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 1 ©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). 22 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. 42 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. 61 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. 75 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