPK6, and OsMKK4 effector constructs were expressed under the control of the

266 CaMV 35S promoter (Figure 5A) and co-transfected with the reporter construct into

267 rice protoplasts. Consistent with a previous study, we found that DST activated the

268 expression of OsCKX2 (Figure 5B) (Li et al., 2013). Notably, co-expression of

CA 269 OsMPK6 and constitutively activated OsMKK4 (OsMKK4 ) increased

270 DST-activated OsCKX2 expression; by contrast, co-expression with only OsMPK6 or

CA 271 OsMKK4 failed to enhance DST-activated OsCKX2 expression (Figure 5B),

272 implying that the OsMKK4-OsMPK6 cascade increases the transcriptional activity of

273 DST through phosphorylation. Consistent with this notion, expression of the

CA 274 constitutively activated form of OsMPK6, OsMPK6 , which carries the Tyr151Cys

275 mutation (Berriri et al., 2012; Xu et al., 2018b), directly increased the DST-induced

276 transcriptional activation to OsCKX2, which depends on the four phosphorylation

4D 277 sites (Figure 5C). Moreover, the quadruple phosphomimetic DST (Thr88Asp,

278 Thr103Asp, Thr112Asp, Thr274Asp) displayed markedly enhanced transcriptional

279 activity (Figure 5C). These results suggest that OsMPK6-mediated phosphorylation

280 enhances the transcriptional activation of OsCKX2 by DST.

281

282 We showed that OsMPK6 is required for rice panicle development and negatively

283 regulates spikelet number (Supplemental Figure 5) and that the expression level of

284 OsCKX2 is significantly reduced in osmpk6 (Figure 3D and 3E), implying that

285 loss-of-function of OsMPK6 attenuates DST-activated OsCKX2 expression. To

286 further explore the genetic interaction between OsMPK6 and DST, we overexpressed

287 DST in the dsg1 mutant, a null mutant of OsMPK6 (Liu et al., 2015). Overexpressing

288 DST suppressed the plant height and spikelet number per panicle phenotypes of the

289 dsg1 mutant in the ZH11 background (Figure 5D and 5E, 5H, 5J and 5K), which is

290 consistent with a previous finding on the effect of DST on rice development (Li et al.,

291 2013), supporting the hypothesis that DST is also a negative regulator of spikelet

292 number and acts downstream of OsMPK6. Moreover, overexpressing OsCKX2 in the

11 293 dsg1 mutant background dramatically decreased plant height and spikelet number per

294 panicle (Figure 5F and 5G, 5I, 5L and 5M), suggesting that OsCKX2 negatively

295 regulates spikelet number and is a downstream effector in the OsMPK6-DST module.

296 Overall, these results indicate that OsMPK6 acts upstream of DST to positively

297 regulate OsCKX2 gene expression.

298

299 The OsER1-OsMKKK10-OsMKK4-OsMPK6 pathway controls spikelet number

300 per panicle and is genetically dependent on the DST-OsCKX2 module

301 Our findings provide the evidence that OsMPK6 interacts with and phosphorylates

302 DST to enhance the expression of OsCKX2, thereby indicating that direct crosstalk

303 occurs between the OsMKKK10-OsMKK4-OsMPK6 cascade and cytokinin

304 metabolism. To determine whether there is a genetic relationship between the MAPK

305 signaling pathway and the DST-OsCKX2 module, we performed a wide array of

306 transgenic experiments using mutants of the

307 OsER1-OsMKKK10-OsMKK4-OsMPK6 pathway. Strikingly, overexpressing DST

308 rescued the spikelet number phenotype of the oser1 mutant (Figure 6A, 6E),

309 indicating that DST is sufficient for OsER1-regulated panicle morphogenesis, which is

310 consistent with that of the dsg1 mutant (Figure 5E, 5K). Overexpressing DST also

311 reduced the number of spikelets in the osmkkk10 mutant (Figure 6B, 6F), which

312 indicates that the regulation of spikelet number by MAPK signaling relies on the

313 action of DST. These results not only imply that the genetic effects of DST on the

314 components of the MAPK cascade are indeed universal, but they also suggest that

315 DST is responsible for MAPK cascade-controlled rice panicle development and

316 genetically acts downstream of the OsER1-OsMKKK10-OsMKK4-OsMPK6

317 pathway.

318

319 We showed that the abundance of OsCKX2 is markedly reduced in the MAPK

320 signaling pathway mutants during the young panicle stage (Figure 3B, 3E). We

321 further confirmed the genetic effect of OsCKX2 in the oser1 and osmkk4 mutant

322 backgrounds. Overexpressing OsCKX2 in the oser1 mutant background significantly

12 323 suppressed the production of spikelets and rescued the panicle architecture phenotype

324 (Figure 6C, 6G), suggesting that OsER1-controlled panicle shaping depends on

325 OsCKX2 expression. Moreover, overexpressing OsCKX2 rescued the spikelet number

326 phenotype of osmkk4 (Figure 6D, 6H), which is similar to the findings for dsg1

327 (Figure 5G, 5M), implying that OsCKX2-regulated cytokinin metabolism, which is

328 required for rice panicle development, acts downstream of the MAPK cascade. These

329 findings strongly support the hypothesis that the

330 OsER1-OsMKKK10-OsMKK4-OsMPK6 pathway is closely associated with

331 cytokinin homeostasis in determining the number of spikelets in rice. Taken together,

332 the results suggest that overexpressing either DST or OsCKX2 rescues the spikelet

333 number phenotypes of the oser1, osmkkk10, and osmkk4 mutants to different extents.

334 They also reveal that the control of spikelet number per panicle by the

335 OsER1-OsMKKK10-OsMKK4-OsMPK6 pathway depends on cytokinin metabolism

336 mediated by the DST-OsCKX2 module.

337

338 DISCUSSION

339 Rice is a model plant from the grass family that has evolved a characteristic

340 inﬂorescence morphology, with complex branches and specialized spikelets. To date,

341 the origin and morphogenesis of the diverse inﬂorescence architectures of grass

342 family members, which are regulated by complex integrated networks in response to

343 developmental cues and environmental signals, have attracted wide interest (Zhang

344 and Yuan, 2014). Notably, the inﬂorescence architecture of grass crops is closely

345 associated with the number of spikelets and ﬁnal grain yield. Increasing molecular

346 evidence suggests that multiple elements play important roles in determining the fate

347 of the reproductive meristem and shaping inﬂorescence architecture in the grass

348 family (Satoh-Nagasawa et al., 2006; Derbyshire and Byrne, 2013; Li et al., 2013; Wu

349 et al., 2016; Huo et al., 2017; Jiao et al., 2018; Gao et al., 2019; Guo et al., 2019). For

350 example, in maize, the trehalose-6-phosphate phosphatase RAMOSA3, which is

351 expressed in discrete domains subtending axillary inflorescence meristems, regulates

13 352 inflorescence branching by modifying sugar signals that move into axillary meristems

353 (Satoh-Nagasawa et al., 2006). In Brachypodium, MORE SPIKELETS1 (MOS1), an

354 APETALA2 transcription factor in the ethylene response factor class, determines

355 meristem fate by regulating the transition to terminal spikelet development

356 (Derbyshire and Byrne, 2013). In rice, upregulating the enoyl-CoA

357 hydratase/isomerase gene NUMBER OF GRAINS1 (NOG1) enhances grain number

358 per panicle by decreasing total fatty acid and linolenic acid contents and endogenous

359 jasmonic acid (JA) levels (Huo et al., 2017). In sorghum, the TCP (Teosinte

360 branched/Cycloidea/PCF) transcription factor MULTISEEDED1 (MSD1) controls

361 pedicellate spikelet number through the JA pathway (Jiao et al., 2018). Nonetheless,

362 the upstream signals that shape inﬂorescence architecture in crops are unclear.

363 Furthermore, although it is well known that the phytohormone cytokinin promotes

364 cell division and plays a fundamental and conserved role in regulating the size and

365 activity of reproductive meristems in plants (Kyozuka, 2007; Zhang and Yuan, 2014),

366 how cytokinin levels are regulated by signals responsible for inflorescence

367 morphogenesis in grasses remains largely unknown.

368

369 Here, we demonstrated that OsER1 acts upstream of the

370 OsMKKK10-OsMKK4-OsMPK6 cascade to control the number of spikelets per

371 panicle in rice and that the OsER1-OsMKKK10-OsMKK4-OsMPK6 pathway is

372 required for regulation of cytokinin metabolism. Furthermore, we found that OsMPK6

373 interacts with and phosphorylates the zinc finger transcription factor DST, enhancing

374 its transcriptional activity and positively regulating the expression of OsCKX2.

375 Several lines of genetic and biochemical evidence strongly support the hypothesis that

376 the OsER1-OsMKKK10-OsMKK4-OsMPK6 pathway negatively regulates the

377 number of spikelets per panicle by modulating cytokinin metabolism, specifically by

378 increasing the transcriptional activity of DST through phosphorylation. Based on the

379 present findings, we propose a working model describing the molecular mechanism

380 underlying the determination of spikelet number per panicle (Figure 7). During

381 panicle morphogenesis, when the active reproductive meristem produces cell-specific

14 382 signaling molecules such as small secreted peptides (SSPs), this triggers OsER1 to

383 activate the OsMKKK10-OsMKK4-OsMPK6 cascade directly or through unknown

384 mediators. The activated OsMKKK10-OsMKK4-OsMPK6 cascade phosphorylates

385 the zinc finger transcription factor DST to enhance its transcriptional activity and

386 increase the expression level of OsCKX2, which results in the degradation of

387 cytokinin, thereby maintaining cytokinin at levels required for spikelet differentiation

388 and formation. Meanwhile, the spatiotemporally activated GSN1 protein acts like a

389 molecular brake to negatively regulate the OsER1-OsMKKK10-OsMKK4-OsMPK6

390 pathway by inactivating OsMPK6. This attenuates the phosphorylation of DST,

391 thereby decreasing the expression of OsCKX2 and increasing cytokinin levels. Hence,

392 the signaling output of the OsER1-OsMKKK10-OsMKK4-OsMPK6-DST-OsCKX2

393 pathway negatively regulated by GSN1 maintains cytokinin homeostasis and

394 ultimately determines spikelet number per panicle in rice.

395

396 The contribution of both the OsER1 receptor and the

397 OsMKKK10-OsMKK4-OsMPK6 cascade to spikelet number per panicle raises the

398 interesting question of how the signaling speciﬁcity of the

399 OsMKKK10-OsMKK4-OsMPK6 cascade in different growth and developmental

400 pathways is maintained. In Arabidopsis, the ER receptor kinase gene is strongly

401 expressed in vegetative and reproductive SAMs and in developing leaf and flower

402 primordia, thereby controlling multiple aspects of plant morphology that are critical

403 for efficient plant responses to environmental changes (Kosentka et al., 2019). For

404 example, ER regulates the size of the SAM and the initiation of leaf primordia and

405 plays an essential role in reproductive organ development, which is closely associated

406 with auxin transport and cytokinin homeostasis (Shpak, 2013). Additionally, the

407 mutation of ER resulted in altered hyponastic petiole growth, circadian leaf

408 movements, and the shade avoidance response (Shpak, 2013). Notably, the function

409 of ER and the molecular mechanism of its action are most well understood in stomata

410 development, during which ER negatively modulates cell fate transitions and

411 contributes to the orientation of asymmetric cell divisions (Pillitteri and Torii, 2012).

15 412 Intriguingly, cell-specific expression of input signaling molecules such as peptide

413 ligands recognized by cell surface receptors could maintain signaling speciﬁcity (Lee

414 and Torii, 2012). In Arabidopsis, several secreted cysteine-rich peptides from the

415 EPIDERMAL PATTERNING FACTOR/EPIDERMAL PATTERNING

416 FACTOR-LIKE (EPF/EPFL) family have been shown to be ligands of ER (Shpak,

417 2013; Kosentka et al., 2019). Nevertheless, the secreted peptides responsible for rice

418 inflorescence morphology are largely unknown (Bessho-Uehara et al., 2016). The

419 specificity of signaling can also be determined by the presence of diverse substrates

420 that are differentially expressed and activated by MAPKs.

421

422 In Arabidopsis, the well-known MAPKs MPK3 and MPK6 play essential roles in

423 both plant growth and development and biotic and abiotic stress responses by

424 phosphorylating downstream partner proteins, which could contribute to the

425 functional specificity of MAPK cascades (Rodriguez et al., 2010). Although yeast

426 two-hybrid screening and high-throughput methods have been used to identify the

427 putative MAPK substrates, only a small number of MPK6 substrates have been

428 verified by functional evidence in Arabidopsis (Hoehenwarter et al., 2013; Xu and

429 Zhang, 2015; Komis et al., 2018). To date, very few substrates of OsMPK6 have been

430 identified in rice (34, 36), which are closely associated with the various functions of

431 OsMPK6 in growth and development. The loss-of-function of OsMPK6 results in

432 smaller grain size but increased grain number per panicle (Liu et al., 2015; Guo et al.,

433 2018; Xu et al., 2018b), thereby disordering panicle morphogenesis. Despite the

434 progress in this ﬁeld, the exact substrates of OsMPK6 involved in determining

435 inflorescence architecture are completely unknown, thus raising the important

436 question of which proteins phosphorylated by OsMPK6 control panicle

437 morphogenesis.

438

439 In this study, we identified DST as a substrate of OsMPK6 by yeast two-hybrid

440 screening and confirmed that OsMPK6 interacts with and phosphorylates DST

441 (Figure 4A to 4E). Furthermore, the phosphorylation of DST enhanced its

16 442 transcriptional activation of OsCKX2 (Figure 5A to 5C). In addition, overexpression

443 of DST rescued the spikelet number per panicle phenotypes of the osmpk6, oser1, and

444 osmkkk10 mutants (Figure 5E, 5K and 6A and 6B, 6E and 6F). The identification of

445 a substrate that is a direct target of the OsER1-OsMKKK10-OsMKK4-OsMPK6

446 pathway furthers our understanding of how the signals perceived on the cell surface

447 activate the reproductive meristem through nuclear signals. Moreover, the finding that

448 the phosphorylation of DST by OsMPK6 promotes the expression of OsCKX2

449 (Figure 5A to 5C) indicates that the OsER1-OsMKKK10-OsMKK4-OsMPK6

450 pathway directly controls the degradation of cytokinin and maintains cytokinin

451 homeostasis, which is required for inflorescence meristem development. This finding

452 reveals specific crosstalk between a receptor-like protein kinase and cytokinin

453 metabolism in plants.

454

455 Cytokinin is a classic phytohormone that plays essential roles in regulating meristem

456 organization and activity and is thus a positive regulator of cell proliferation in the

457 SAM (Zhao et al., 2010; Perales and Reddy, 2012). Notably, the level of cytokinin in

458 the meristem depends on its metabolic degradation, which is catalyzed by multiple

459 CKX enzymes. Thus, an increase in cytokinin levels in the SAM due to a mutation in

460 a CKX gene could enhance meristem activity (Bartrina et al., 2011). However, the

461 signaling pathway upstream of cytokinin metabolism regulation remains elusive. In

462 Arabidopsis, the control of stem-cell fate in the SAM relies on a negative feedback

463 loop involving the secreted peptide CLAVATA3 (CLV3) and the homeodomain

464 transcription factor WUSCHEL (WUS) (Zhao et al., 2010; Schaller et al., 2015). In

465 fact, it has been proposed that active cytokinin, together with WUS-induced CLV3

466 expression, acts as a positional cue in the SAM that regulates the dynamic positioning

467 of WUS expression such that it corresponds to the maximum cytokinin activity

468 (Schaller et al., 2015). Interestingly, an Arabidopsis mutant lacking ER family genes

469 displays significant upregulation of cytokinin-responsive genes in the SAM and an

470 increased stem cell population with hyperinduction of CLV3 expression in response to

471 cytokinin, indicating that ER genes regulate stem cell homeostasis by buffering

17 472 cytokinin responsiveness (Uchida et al., 2013). These results support the hypothesis

473 that the ER signaling pathway regulates SAM organization by affecting cytokinin

474 accumulation and responses through unknown molecular mechanism.

475

476 Here, we demonstrated that the OsER1-OsMKKK10-OsMKK4-OsMPK6 pathway

477 directly regulates OsCKX2 expression through phosphorylation of DST, illustrating

478 that crosstalk between an ER receptor and cytokinin underlies the activity of the

479 reproductive meristem as well as inflorescence development. Our findings provide a

480 precise mechanistic understanding of how receptor signaling modulates

481 phytohormone metabolism in plant. Nonetheless, the specific molecular mechanism

482 controlled by OsER1 responsible for grain shape remains unclear, which might

483 depend on the diverse downstream substrates of OsMPK6. Hence, more interacting

484 proteins of OsMPK6 that together contribute to panicle architecture plasticity by

485 coordinating grain number per panicle and grain size should be identified in the

486 future.

487

488 Taken together, our findings reveal that the OsER1 receptor acts upstream of the

489 OsMKKK10-OsMKK4-OsMPK6 cascade to negatively regulate the number of

490 spikelets per panicle and that the OsER1-OsMKKK10-OsMKK4-OsMPK6 pathway

491 controls cytokinin metabolism by phosphorylating DST and enhancing OsCKX2

492 expression, thereby providing a framework for how perceived developmental signals

493 control phytohormone metabolism to shape the plant inflorescence. Furthermore,

494 these findings provide important insights into the developmental plasticity of the

495 inflorescence and a potential means to breed high-yielding crop varieties with more

496 grains per panicle by genetically manipulating the

497 OsER1-OsMKKK10-OsMKK4-OsMPK6-DST-OsCKX2 pathway. Nevertheless,

498 little is known about the specific small secreted peptides that associate with the

499 OsER1 receptor and are required for panicle development. In addition, information

500 about the specific substrates of OsMPK6 is still lacking, which limits our

501 understanding of the OsMKKK10-OsMKK4-OsMPK6 cascade in rice. Moreover, the

18 502 molecular mechanism of signal transduction from OsER1 to OsMKKK10 and the

503 genetic association between OsER1 and its close homolog OsER2 (LOC_Os06g03970)

504 remain unclear. Further identification of the proteins associated with the

505 OsER1-OsMKKK10-OsMKK4-OsMPK6 pathway will ultimately uncover the

506 genetics basis of panicle development in rice.

507

508 METHODS

509 Plant materials and growth conditions

510 The oser1 and osckx2 mutants were isolated by CRISPR/Cas9 gene editing of the elite

511 indica rice (Oryza sativa) variety Fengaizhan-1(FAZ1) and the japonica variety

512 Zhonghua-11 (ZH11), respectively. All rice plants were cultivated in experimental

513 fields in Shanghai (China) or Lingshui (Hainan Province, China) under natural growth

514 conditions.

515

516 Plasmid construction and plant transformation

517 The full-length genomic sequence of OsER1 was amplified from the wild-type FAZ1

518 and cloned into the plant binary vector pCAMBIA1300 to produce the

519 complementation construct pCAMBIA1300-gOsER1. To generate overexpression

520 constructs, the full-length coding sequences of OsER1, DST, and OsCKX2 were

521 amplified from FAZ1 and cloned into the plant binary vector pCAMBIA1301 under

522 the control of the Ubiquitin promoter. To produce constitutively activated

CA CA 523 OsMKK4 and OsMKKK10 , the full-length coding sequence of OsMKK4

524 harboring the Thr238Asp and Ser244Asp mutations and a modified OsMKKK10

525 sequence harboring a deletion mutation (Xu et al., 2018b) were cloned into

526 pCAMBIA1301. The gene knockout constructs for CRISPR/Cas9 gene editing of

527 OsER1 and OsCKX2 were designed as previously described (Ma et al., 2015). A.

528 tumefaciens-mediated transformation in rice with the strain EHA105 was performed

529 as previously described (Hiei et al., 1994). All constructs used in the study were

530 produced using NEBuilder HiFi DNA Assembly Master Mix (NEB) and were

531 confirmed by sequencing. The PCR primer sets are listed in Supplemental Data Set 1.

19 532

533 RNA extraction and qRT-PCR

534 Total plant RNA was extracted individually from diverse rice tissues using TRIzol

535 Reagent (Invitrogen). Reverse transcription was carried out with ReverTra Ace qPCR

536 RT Master Mix with gDNA Remover (Toyobo) with 500 ng total RNA. qRT-PCR

537 analysis was performed with the ABI 7300 Real Time PCR System using Fast Start

538 Universal SYBR Green Master Mix and ROX (Roche). The rice UBQ5 gene was used

539 as an internal reference to normalize gene expression data, which were analyzed using

-ΔΔCT 540 the 2 method.

541

542 Nucleus isolation and assessment of ploidy

543 Isolation of cell nuclei and ploidy assessment were performed as described previously

544 (Guo et al., 2018). In brief, young rice panicles (0.1-0.3 cm) were selected and soaked

545 in nuclear isolation solution and then in staining solution (Beckman), after which the

546 tissues were chopped with a sharp blade. The nuclei suspension was loaded into a

547 Beckman MoFlo for flow cytometric analysis after filtering through a 40-µm nylon

548 filter, and the ploidy levels of approximately 10,000 nuclei were recorded for each

549 assay. The numbers of diploid and tetraploid nuclei were recorded, and FCS Express

550 4 software was used to calculate the relative proportions of cells in the G1, G2/M, and

551 S phases.

552

553 Measuring endogenous cytokinin levels

554 To assay endogenous cytokinin levels, all rice plants were cultivated in experimental

555 fields for approximately five weeks. Next, freshly initiated 0.1-0.5-cm long panicles

556 were harvested, and approximately 1-g samples were pooled for measurements, with

557 three independent biological repeats per sample. Quantification of endogenous

558 cytokinins was performed as previously described (Wu et al., 2016).

559

560 Yeast two-hybrid assays

561 The Y2H Gold-Gal4 system (Clontech) was used to perform yeast two-hybrid assays.

20 562 The full-length coding sequences of OsMPK6 and DST were inserted into the

563 pGBKT7 and pGADT7 vectors to prepare the bait and prey constructs, respectively,

564 which were transformed into yeast strain Y2H Gold. For yeast two-hybrid screening

565 of OsMPK6 interacting proteins, the yeast cells were cultured on SD/-Trp medium,

566 and 500-ml cultures were grown to prepare competent cells for transformation with

567 the pGADT7 plasmid library as previously described (Chen et al., 2019). After

568 transformation, yeast cells containing the pGADT7 plasmid library were cultured on

569 SD/-Trp-Leu-His or SD/-Trp-Leu-His-Ade medium containing X-ɑ-gal at 30 °C in

570 the dark for three days, after which the positive colonies were selected, and the inserts

571 in the pGBKT7 vectors were sequenced. For the specific yeast two-hybrid assays, the

572 bait and prey constructs were co-transformed into yeast strain Y2H Gold according to

573 the manufacturer’s instructions (Clontech). The yeast cells were cultured on different

574 media for observation. SD/-Trp is yeast culture medium without tryptophan.

575 SD/-Trp-Leu-His is yeast culture medium without tryptophan, leucine, and histidine.

576 SD/-Trp-Leu-His-Ade is culture medium without tryptophan, leucine, histidine, and

577 adenine. The PCR primers used for the yeast two-hybrid assays are listed in

578 Supplemental Data Set 1.

579

580 Bimolecular fluorescence complementation assays

581 The full-length coding sequences of OsMPK6, DST, and OsHAL3 were cloned into

582 pCAMBIA1300S-YC and pCAMBIA1300S-YN to produce the cYFP-protein and

583 nYFP-protein constructs, respectively. Leaves of five-week-old N. benthamiana

584 plants were co-infiltrated with A. tumefaciens strain GV3101 carrying the two

585 constructs. After infiltration, the plants were grown in the dark for 48 h, and

586 fluorescence signals were observed under an LSM 880 confocal laser-scanning

587 microscope (Carl Zeiss). PCR primers used for BiFC are listed in Supplemental Data

588 Set 1.

589

590 Pull-down assays

591 The pull-down assays were performed as previously described (Chen et al., 2019). In

21 592 brief, the coding sequence of OsMPK6 was inserted into the pMAL-c5x vector, and

593 the coding region of DST was cloned into the pET-32a vector. The recombinant

594 pMAL-c5x construct was transformed into Escherichia coli TB1 competent cells, and

595 the pET-32a expression plasmid was transformed into E. coli BL21 (DE3) competent

596 cells, which were cultured at 37 °C until the OD600 of the cell culture was 0.5.

597 Recombinant protein production was then induced with 1 mM IPTG for 36 h at 12 °C.

598 Next, E. coli cells expressing MBP-OsMPK6 and MBP were lysed by high-pressure

599 cell disruption (Constant Systems) and centrifuged. The supernatant was incubated

600 with amylose resin (NEB) for 30 min at 4 °C and washed five times with MBP

601 column buffer. Supernatant containing DST-His protein was incubated with

602 approximately the same amount of MBP-OsMPK6 and MBP binding beads for 2 h at

603 4 °C. The beads were washed five times with MBP column buffer, and the eluted

604 mixture was resuspended in SDS loading buffer and boiled for 5 min, separated by 10%

605 SDS-PAGE, and immunoblotted with anti-MBP (NEB, E8032) and anti-His

606 (ABclonal, AE028) antibodies. PCR primers used for the pull-down assays are listed

607 in Supplemental Data Set 1.

608

609 Co-immunoprecipitation

610 The coding sequence of DST was cloned into the pCAMBIA1306-Flag (3x) plasmid

611 to produce the Pro35S:DST-Flag vector. The OsMPK6 coding sequence was cloned

612 into pCAMBIA1301-Myc (7x)-His (6x) to generate the Pro35S:OsMPK6-Myc vector.

613 The Pro35S:DST-Flag and Pro35S:OsMPK6-Myc constructs in A. tumefaciens strain

614 GV3101 were transiently co-expressed in N. benthamiana leaf cells. The protein

615 extraction and immunoblot assays were performed as previously described using

616 anti-Flag (CST, 14793) and anti-Myc (CST, 2276) antibodies (Guo et al., 2018). The

617 PCR primers used for Co-IP are listed in Supplemental Data Set 1.

618

619 In vitro phosphorylation assays

CA 620 The coding sequences of OsMPK6 and OsMPK4 were inserted into the pMAL-c5x

621 vector, and the coding region of DST was cloned into the pET-32a vector to produce

22 622 fusion proteins, which were used in the phosphorylation assays. In vitro

623 phosphorylation assays were performed as previously described (Zhang et al., 2017).

624 In brief, recombinant MBP-OsMPK6 (20 µg) was activated by incubation with

CA 625 recombinant MBP-OsMKK4 (0.5 µg) in the presence of 50 µM ATP in 100 µL of

626 reaction buffer (25 mM HEPES, pH 7.5, 10 mM MgCl2, and 2 mM dithiothreitol) at

627 25 °C for 1 h. Activated MBP-OsMPK6 was then used to phosphorylate recombinant

4A 628 His-DST and His-DST proteins (1:20 enzyme to substrate ratio) in the same

629 reaction buffer with 200 µM ATP. The reactions were stopped by adding SDS loading

630 buffer. After phosphorylation, one half of the protein products were separated by 10%

631 SDS-PAGE, followed by staining with Coomassie Brilliant Blue. The other half of

632 the proteins were separated for immunoblotting analysis using

633 anti-Phospho-Threonine (CST, 9391), anti-His (ABclonal, AE028), anti-OsMPK6

634 (Beijing Protein Innovation, AbP80140-A-SE), anti-OsMKK4 (Beijing Protein

635 Innovation, AbP80132-A-SE), and anti-Phospho-p44/42 (CST, 4370) antibodies. PCR

636 primers used for in vitro phosphorylation assays are listed in Supplemental Data Set 1.

637

638 Dual-luciferase transient expression assays in rice protoplasts

639 The dual-luciferase transient expression assays were performed as previously

640 described (Su et al., 2016). The promoter of OsCKX2 was ampliﬁed from FAZ1

641 genomic DNA and cloned upstream of the ﬁreﬂy luciferase (LUC) coding region to

642 form the LUC reporter vector. The reporter vector included two expression cassettes:

643 the target promoter driving the LUC reporter gene and a CaMV 35S promoter driving

644 the Renilla luciferase (REN) gene as an internal control. To construct the different

645 effector vectors, the full-length coding sequences of DST, OsMKK4, and OsMPK6

646 were ampliﬁed and cloned into the vectors driven by the CaMV 35S promoter. The

647 plasmids were puriﬁed using a HiSpeed Plasmid Midi Kit (Qiagen) and introduced

648 into rice protoplasts by polyethylene glycol-mediated transformation. Transformed

649 protoplasts were incubated at 22 °C for 16 h in the dark. The LUC and REN luciferase

650 activities were measured using a Dual-Luciferase Reporter Assay System (Promega)

651 according to the manufacturer’s instructions. The LUC/REN ratio represents relative

23 652 transcriptional activity. The PCR primers are listed in Supplemental Data Set 1.

653

654 Plant protein extraction and immunoblot analysis

655 Protein extraction and immunoblot assays were performed as previously described

656 (Guo et al., 2019). Young panicle samples were harvested, and soluble proteins were

657 extracted with a Plant Total Protein Extraction Kit (Sigma) according to the

658 manufacturer’s instructions. In brief, the pooled young panicles were ground to a ﬁne

659 powder in liquid nitrogen. The powder was rinsed with methanol solution, followed

660 by acetone. The supernatant from the ﬁnal extraction with acetone was removed, and

661 the samples were dried and dissolved. Proteins were denatured by the addition of

662 concentrated SDS loading buffer, followed by boiling for 5 min, and separated by 10%

663 SDS-PAGE. The endogenous protein levels of OsER1, OsMKK4, OsMPK6,

664 OsMPK3, OsCKX2, and GSN1 were visualized by immunoblot analysis using

665 anti-OsER1 (ABclonal, WG02338), anti-OsMKK4 (Beijing Protein Innovation,

666 AbP80132-A-SE), anti-OsMPK6 (Beijing Protein Innovation, AbP80140-A-SE),

667 anti-OsMPK3 (Beijing Protein Innovation, AbP80147-A-SE), anti-OsCKX2 (Beijing

668 Protein Innovation, AbP80080-A-SE), and anti-GSN1 (ABclonal, WG01927)

669 antibodies, respectively. Phosphorylated OsMPK6 was visualized by immunoblot

670 analysis using anti-Phospho-p44/42 antibody (CST, 4370). The loading control was

671 visualized using anti-Actin antibody (Abmart, M20009).

672

673 Statistical analysis

674 For panicle phenotype analysis, gene expression assays, comparison of endogenous

675 cytokinin levels, and dual-luciferase transient expression assays, statistical analysis

676 was carried out as described in the figure legends. Significant differences were

677 determined with paired two-tailed Student's t-test. All analyses were performed using

678 GraphPad Prism 8 software.

679

680 Accession numbers

681 Sequence data from this article can be found in the MSU Rice Genome Annotation

24 682 Project Database under the following accession numbers: OsER1, LOC_Os06g10230;

683 OsMKKK10, LOC_Os04g47240; OsMKK4, LOC_Os02g54600; OsMPK6,

684 LOC_Os06g06090; DST, LOC_Os03g57240; OsCKX2, LOC_Os01g10110; GSN1,

685 LOC_Os05g02500; OsMPK3, LOC_Os03g17700.

686

687 Supplemental Data

688 Supplemental Figure 1. Deletion of OsER1 causes an increase in spikelet number

689 per panicle phenotype resembling that of the osckx2 mutant in the japonica variety

690 ZH11.

691 Supplemental Figure 2. Genotyping of the oser1 and osckx2 mutants.

692 Supplemental Figure 3. Overexpression of OsER1 has no effect on spikelet number

693 per panicle.

694 Supplemental Figure 4. OsER1 regulates localized cell proliferation during young

695 panicle developmental.

696 Supplemental Figure 5. The GSN1-OsMPK6 module regulates spikelet number per

697 panicle in rice.

698 Supplemental Figure 6. OsER1 and the OsMKKK10-OsMKK4-OsMPK6 cascade

699 negatively regulate the number of spikelets per panicle.

700 Supplemental Data Set 1. Primers used in this study.

701

702 ACKNOWLEDGEMENTS

703 We are grateful for the support of Shanghai Post-doctoral Excellence Program and

704 SA-SIBS Scholarship Program. We thank Min Shi (CAS Centre for Excellence in

705 Molecular Plant Sciences, CAS) for technical support. We thank Professor Fan Chen

706 (Institute of Genetics and Developmental Biology, CAS) for the dsg1 mutant. We also

707 thank Wuhan Greensword Creation Technology Co., Ltd. for help with cytokinin

708 measurements. This work was supported by grants from the National Natural Science

709 Foundation of China (31788103), Chinese Academy of Sciences (XDB27010104,

710 QYZDY-SSW-SMC023), Guangdong Laboratory of Lingnan Modern Agriculture,

711 Ministry of Science and Technology of China (2016YFD0100902), China

25 712 Postdoctoral Science Foundation (2018M642102), Science and Technology

713 Commission of Shanghai Municipality (18JC1415000), and CAS-Croucher Funding

714 Scheme for Joint Laboratories and National Key Laboratory of Plant Molecular

715 Genetics.

716

717 AUTHOR CONTRIBUTIONS

718 H.X.L. conceived and supervised the project, and H.X.L. and T.G. designed the

719 experiments. T.G. and Z.Q.L. performed most of the experiments. J.X.S., W.W.Y.,

720 N.Q.D., and H.X.L. performed some of the experiments. T.G. and H.X.L. analyzed

721 data and wrote the manuscript.

722

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31 A B C

FAZ1 oser1 FAZ1 oser1 E D

FAZ1 oser1 FAZ1 OsER1com F ** G H I ** ** Yield per plant (g) Yield (g) per plant Spikelet number (mm) Grain length Grain width (mm) Grain width

FAZ1 oser1 FAZ1 oser1 FAZ1 oser1 FAZ1 oser1 J K L M

** Setting percentage percentage Setting Spikelet number (mm) Grain length Grain width (mm) Grain width

FAZ1 oser1 FAZ1 OsER1Com FAZ1 OsER1Com FAZ1 OsER1Com

N kDa FAZ1 oser1 OsER1Com 135- Anti-OsER1 100- 48- -Phos-OsMPK6 -Phos-OsMPK3 35- Anti-Phos-p44/42 48 - Anti-OsMPK6 35- 48 - Anti-OsMPK3 35- 48 - Anti-Actin 35-

Figure 1. OsER1 is responsible for rice panicle morphogenesis and negatively regulates spikelet number per panicle. (A) Plant architecture of wild-type FAZ1 and mutant oser1 plants at the reproductive stage. Scale bar, 10 cm. (B) Rice panicles from FAZ1 and oser1 plants. Scale bar, 5 cm. (C) Brown rice grains from FAZ1 and oser1. Scale bar, 2 mm. (D) Comparison of panicles between FAZ1 and the complementation line OsER1com. Scale bar, 5 cm. (E) Comparison of mature paddy rice grains between FAZ1, oser1, and OsER1com. Scale bar, 2 mm. (F) to (J) Comparisons of average spikelet number per panicle (n=15 plants) (F), grain length (n=15 plants) (G), grain width (n=15 plants) (H), yield per plant (n=10 plants) (I), and seed setting percentage (n=15 plants) (J) between FAZ1 and oser1. (K) to (M) Comparisons of average spikelet number per panicle (n=15 plants) (K), grain length (n=15 plants) (L), and grain width (n=15 plants) (M) between FAZ1 and the complementation line OsER1com. Values are given as the mean ± SD. **P < 0.01 compared with the wild type using Student’s t-test. (N) The phosphorylation level of OsMPK6 was reduced in the oser1 mutant but restored in OsER1com. Samples were prepared from 0.1-0.5 cm young panicles pooled from different plants and subjected to immunoblot analysis with the anti-OsER1, anti-Phospho-p44/42, anti-OsMPK6, and anti-OsMPK3 antibodies. The anti-Actin antibody was used as the loading control. A #1 #2 C #1 #2

gsn1 gsn1 oser1 gsn1 oser1 oser1 oser1/OsMKK4CA oser1/OsMKK4CA B #1 D #1 #2

FAZ1 oser1 GSN1OE oser1 oser1/OsMKKK10CA oser1/OsMKKK10CA E F G H ** ** ** ** ** ** ** ** Spikelet number Spikelet number Spikelet number Spikelet number

#1 #2 #1 #2 #1 #2 gsn1 oser1 oser1/OsMKK4CA oser1/OsMKKK10CA

I J OE gsn1 oser1 GSN1 kDa kDa #1 #2 48- -Phos-OsMPK6 48 Phos-OsMPK6 Anti-Phos-p44/42 - - Anti-Phos-p44/42 35- 35- 100 100 - Anti-GSN1 - Anti-GSN1 75- 75- 48 48 - Anti-OsMPK6 - Anti-OsMPK6 35- 35- 48 48 - Anti-Actin - Anti-Actin 35- 35- K L

kDa kDa 48- -Phos-OsMPK6 48- -Phos-OsMPK6 35- Anti-Phos-p44/42 35- Anti-Phos-p44/42 48 135 - Anti-OsMKK4 - Anti-Flag 35- 100- 48 48 - Anti-OsMPK6 - Anti-OsMPK6 35- 35- 48 - Anti-Actin 48- Anti-Actin 35- 35-

Figure 2. OsER1 acts upstream of the OsMKKK10-OsMKK4-OsMPK6 cascade to negatively regulate spikelet number per panicle. (A) Panicles from gsn1 mutant and gsn1 oser1 double mutant rice plants. Scale bar, 5 cm. (B) Comparison of panicles between FAZ1 and oser1 and GSN1 overexpression line (GSN1OE). Scale bar, 5 cm. (C) Panicles from oser1 mutant and oser1 plants expressing constitutively active OsMKK4 (oser1/OsMKK4CA). Scale bar, 5 cm. (D) Panicles from oser1 mutant and oser1 plants expressing constitutively active OsMKKK10 (oser1/OsMKKK10CA). Scale bar, 5 cm. (E) to (H) Comparisons of average spikelet number per panicle for gsn1 and gsn1 oser1 double mutant plants (n=10 plants) (E), FAZ1 and oser1 and GSN1OE (n=10 plants) (F), oser1 and oser1/OsMKK4CA lines (n=10 plants) (G), and oser1 and oser1/OsMKKK10CA lines (n=10 plants) (H). Values are given as the mean ± SD. **P < 0.01 compared with the wild type using Student’s t-test. (I) to (L) Comparisons of the phosphorylation levels of OsMPK6 in oser1 mutant and gsn1 oser1 double mutant (I), in the oser1 and GSN1OE lines (J), in the oser1 and oser1/OsMKK4CA lines (K), and in the oser1 and oser1/OsMKKK10CA lines (L). Proteins extracted from pooled 0.1-0.5 cm young panicles were subjected to immunoblot analysis with anti-Phospho-p44/42, anti-OsMPK6, anti-OsMKK4, anti-GSN1, or anti-Flag antibodies. The anti-Actin antibody was used as the loading control. A B C FAZ1 oser1 FAZ1 oser1 kDa ** 63- 48- ** Anti-OsCKX2 ** OsCKX2

of of ** FAZ1 oser1 48 ** 35- Relative Relative expression level - (ng/g) content Cytokinin ** ** Anti-Actin iP iPR tZ tZR cZ cZR DHZ DHZR

D F FAZ1 osmkkk10 osmkk4 osmpk6

** ** ** ** ** ** OsCKX2

of of ** ** ** Cytokinin (ng/g) content Cytokinin

Relative Relative expression level ** * ** * **

iP iPR tZR DHZR

E G 6 ** FAZ1 oser1 FAZ1 osmkkk10 osmkk4 osmpk6 kDa 63- 4 ** 48- Anti-OsCKX2 ** ** 2 ** FAZ1 osmkkk10 osmkk4 osmpk6 48 35- - Relative expression level 0 Anti-Actin OsRR1 OsRR2 OsRR3 OsRR4 OsRR5 OsRR6 OsRR7

Figure 3. The OsER1-OsMKKK10-OsMKK4-OsMPK6 pathway is required to modulate cytokinin metabolism. (A) Relative expression levels of OsCKX2 in FAZ1 and the oser1 mutant (n=3 pools, and five 0.1-0.5 cm young panicles per pool). The UBQ5 gene was used as the internal reference to normalize gene expression data. (B) The levels of OsCKX2 protein in young panicles of FAZ1 and the oser1 mutant. Proteins extracted from pooled 0.1-0.5 cm young panicles were subjected to immunoblot analysis using the anti-OsCKX2 antibody. The anti-Actin antibody was used as the loading control. (C) Comparison of endogenous cytokinin (iP, iPR, tZ, tZR, cZ, cZR, DHZ, and DHZR) levels in young panicles (0.1-0.5 cm) between FAZ1 and the oser1 mutant (n=3 pools, and 20 young panicles per pool). (D) Relative expression levels of OsCKX2 in FAZ1, osmkkk10, osmkk4, and osmpk6 (n=3 pools, and five 0.1-0.5 cm young panicles per pool). The UBQ5 gene was used as the internal reference to normalize gene expression data. (E) The levels of OsCKX2 protein in young panicles of FAZ1, osmkkk10, osmkk4, and osmpk6. Proteins extracted from pooled 0.1-0.5 cm young panicles were subjected to immunoblot analysis using the anti- OsCKX2 antibody. The anti-Actin antibody was used as the loading control. (F) Comparison of endogenous cytokinin (iP, iPR, tZR, and DHZR) levels in young panicles (0.1-0.5 cm) between FAZ1 and the osmkkk10, osmkk4, and osmpk6 mutants (n=3 pools, and 20 young panicles per pool). (G) Relative expression levels of the cytokinin signal transduction-related genes OsRRs in FAZ1 and the oser1 mutant (n=3 pools, and five 0.1-0.5 cm young panicles per pool). The UBQ5 gene was used as the internal reference to normalize gene expression data. Values are given as the mean ± SD. *P < 0.05; **P < 0.01 compared with the wild type using Student’s t-test. A SD/-Trp-Leu-His SD/-Trp-Leu-His-Ade C 1 10-1 10-2 1 10-1 10-2

BK-OsMPK6/AD nYFP-OsMPK6+ cYFP-DST BK/AD-OsMPK6

BK-DST/AD

BK/AD-DST cYFP-OsMPK6+ BK-OsMPK6/AD-DST nYFP-DST

BK-DST/AD-OsMPK6

BK-53/AD-T nYFP-OsMPK6 BK-Lam/AD-T +cYFP-OsHAL3

B MBP + - + - D DST-Flag + - + MBP-OsMPK6 - + - + His-DST + + + + OsMPK6-Myc - + + WB kDa WB kDa Anti-His 63 48 - Anti-Flag - His-DST- 48 35 - Input - Anti-MBP -63 100 Anti-Myc - 48 MBP-OsMPK6 75 - - - 48 Anti-Flag - -48 IP -35 Flag -63 MBP 35 Anti-Myc - - -48

Input Pull down E MBP-OsMKK4CA - - + + + + MBP-OsMPK6 - + - + + + His-DST + + + - + - His-DST4A - - - - - + WB kDa -68 Anti-pThr -52 -68 Anti-His -52 100 Anti-OsMPK6 - -68 100 Anti-OsMKK4 - -68 100 Anti-Phos-p44/42 - -68

OsMPK6 -OsMKK4 CBB -DST

Figure 4. OsMPK6 interacts with and phosphorylates DST. (A) Yeast two-hybrid assays indicating that OsMPK6 interacts with DST. The yeast cells were cultured on SD/-Trp-Leu-His (medium without tryptophan, leucine, and histidine) or SD/-Trp-Leu-His-Ade (medium without tryptophan, leucine, histidine, and adenine) containing X-ɑ-gal. BK (pGBKT7) and AD (pGADT7) are the bait and prey vectors, respectively. (B) In vitro pull-down assays of His-tagged DST using MBP-tagged OsMPK6. Pull-down was verified by immunoblotting with anti-His and anti-MBP antibodies. (C) OsMPK6 associates with DST, as shown by BiFC assays in N. benthamiana leaf cells. OsMPK6 and DST were both fused to the N-terminal fragment of YFP (nYFP) and the C-terminal fragment of YFP (cYFP). nYFP-OsMPK6 and cYFP-DST or cYFP-OsMPK6 and nYFP-DST were then co-expressed in N. benthamiana leaves. The nYFP-OsMPK6 and cYFP-OsHAL3 fusion proteins were used as negative controls. (D) Co-immunoprecipitation assays indicating that OsMPK6 interacts with DST in planta. Pro35S:DST- Flag and Pro35S:OsMPK6-Myc fusions were co-expressed in N. benthamiana leaves. Proteins were extracted (Input) and immunoprecipitated (IP) with Flag beads. The immunoblot assays were performed using anti-Flag and anti-Myc antibodies. (E) OsMPK6 phosphorylates DST in vitro. The MBP-OsMKK4CA, MBP-OsMPK6, His-DST, and His-DST4A fusion proteins were expressed in E. coli and purified. The in vitro phosphorylation reactions were performed using the purified proteins. DST phosphorylation was detected with the anti-Phospho- Threonine antibody. Recombinant OsMKK4, OsMPK6, and DST were detected using anti-OsMKK4, anti- OsMPK6, and anti-His, respectively. Phosphorylated OsMPK6 was detected with anti-Phospho-p44/42. The gel stained with Coomassie brilliant blue (CBB) was used as a loading control. A B C ** ** Reporter 35S REN ProOsCKX2 LUC **

Effectors 35S DST

35S DST4A ** ** 35S DST4D Relative activity Relative LUC Relative activity Relative LUC

35S OsMKK4CA

35S OsMPK6

35S OsMPK6CA

D F H #1 #2 #1 #2 kDa 63 - Anti-OsCKX2 48 48- - Anti-Flag 35- 48 - Anti-Actin dsg1 dsg1/DSTOE dsg1 dsg1/OsCKX2OE 35- E #1 #2 G #1 #2 I

kDa 63- Anti-OsCKX2 48- 48 - Anti-Actin 35- dsg1 dsg1/DSTOE dsg1 dsg1/OsCKX2OE

J K L M

** ** ** ** ** ** ** ** Plant height (cm) Plantheight Plant (cm) height Spikelet number Spikelet number

#1 #2 #1 #2 #1 #2 #1 #2 dsg1/DSTOE dsg1/DSTOE dsg1/OsCKX2OE dsg1/OsCKX2OE

Figure 5. OsMPK6-mediated phosphorylation of DST enhances its transcriptional activity and positively regulates OsCKX2 expression. (A) Schematic diagram of the reporter and effector constructs used in the dual luciferase assay system. (B) and (C) Transcriptional activation of OsCKX2 is enhanced by OsMKK4-OsMPK6 cascade-mediated DST phosphorylation (B), and constitutively activated DST (DST4D) (C) in rice protoplasts. Protoplasts were co-transformed with the reporter and different effector constructs and kept in the dark for 16 h. The relative ratios of ﬁreﬂy luciferase (LUC) to renilla luciferase (REN) activity are shown (n=3 independent biological repeats). Values are given as the mean ± SD. **P < 0.01 compared with the control using Student’s t-test. (D) Plant architecture of dsg1 mutant and dsg1 plants overexpressing DST (dsg1/DSTOE) at the reproductive stage. Scale bar, 10 cm. (E) Panicle architecture of dsg1 mutant and dsg1/DSTOE plants at the reproductive stage. Scale bar, 5 cm. (F) Plant architecture of dsg1 mutant and dsg1 plants overexpressing OsCKX2 (dsg1/OsCKX2OE) at the reproductive stage. Scale bar, 10 cm. (G) Panicle architecture of dsg1 mutant and dsg1/OsCKX2OE plants at the reproductive stage. Scale bar, 5 cm. (H) and (I) DST protein levels in the young panicles of dsg1/DSTOE (H), and OsCKX2 protein levels in the young panicles of dsg1/OsCKX2OE (I). Proteins extracted from pooled 0.1-0.5 cm panicles were subjected to immunoblot analysis using anti-Flag and anti-OsCKX2 antibodies. The anti-Actin antibody was used as the loading control. (J) Comparison of average plant height between dsg1 and the dsg1/DSTOE transgenic lines (n=8 plants). (K) Comparison of average spikelet number per panicle between dsg1 and the dsg1/DSTOE lines (n=8 plants). (L) Comparison of average plant height between dsg1 and the dsg1/OsCKX2OE transgenic lines (n=8 plants). (M) Comparison of average spikelet number per panicle between dsg1 and the dsg1/OsCKX2OE lines (n=8 plants). Values are given as the mean ± SD. **P < 0.01 compared with the control using Student’s t-test. A #1 #2 B #1 #2

FAZ1 oser1 oser1/DSTOE FAZ1 osmkkk10 osmkkk10/DSTOE C #1 #2 D #1 #2

FAZ1 oser1 oser1/OsCKX2OE FAZ1 osmkk4 osmkk4/OsCKX2OE

E F G H

** ** ** ** ** ** ** ** Spikelet number Spikelet number Spikelet number Spikelet number

#1 #2 #1 #2 #1 #2 #1 #2 oser1/DSTOE osmkkk10/DSTOE oser1/OsCKX2OE osmkk4/OsCKX2OE

Figure 6. The OsER1-OsMKKK10-OsMKK4-OsMPK6 pathway controls DST-OsCKX2 module- dependent regulation of spikelet number per panicle. (A) Panicle architecture of oser1 mutant and oser1 plants overexpressing DST (oser1/DSTOE) at the reproductive stage. Scale bar, 5 cm. (B) Panicle architecture of osmkkk10 mutant and osmkkk10 plants overexpressing DST (osmkkk10/DSTOE) at the reproductive stage. Scale bar, 5 cm. (C) Panicle architecture of oser1 mutant and oser1 plants overexpressing OsCKX2 (oser1/OsCKX2OE) at the reproductive stage. Scale bar, 5 cm. (D) Panicle architecture of osmkk4 mutant and osmkk4 plants overexpressing OsCKX2 (osmkk4/OsCKX2OE) at the reproductive stage. Scale bar, 5 cm. (E) to (H) Comparisons of average spikelet number per panicle between oser1 mutant and oser1/DSTOE lines (n=8 plants) (E), osmkkk10 and osmkkk10/DSTOE lines (n=8 plants) (F), oser1 and oser1/OsCKX2OE lines (n=8 plants) (G), and osmkk4 and osmkk4/OsCKX2OE lines (n=8 plants) (H). Values are given as the mean ± SD. **P < 0.01 compared with oser1, osmkkk10 or osmkk4 using Student’s t-test. Figure 7. Proposed working model of the role of the OsER1-OsMKKK10-OsMKK4-OsMPK6-DST- OsCKX2 pathway in determining spikelet number per panicle in rice. During panicle morphogenesis, when the active reproductive meristem produces cell-specific signaling molecules such as small secreted peptides (SSPs), the RLK OsER1 binds to the signals and activates the OsMKKK10-OsMKK4-OsMPK6 cascade directly or through unknown mediators. The activated OsMKKK10-OsMKK4-OsMPK6 cascade phosphorylates the transcription factor DST to enhance its transcriptional activity and positively increase the expression level of OsCKX2. This results in the degradation of cytokinin, thereby maintaining proper cytokinin levels for spikelet differentiation and formation. Meanwhile, the spatiotemporally activated GSN1 protein acts like a molecular brake to negatively regulate the OsER1-OsMKKK10-OsMKK4-OsMPK6 pathway by inactivating OsMPK6, which attenuates the phosphorylation of DST, thereby decreasing the expression of OsCKX2 and increasing cytokinin levels. ERECTA1 Acts Upstream of the OsMKKK10-OsMKK4-OsMPK6 Cascade to Control Spikelet Number by Regulating Cytokinin Metabolism in Rice Tao Guo, Zi-Qi Lu, Jun-Xiang Shan, Wang-Wei Ye, Nai-Qian Dong and Hong-Xuan Lin Plant Cell; originally published online July 2, 2020; DOI 10.1105/tpc.20.00351

This information is current as of July 2, 2020

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