OsBSK3 and OsBSK2 regulate grain size and leaf angle via MAPK signaling pathway in rice

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OsBSKs (BRASSINOSTEROID-SIGNALING KINASES) and OsMAPKs (MITOGEN ACTIVATED PROTEIN KINASE) are known to play essential roles in plant growth, development, and stress responses. However, the potential crosstalk between these pathways and their specific roles in regulating grain size and leaf angle remain largely unexplored in rice. Here, we characterized OsBSKs regulate grain size and leaf angle in rice, and among these, OsBSK2 and OsBSK3 may play more critical roles. The grain size and leaf angle in osbsk3 and osbsk2 mutants are significantly smaller, whereas the OsBSK3 -overexpressing lines ( OsBSK3 -OEs) exhibit considerably larger grain size and leaf angle compared to the others. Furthermore, both OsBSK3 and OsBSK2 interact with OsMKKK10, indirectly activating OsMAPK6 in plant cells. Notably, mutations in MAPK cascade components, such as smg2-1 ( osmkkk10 mutant), smg1-1 ( osmkk4 mutant), and dsg1 ( osmapk6 mutant), resulted in significantly reduced leaf angles. Moreover, these mutations were able to rescue the increased grain size and leaf angle in OsBSK3 overexpression lines. Additionally, we also identified OsWRKY53 as a potential downstream target of the OsBSKs-OsMKKK10-OsMKK4-OsMAPK6 cascade in regulation of grain size and leaf angle. Taken together, the above results not only highlight the essential and specific roles of OsBSK3 and OsBSK2 in regulating rice grain size and leaf angle, but also reveal the mechanism which OsBSK3/OsBSK2 mediating MAPK cascade to regulate grain size and leaf angle, OsBSK3 and OsBSK2 may act as key mediator of crosstalk between BR and MAPK signaling. MAPK cascades OsBSK3 OsBSK2 Grain size Leaf angle Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Rice ( Oryza sativa L.) is a staple food crop, providing sustenance to over half of the global population (Wang and Li, 2011 ). Increasing rice yield is crucial for ensuring global food security. Grain size and leaf angle are the key agronomic traits that determine grain yield in crops. Erect leaves facilitate high-density planting, and grain size is directly correlated with yield per plant (Liu et al., 2021 ). So far, several genes regulating rice grain size and leaf angle have been identified (Fan et al., 2006 ; Li et al., 2011 ; Ishimaru et al., 2013 ; Liu et al., 2017 ; Qiao et al., 2017 ; Liu et al., 2021 ). However, the genetic and molecular mechanisms that determine these traits are still largely unknown. Identifying the genetic components that regulate rice grain size and leaf angle holds promise for crop improvement. Recent advances have identified several signaling pathways that controlling grain size in rice, including the ubiquitin-proteasome pathway, G-protein signaling, mitogen-activated protein kinase (MAPK) signaling, some transcriptional regulators, and phytohormone perception and homeostasis, such as Brassinosteroid (BR) signaling and auxin signaling (Li et al., 2019a ; Ren et al., 2023 ). Leaf angle, the inclination angle between the vertical culm and leaf blade, contributes to rice architecture. Suitably compact plants with erect leaves exhibit enhanced photosynthetic efficiency under dense planting conditions, thereby increasing yields (Liu et al., 2021 ). Various plant hormones, such as BRs, auxin, and gibberellic acid, are involved in controlling leaf angle (Luo et al., 2016 ; Nolan et al., 2020 ; Liu et al., 2021 ). Phytohormones signaling and crosstalk regulate grain size and leaf angle in rice, BRs and auxin are reported to simultaneously regulate these two traits (Luo et al., 2016 ; Jang et al., 2017 ; Qiao et al., 2017 ). Receptor-like protein kinases (RLKs) constitute an important kinase family in plants. Some RLKs, called cytoplasmic receptor-like kinases (RLCKs), lack extracellular domains (Shubha et al., 2008 ; Liang and Zhou, 2018 ). BRASSINOSTEROID-SIGNALING KINASES (BSKs) belong to the RLCK XII superfamily, BR induces the BR receptor BR INSENSITIVE1 (BRI1) to phosphorylate and activate BSKs, which then phosphorylate BRI1-SUPPRESSOR1 (BSU1) to transduce the BR signal in Arabidopsis (Tang et al., 2008 ; Kim et al., 2009 ). The BSKs gene originated in embryophytes and plays a role in various stages of plant development (Li et al., 2019b ). Rice contains five OsBSK members, OsBSK1-1 likely serves as a scaffold protein, directly bridging OsBRI1 and OsGSK2 to positively regulate BR signaling (Tian et al., 2023 ). OsBSK1-2 functions as an important regulator in rice plant immunity, and silencing OsBSK1-2 decreased resistance to M. oryzae (Li et al., 2024 ). OsBSK2, a putative brassinosteroid-signalling kinase, positively controls grain size and can form homodimers or heterodimers with OsBSK3 and OsBSK4 in rice (Yuan et al., 2022 ). OsBSK3 is a positive regulator of BR signaling in rice, OsBRI1 interacts directly with and phosphorylates OsBSK3, disrupting the interaction between its TPR and kinase domains (Zhang et al., 2016 ). Additionally, AtBSK3 promotes root elongation by interacting with AtBAK1 under mild nitrogen deficiency conditions in Arabidopsis (Jia et al., 2019 ). However, although certain functions of OsBSKs have been reported in rice, their overall roles and mechanisms remain poorly understood currently. The MAPK cascades consist of protein kinases (MAPKs), MAPK kinases (MAPKKs), and MAPKK kinases (MAPKKKs). In cellular activity, upstream signals recognition activates MAPKKKs, further phosphorylate MAPKs to amplify the signal through the cascade pathway (Liu et al., 2021 ). MAPK cascades play different roles in plant growth, morphogenesis, signal transduction, and responses to biological and abiotic stress by phosphorylating various substrates (Zhang and Zhang, 2022 ). The single mutants osmkkk10 , osmkk4, osmapk6 exhibit small and light grains. The OsMPKKK10-OsMKK4-OsMAPK6 cascade functions in a common pathway to control grain size and spikelet shape in rice. Additionally, OsWRKY53 acts genetically downstream of this cascade to control leaf angle and seed size (Xu et al., 2018 ; Guo et al., 2020 ; Tian et al., 2021 ). Besides, the OsYDA1/OsYDA2-OsMKK4-OsMPK6 cascade plays a crucial role in tapetal development and male gametophyte fertility (Zeng et al., 2024 ). Studies report that AtMKK4/5-AtMPK3/6 negatively regulate freezing tolerance, while AtMEKK1-AtMKK2-AtMAPK4 positively regulate it in Arabidopsis (Teige et al., 2004 ; Li et al., 2017 ; Zhao et al., 2017 ). However, cold activates OsMAPK3/6, which then phosphorylates and stabilizes OsICE1 to positively regulate cold tolerance at the seedling stage in rice (Zhang et al., 2017 ; Liu et al., 2024 ). Moreover, OsMAPK6 also interacts with and phosphorylates OsWRKY78, enhancing its stability to regulate rice panicle exsertion (Mei et al., 2024 ). The pathogen-inducible OsMPKK10.2-OsMPK6 cascade phosphorylates the Raf-like kinase OsEDR1, inhibiting its scaffold function to promote rice disease resistance (Ma et al., 2021 ). Recent studies have shown that YDA-MKK4/MKK5-MPK3/MPK6 can regulate stomatal development (Li et al., 2014 ; Wu et al., 2024 ). Furthermore, rice Salt Intolerance 1 (OsSIT1) is reported to act upstream of MAPK6/3, regulating the accumulation of ROS under salt stress (Li et al., 2014 ). Although MAPK cascades have been reported to be involved in regulating various biological functions, but their role of MAPK cascades in regulating leaf angle are still largely unknown. Similarly, the molecular mechanism by how MAPK perceives and transmits upstream signals to regulate rice grain size and leaf angle is poorly understood. In this study, we found that OsBSKs regulate rice grain size and leaf angle, with OsBSK3 and OsBSK2 playing more critical roles. The grain size and leaf angle in osbsk3 and osbsk2 mutants are significantly smaller, whereas the OsBSK3 -overexpressing lines ( OsBSK3 -OEs) exhibit considerably larger grain size and leaf angle compared to the others. Both OsBSK3 and OsBSK2 can bind to OsMKKK10, indirectly activating OsMAPK6. Furthermore, mutations in MAPK cascade components, such as smg2-1 ( osmkkk10 mutant), smg1-1 ( osmkk4 mutant), and dsg1 ( osmapk6 mutant) showed an erect leaf angle. Moreover, these mutations were able to rescue the increased grain size and leaf angle in OsBSK3 overexpression lines. Additionally, OsWRKY53 may a target of the OsBSKs-OsMKKK10-OsMKK4-OsMAPK6 cascade that regulates rice grain size and leaf angle. Taken together, these results suggest that OsBSK3 and OsBSK2 regulate rice grain size and leaf angle via the OsMKKK10-OsMKK4-OsMAPK6-OsWRKY53 cascade, OsBSK3 and OsBSK2 may mediate the crosstalk between BR and MAPK signal. Results OsBSK3 and OsBSK2 play a more critical role in regulating grain size and leaf angle compared to other OsBSKs gene. The BSKs family genes play a crucial role in regulating plant growth and development, including plant defense and BR signaling responses (Shubha et al., 2008 ; Bi et al., 2018 ). In our previous study, we generated several mutants of the OsBSK family members. We found that most osbsks mutants have smaller grain size, while the OsBSKs-OE ( OsBSKs overexpression lines) have larger grain size (Fig. 1 A- 1 F). Surprisingly, compared to other OsBSKs members, osbsk3 and osbsk2 have significantly shorter grain length and width (Fig. 1 A- 1 F). These indicate that OsBSKs members have functional redundancy in regulating grain size, and OsBSK3 and OsBSK2 may play a more critical role. Subsequently, we observed their plant architecture, and found that the leaf angle of osbsk2 and osbsk3 decreased significantly, while the leaf angle of OsBSK3-OE and OsBSK4-OE increased significantly (Fig. 1 G- 1 L). This suggests that OsBSKs family genes also play an important role in regulating rice leaf angle. To further verify this result, we generated multiple independent osbsk3 , osbsk2 and OsBSK3-OEs plants for careful observation. Through years of continuous statistical analysis of the leaf angle of these mutants grown in the fields, we confirmed that osbsk3 and osbsk2 indeed have a decreased leaf angle, while OsBSK3-OEs have an significantly increased leaf angle (Supplemental Fig. 1–3). Collectively, these findings imply that OsBSKs play an essential role in regulating rice grain size and leaf angle, with OsBSK3 and OsBSK2 potentially playing a more pivotal role. OsBSK3 and OsBSK2 may also be involved in other signaling pathways beyond brassinosteroid (BR) signaling. OsBSK3 and OsBSK2 interact with OsMKKK10 To investigate the molecular mechanism by which OsBSK2 and OsBSK3 regulate rice grain size and leaf angle, we conducted RNA-Seq analysis on 3 cm young spike from osbsk3 mutant and control plants, identifying 1834 differently expressed genes (Supplemental Fig. 4A and 4B). KEGG pathway analysis revealed significant enrichment of these genes in the MAPK signaling pathway, suggesting a crucial role for OsBSK3 in this pathway (Supplemental Fig. 4C). Given that previous reports have shown multiple members of RLCKs can bind to MKKKs, we performed the LCI (luciferase complementary imaging) assay to examine the interactions between OsBSKs and OsMKKKs. Constructs expressing OsBSK3 and OsBSK2 fused to the C-terminal part of firefly luciferase (cLUC-OsBSK2 and cLUC-OsBSK3) and OsMKKKs fused to the N-terminal part (nLUC-OsMKKKs) were generated. Strong LUC activity was observed when nLUC-OsMKKK10 were co-infiltrated with cLUC-OsBSK3 or cLUC-OsBSK2 into N. benthamiana leaves, indicating interactions between OsMKKK10 and both OsBSK3 and OsBSK2 in planta (Fig. 2 A and 2 B). These interactions were confirmed using BiFC (Bimolecular Fluorescence Complementation) assay, which showed strong GFP fluorescence in N. benthamiana leaves co-transformed with cGFP-OsOsBSK3 and nGFP-OsMKKK10, as well as cGFP-OsOsBSK2 and nGFP-OsMKKK10 (Fig. 2 C). Further validation using a yeast two-hybrid assay demonstrated that OsBSK2 interacts with the kinase domain of OsMKKK10 in vitro (Fig. 2 D and 2 E). The interaction between OsBSK2 and OsMKKK10 was further validated through Co-IP (co-immunoprecipitation) in rice protoplasts (Fig. 2 F). Collectively, these results indicate that OsMKKK10 interacts with both OsBSK3 and OsBSK2. OsBSK3 and OBSK2 activating OsMAPK6 Despite interactions between OsBSK3, OsBSK2 with OsMKKK10, we were unable to show whether OsBSK3 and OsBSK2 can phosohorylate OsMKKK10 in vitro due to difficulties in detecting their kinase activity (Supplemental Fig. 5). Given that MAPK cascades function downstream of RLK signaling in Arabidopsis and the OsMKKK10-OsMKK4-OsMAPK6 function in a cascade during control of grain size and weight in rice ((Xu and Zhang, 2015 ; Xu et al., 2018 ). We investigated whether OsBSK3 and OsBSK2 can indirectly regulate OsMAPK6 activity. In a rice protoplasts transient expression assay, co-transformed the constructs expressing either Flag-OsBSK3 or Flag-OsBSK2 significantly enhanced the phosphorylation level of OsMAPK6, as detected by an anti-pTEpY (anti phospho-p44/42 MAPK) antibody (Fig. 3 A), which specifically recognizes the phosphorylated TEY loop motif of MAPK, detecting phosphorylated OsMAPK6 (Liu et al., 2021 ). This suggests that both OsBSK3 and OsBSK2 can activate OsMAPK6 in plant cells. To further verify this result, we assayed the phosphorylation level of OsMAPK6 in the 3 cm young spike of OsBSK3- OE, OsBSK2- OE, osbsk3 and osbsk2 plants. We found that gain-of-function of OsBSK3 and OsBSK2 increased the phosphorylation of OsMAPK6, while loss-of-function suppressed it (Fig. 3 B- 3 E). Overall, these results indicate that OsBSK3 and OBSK2 can activate OsMAPK6 in vivo. The OsMKKK10-OsMKK4-OsMAPK6 cascade plays a mediating role in the control of rice leaf angle The MAPK cascade pathway plays a crucial role in plant growth, development, and the response to both biotic and abiotic stresses (Zhang et al., 2018 ). Specifically, the OsMKKK10-OsMKK4-OsMAPK6 cascade regulates rice grain size and weight (Xu et al., 2018 ). additionally, ER1 functions upstream of the OsMKKK10-OsMKK4-OsMAPK6 cascade to regulate spikelet number by modulating cytokinin metabolism (Guo et al., 2020 ). To investigate whether the OsMKKK10-OsMKK4-OsMAPK6 cascade can regulate rice leaf angle, we conducted field trails in Hainan and Harbin using smg2-1 (a small grain 2 − 1 mutant, which is a osmkkk10 mutant), smg1-1 (a small grain 1–1 mutant, which is a osmkk4 mutant), and dsg1 (a dwarf and small grains 1 mutant, which is a weak allele of OsMAPK6 ) (Liu et al., 2015 ; Xu et al., 2018 ). Our results showed that smg2-1 , smg1-1 , and dsg1 exhibited significantly more erect leaf angle compared to their respective controls, regardless of whether they were planted in Hainan or Harbin (Fig. 4 A- 4 F). These findings suggest that OsMKKK10, OsMKK4, and OsMAPK6 regulate leaf angle development in rice. OsBSK3 and the OsMKKK10-OsMKK4-OsMAPK6 cascade may potentially function within a shared pathway to regulate rice grain size and leaf angle As described above, OsBSK3 and OsBSK2 can interact with OsMKKK10, activating the OsMAPK6 in vivo (Figs. 2 and 3 ). Both OsBSK3, OsBSK2 and OsMKKK10-OsMKK4-OsMAPK6 can regulate rice grain size and leaf angle (Xu et al., 2018 ) (Figs. 1 and 4 ). We investigated whether OsBSKs function genetically in a common signaling cascade with OsMKKK10-OsMKK4-OsMAPK6 to regulate rice grain size and leaf angle. First, we obtained OsBSK3- OEs under KYJ (genetic background of smg2-1 ), SF43 (genetic background of smg1-1 ), and ZH11 (genetic background of dsg1 ) (Supplemental Fig. 6A-6C). We found OsBSK3- OEs in different genetic backgrounds still display increased grain size and leaf angle, consistent with above mentioned (Supplemental Fig. 6D -6L). To further explore this, we overexpressed the OsBSK3 in smg2-1 , smg1-1 and dsg1 , generating OsBSK3- OE smg2-1 , OsBSK3- OE smg1-1 , and OsBSK3- OE dsg1 lines respectively (Fig. 5 A- 5 C; Supplemental Fig. 7A to 7C). It was shown that OsBSK3- OE smg2-1 , OsBSK3- OE smg1-1 , and OsBSK3- OE dsg1 lines show similar grain size and leaf angle to s mg2-1 , smg1-1 , and dsg1 (Fig. 5 D- 5 O). implying that these mutations were able to rescue the increased grain size and leaf angle in OsBSK3 overexpression lines, OsBSK3 may act upstream of the OsMKKK10-OsMKK4-OsMAPK6 cascade in regulating rice grain size and leaf angle. OsWRKY53 may a target of the OsBSKs-OsMKKK10-OsMKK4-OsMAPK6 cascade that regulates rice grain size and leaf angle Our previously reported data indicates that the OsMKK4-OsMAPK6 cascade phosphorylates and activates OsWRKY53, and the OsMKK4, OsMAPK6, and OsWRKY53 cascade might function in the same pathway to regulate rice grain size (Tian et al., 2017 ; Tian et al., 2021 ). We then asked whether OsWRKY53 is a downstream component of OsBSKs-regulated signaling in regulating grain size and leaf angle. We therefore Knocking out OsWRKY53 in N390 , which overexpressing the kinase domain (amino acids 1-390) of OsBSK3 (N390), and obtained N390 oswrky53 lines (Zhang et al., 2016 , plant physiology) (Fig. 6 A). We found that N390 oswrky53 lines show similar grain size and leaf angle to oswrky53 , oswrky53 can fully rescue the increased grain size and leaf angle in N390 (Fig. 6 A-E). To further verify this result, we overexpressed OsWRKY53 in the osbsk3 background, generating osbsk3 OsWRKY53-OE lines (Fig. 6 F). Phenotypic analysis demonstrated that OsWRKY53 overexpression fully rescued the reduced leaf angle and grain size in osbsk3 mutants (Fig. 6 G-J). These results implied that OsWRKY53 is a key downstream target of the OsBSKs-OsMKKK10-OsMKK4-OsMAPK6 cascade in regulating seed size and leaf angle in rice. Discussion In this study, we elucidate the regulatory roles of OsBSK3 and its homolog OsBSK2 in controlling grain size and leaf angle through the OsMKKK10-OsMAPK4-OsMAPK6-OsWRKY53 signaling cascade. First, OsBSKs regulate grain size and leaf angle, with OsBSK2 and OsBSK3 playing more critical roles. The grain size and leaf angle in osbsk3 and osbsk2 are more smaller, while OsBSK3- overexpressing lines ( OsBSK3- OEs) have more larger grain size and leaf angle (Fig. 1 and Supplemental Fig. 1–3). Second, both OsBSK3 and OsBSK2 can bind to OsMKKK10, directly activate OsMAPK6 in vivo (Fig. 2 , 3 and Supplemental Fig. 4, 5). Third, the leaf angle in smg2-1 ( osmkkk10 mutant), smg1-1 ( osmkk4 mutant), and dsg1 ( osmapk6 mutant) showed a significant decreased (Fig. 4 ). Fourth, mutation in MAPK component (s mg2-1 , smg1-1 , and dsg1 ) can rescue the increased grain size and leaf angle in OsBSK3 overexpression line (Fig. 5 and Supplemental Fig. 6, 7). Lastly, OsWRKY53 may a target of the OsBSKs-OsMKKK10-OsMKK4-OsMAPK6 cascade in regulating rice grain size and leaf angle. Knockout OsWRKY53 can fully complement the increased leaf angle and grain size in N390 , and OsWRKY53 overexpression can also rescue the decreased leaf angle and grain size in osbsk3 (Fig. 6 and Supplemental Fig. 8, 9). Collectively, these results indicate that OsBSK3 and OsBSK2 regulate rice grain size and leaf angle through the OsBSK3/OsBSK2-OsMKKK10-OsMAPK4-OsMAPK6 cascade, OsBSK2 and OsBSK3 may mediate the crosstalk between BR and MAPK (Fig. 7 ). OsBSK3 and OsBSK2 are functional kinases that indirectly activate MAPK6 The RLCK-XII subfamily in Arabidopsis comprise 12 members, each containing a kinase domain at the N-terminal and tetratricopeptide repeat (TPR) domains at the C terminus (Tang et al., 2008 ). OsBSK3 shares 72% homology with AtBSK3 and process all key features of AtBSK3 (Zhang et al., 2016 ). A protein structure analysis of AtBSK8 indicated that AtBSKs are constitutively inactive protein kinases (Grütter et al., 2013 ). However, AtBSK1 was reported to exhibit weak Mn 2+ -dependent kinase activity (Shi et al., 2013 ). AtBSK1 physically interacts with MPK15 and enhances fungal resistance partly by promoting MPK15 Ser-511 phosphorylation in plant cells (Shi et al., 2022 ). OsBSK3 was reported has a weak autophosphorylation activity, which requiring 1 week exposure of the dired gel under a storage phosphor screen to obtain a clear image, and its kinase activity is crucial for BR signaling function in rice (Zhang et al., 2016 ). In this study, although it is difficult to detect the kinase activity of OsBSK3 and OsBSK2 using BTL-104 in vitro, but OsBSK3 and OsBSK2 can activate OsMAPK6 in rice protoplasts and rice young spikes directly (Fig. 3 and supplemental Fig. 5). These results suggest that OsBSK3 and OsBSK2 are functional and might promote OsMAPK6 phosphorylation in an OsMKKK10-dependent manner or through an unknown mechanism, which need further investigation. OsBSK3 and OsBSK2 may mediate the crosstalk between BR and MAPK signal Cell surface receptor kinases recognize extracellular signals, such as hormones and environmental cues, thereby regulating multiple processes, and involved in plant growth, development and stress response. Although BSKs have been reported as regulatory factors in the BR signal transduction pathway, leading to activation of the protein phosphatase BSU1, then dephosphorylates and inhibits the GSK3/Shaggy-like kinase BRI1 INSENSITIVE2 (BIN2), thereby activating BR signal response (Tang et al., 2008 ; Zhang et al., 2016 ; Kim et al., 2009 ). However, their role remains incompletely understood in rice, it is currently unclear whether OsBSKs are involved in other signaling pathways, such as MAPK, gibberellin and auxin signaling. In this study, we found that OsBSKs can regulate rice grain size and leaf angle, with OsBSK2 and OBSK3 may play more critical roles (Fig. 1 and supplemental Fig. 1–3). These indicated that OsBSK2 and OsBSK3 may also be involved in other signaling pathways besides BR signal. Subsequently, we found that mutation in MAPK component (s mg2-1 , smg1-1 , and dsg1 ) could suppress the enhanced grain size and leaf angle phenotypes caused by OsBSK3 overexpression (Fig. 5 and Supplemental Fig. 6, 7). These above results suggest that OsBSK3 and OsBSK2 regulate multiple biological processes through distinct signaling pathways, and may play a role in mediating the crosstalk between MAPK and BR signaling pathways to control grain size and leaf angle in rice. OsBSK3 and OsBSK2 may be the upstream messenger of MAPK cascade in regulating leaf angle and grain size RLCKs sense external signals and transmit to MAPK cascades, regulating multiple processes of plant growth and development. RLCK185, transmits immune signaling from the PAMP receptor OsCERK1 to an MAPK signaling cascade by interacting with OsMAPKKKs (Wang et al., 2017 ). Receptor-like cytoplasmic kinases VII (RLCK VII), which act downstream of PRRs, directly phosphorylate MAPKKK5 at Ser-599, and this phosphorylation is essential for pattern-triggered MPK3/6 activation, defense gene expression, and disease resistance (Bi et al., 2018 ). BSK1 enhances MPK15 phosphorylation at Ser-511, regulating fungal resistance in Arabidopsis (Shi et al., 2022 ). OsBSK1-2 mediates disease resistance in rice through the MAPKKK16/18/19-MAPKK4/5-MAPK3/6 cascade (Li et al., 2024 ). In this study, we found that OsBSK3 and OsBSK2 physically interact with OsMKKK10, and directly activate OsMAPK6 in plant cells. This suggests that OsBSK3 and OsBSK2 may be the upstream signal transmitter of the OsMKKK10-OsMKK4-OsMAPK6 cascade in regulating grain size and leaf angle in rice. Materials and methods Plant materials and growth conditions Rice ( Oryza sativa ) cultivar Longjing 11 ( Oryza sativa ssp. japonica) was used for generate OsBSK3 and OsBSK2 transgenic plants. Plants were grown in the field (natural long day conditions). Rice cultivar Kuanyejing (KYJ), SF43, and Zhonghua 11 ( Oryza sativa ssp. japonica ) were used as wild type control to compare with overexpression plants or diverse mutants in relative background. OsMKKK10 mutant small grain 2 ( smg2-1 ) (Xu et al., 2018 ), OsMKK4 mutant small grain 1 ( smg1-1 ) (Duan et al., 2014 ), OsMAPK6 mutant dwarf and small grain ( dsg1 ) (Liu et al., 2015 ), N390 (which overexpressing the kinase domain (amino acids 1-390) of OsBSK3 ) (Zhang et al., 2016 ), oswrky53 (Tian et al., 2017 ), and osbsk3 were used to develop double mutant. The Ubiquitin pro : OsBSK3 was transformed into smg2-1 , smg1-1 and dsg1 to generate OsBSK3-OE smg2-1 , OsBSK3-OE smg1-1 and OsBSK3-OE dsg1 , respectively. Ubiquitin pro : OsWRKY53 was transformed into osbsk3 to generate osbsk3 OsWRKY53OE (Tian et al., 2017 ). The N390 oswrky53 line was generated through hybridization between N390 and oswrky53. Plants were grown in the field, natural long day condition (Harbin) and natural short day condition (Hainan). For all these mutants, the expression level and mutation site of corresponding genes were examined by RT-qPCR, western blot, and DNA sequence. Total RNA isolation and RT-qPCR analysis Total RNA was extracted from leaves using TRIzol (Invitrogen) and then subjected to DNase I digestion. cDNA was synthesized from the extracted total RNA using Superscript II Reverse Transcriptase (Invitrogen). Real-time PCR was performed with a Lightcycler 480 using SYBR Green PCR master mix (Takara). All expressions were standardized for the ubiquitin gene ( Os01g0328400 ). The primers used are listed in Supplementary Data 1. Three biological replicates were performed for each analysis. LUC complementary imaging (LCI) assay As shown in Supplemental Data 1, cLUC-OsBSK3, cLUC-OsBSK2, and nLUC-OsMKKK10 constructs were generated. Agrobacteria harboring different construct combinations infiltrate into N. benthamiana leaves. Finally, 60 hours after infiltration, the LUC activity of the N. benthamiana infiltrated leaves was analyzed using Chemiluminescence imaging (Tanon 5200). The assay was conducted in three biological replicates, with three leaves per replicate. Bimolecular fluorescence complementation (BiFC) assay For BiFC assay, OsMKKK10 was fused with partial GFP to generate nGFP-OsMKK10, while OsBSK3 and OsBSK2 were combined with partial GFP to generate cGFP-OsBSK3 and cGFP-OsBSK2, as described in Supplemental Data 1. Transform these vectors into Agrobacterium strain GV3101 , after different combinations, co-injected into the young leaves of N . benthamiana separately. Subsequently, fluorescence emission was observed under confocal microscopy (Leica) after 60 h. The assay was conducted in three biological replicates, with three leaves per replicate. Yeast two-hybrid assay Cloning the coding sequences of OsBSK3 and OsBSK2 into the EcoRI and PstI sites of pGBKT7 vector to generate BD-OsBSK3 and BD-OsBSK2 constructs. And then, the coding sequence of OsMKKK10KD was cloned into the EcoRI and XhoI sites of pGADT7 vector to generate the AD-OsMKKK10KD construct. The obtained constructs were transformed into yeast strain Y2H Gold. The presence of plasmids was confirmed by growth on the synthesis limited (SD) medium lacking Trp and Leu (SD-Trp-Leu). Next, the positive yeast clones were suspended in liquid SD-Trp-Leu medium (OD600 = 1.0) to assess protein interactions. Finally, the suspension cells and their dilutions were plated onto SD-Trp-Leu-His and SD-Trp-Leu-His-Ade medium. Protein interactions were observed after incubation at 30 ℃ for three days. Co-Immunoprecipitation (Co-IP) assays 35S pro : FLAG-OsBSK2 , 35S pro : FLAG-GFP , and 35S pro : MYC-OsMKKK10KD constructs were made as described in Supplemental Data Set 1 for Co-IP assay, These plasmids were co expressed transiently in rice protoplasts, as shown in the combination. Total protein was extracted using the lysis buffer (50 mM Tris HCl at pH 7.5, 150 mM NaCl, 0.5 mM EDTA at pH 8.0, 10% glycerol, 0.5% Triton X-100) with freshly added protease inhibitor cocktail (Roche, 11873580001), PMSF (phenylmethylsulfonyl fluoride, Roche, 10837091001), and MG132 (Sigma Aldrich, M8699). Before the Co-IP assays, incubating 20 µl of protein-A/G magnetic beads (GenScript, L00277) with 2 µl anti-Flag (Abmart, M20008) for 2 hours, gently rotate at 4°C, then adding an equal amount of total protein extractions and incubating at 4°C for 2 hours. Washing the magnetic beads multiple times with the washing buffer (50 mM Tris HCl at pH 7.5, 100 mM NaCl, 0.5 mM EDTA at pH 8.0, 0.1% Triton X-100). The immunoprecipitates were eluted with SDS sample buffer, separated on SDS-PAGE gel, transferred to PVDF membrane (Millipore 0.45um, IPVH00010), and detected with anti FLAG (Abmart, M20008) and anti MYC (ThermoFisher, 9E10) antibodies respectively. For all Co-IP assays, we conducted at least three independent replications. RNA-Seq and data analysis For Illumina sequencing, collecting tiller buds from LJ11 and osbsk3 plants. The extraction and detection of total RNA, library preparation, and Illumina sequencing were performed by Novogene Bioinformatics Technology Co., Ltd., Beijing, China using the Illumina HiSeq 2500 platform. The reference genome and gene information of Rice cultivar Nipponbare can be downloaded from IRGSP-1.0 ( http://rapdb.dna.affrc.go.jp/download/irgsp.html ). Using Hisat 2 for mapping quality and saturation analysis. Using DESeq2 to detect differentially expressed genes, with the absolute fold change > 1.5, FDR-adjusted P < 0.05. The GO analysis was carried out by the PANTHER classification system ( www.pantherdb.org ) with FDR ≤ 0.05, and get the GO annotations based on biological process. In vitro kinase assay As described in Supplementary Data 1, constructs were prepared for expressing GST-OsBSK3S215E, GST-OsBSK2, and GST-OsMKKK10. Purify the fusion protein according to the manufacturer's instructions. Subsequently, the purified protein was incubated with phosphorylation buffer (25 mM Tris-HCl at pH 7.4, 12 mM MgCl 2 , 1 mM DTT, and 1 mM ATP) at 30 ℃ for 45 minutes, and then boiled in SDS loading buffer. And according to the manufacturer's instructions, using Phos-tag Biotin BTL-104 (Wako, Richmond, VI, USA; 301-93531) for detect. In vivo MAPK activity assay The wild-type LJ11, OsBSK3-OE , OsBSK2-OE , osbsk3 and osbsk2 plants were grown in a greenhouse, and collect their 3 cm young spikles separately. Then, following the manufacturer's instructions, extract total protein from the plant and the rice protoplast using SDS Lysis solution (Beyotime, P0013G) with added protease inhibitor cocktail (Roche, 11206893001), 50 µM MG132, 1 mM PMSF, and phosphatase inhibitor cocktail (Sigma-Aldrich, P2850) respectively. The proteins were separated by 8% SDS-PAGE and detected by immunoblotting with anti-Phospho-p44/42 MAPK (Cell Signaling Technology, #9101) and anti-ACTIN (Abmart, M20009M) antibodies. In addition, 35S pro : FLAG-OsBSK2 , 35S pro : FLAG-OsBSK3 , 35S pro : GFP-FLAG , and 35S pro : MYC-OsMAPK6 constructs were made as described in Supplemental Data Set 1 for MAPK activity assays, these plasmids were co expressed transiently in rice protoplasts as indicated combination. And perform in vivo MAPK activity assay according to the above method, detecting by immunoblotting with anti-Phospho-p44/42 MAPK (Cell Signaling Technology, #9101), anti FLAG (Abmart, M20008) and anti MYC (ThermoFisher, 9E10) antibodies respectively. Statistical analysis Student’s two-tailed t-test was used to compare the means between two samples/variables, while the one-way ANOVA analyses were used for multiple pairwise comparisons. Tukey’s test at P < 0.05 significance level separated the differences between the means. All the analyses were performed using SPSS software. Student's two tailed t-test was used to compare the means between two samples/variables, while the one-way ANOVA analyses was used for multiple pairwise comparisons. Tukey’s test at P < 0.05 significance level separated the differences between the means. All analyses were conducted using SPSS software. Accession numbers DNA sequence data from this article can be found in gramene ( https://www.gramene.org/ ) under the following accession numbers: Ubiquitin (Os01g0328400), OsMAPK6 (Os06g0154500), OsMKKK10 (Os04g0559800), OsBSK3 (Os04g0684200), OsBSK2 (Os10g0571300), OsBSK1-2 (Os10g0542800), OsBSK4 (Os03g0825300). Declarations Acknowledgements We would thank Prof. Yunhai Li, Prof. Fan Chen and Prof. Wenqiang Tang for providing the relative mutants and vectors. This study was supported by National Key Research and Development Program of China (Grant No. 2024YFD1201001), Youth Innovation Promotion Association CAS (Grant No. 2021229), Young Scientist Group Project of Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences (Grant No. 2023QNXZ02). Author contributions X.T., Q.B., and C. L. conceived and supervised the project. X.T., Q.B., and X.J. analyzed the data and wrote the article. X.J. and L.F. performed most of the experiments. J.L., C.C., Y.L., W. 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Zhao, C.Z., Wang, P.C., Si, T., Hsu, C.C., Wang, L., Zayed, O., Yu, Z.P., Zhu, Y.F., Dong, J., Tao, W.A., and Zhu, J.K. (2017). MAP Kinase Cascades Regulate the Cold Response by Modulating ICE1 Protein Stability. Developmental cell 43, 618. Supplementary Files Supplementalfigure.pdf supplementalData1.xlsx Cite Share Download PDF Status: Published Journal Publication published 20 Apr, 2025 Read the published version in Theoretical and Applied Genetics → Version 1 posted Editorial decision: Major revisions 25 Feb, 2025 Reviewers agreed at journal 07 Feb, 2025 Reviewers invited by journal 07 Feb, 2025 Editor assigned by journal 03 Feb, 2025 First submitted to journal 01 Feb, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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LJ11and \u003cem\u003eosbsks \u003c/em\u003emutants respectively. Data are shown as means±SE (n = 20).\u003c/p\u003e\n\u003cp\u003e(D) Grain morphology in LJ11 and \u003cem\u003eOsBSKs\u003c/em\u003e-OE plants (Scale bar = 5 mm).\u003c/p\u003e\n\u003cp\u003e(E) and (F) Quantification of grain length (E) and grain width (F) in LJ11and \u003cem\u003eOsBSKs\u003c/em\u003e-OE plants respectively. Data are shown as means±SE (n = 20).\u003c/p\u003e\n\u003cp\u003e(G) The gross morphology of LJ11\u003cem\u003e \u003c/em\u003eand \u003cem\u003eosbsks\u003c/em\u003e at heading stage.\u003c/p\u003e\n\u003cp\u003e(H) The lamina joint of flag leaves in LJ11\u003cem\u003e \u003c/em\u003eand \u003cem\u003eosbsks\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e(I) Quantification of lamina angles of the flag leaf in LJ11\u003cem\u003e \u003c/em\u003eand \u003cem\u003eosbsks\u003c/em\u003e. Data are shown as means±SE (n = 30)\u003c/p\u003e\n\u003cp\u003e(J) The gross morphology of LJ11\u003cem\u003e \u003c/em\u003eand \u003cem\u003eOsBSKs\u003c/em\u003e-OE plants at heading stage.\u003c/p\u003e\n\u003cp\u003e(K) The lamina joint of flag leaves in LJ11\u003cem\u003e \u003c/em\u003eand \u003cem\u003eOsBSKs\u003c/em\u003e-OE plants.\u003c/p\u003e\n\u003cp\u003e(L) Quantification of lamina angles of the flag leaf in LJ11\u003cem\u003e \u003c/em\u003eand \u003cem\u003eOsBSKs\u003c/em\u003e-OE. Data are shown as means±SE (n = 30).\u003c/p\u003e\n\u003cp\u003eIn (A)-(F), Each dot represents the result from one biological replicate, error bars indicate means±SE. Statistically significant differences are indicated by different lowercase letters (P \u0026lt; 0.05, one-way ANOVA with Tukey’s significant difference test). In (G)-(L), the asterisks indicate significant differences compared with the control (**P \u0026lt; 0.01, *P\u0026lt; 0.05, Student's t-test).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5897621/v1/031f6f717dbbbec0310ffca5.png"},{"id":75882825,"identity":"54dcb270-aac4-4044-b054-0dd29bf6db4c","added_by":"auto","created_at":"2025-02-10 08:45:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1759442,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBoth OsBSK3 and OsBSK2 can be combined with OsMAKKK10.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) LCI assay indicates that OsBSK3 interacts with OsMKKK10 in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. Co-transformation of cLUC-OsBSK3 and nLUC-OsMAKKK10, leading to the reconstitution of LUC signal, whereas no signal was detected when cLUC-OsBSK1-2 (OsBSK1-2 is homologous to OsBSK3) and nLUC-OsMKKK10, cLUC and nLUC-OsMKKK10, and cLUC-OsBSK3 and nLUC were co-expressed. In each experiment, at least 5 independent \u003cem\u003eN. benthamiana \u003c/em\u003eleaves were infiltrated and analyzed.\u003c/p\u003e\n\u003cp\u003e(B) LCI assay indicates that OsBSK2 interacts with OsMKKK10 in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. Co-transformation of cLUC-OsBSK2 and nLUC-OsMAKKK10, leading to the reconstitution of LUC signal, whereas no signal was detected when cLUC-OsBSK1-2 (OsBSK1-2 is homologous to OsBSK2) and nLUC-OsMKKK10, cLUC and nLUC-OsMKKK10, and cLUC-OsBSK2 and nLUC were co-expressed. In each experiment, at least 5 independent \u003cem\u003eN. benthamiana \u003c/em\u003eleaves were infiltrated and analyzed.\u003c/p\u003e\n\u003cp\u003e(C) BiFC assay indicates that both OsBSK3 and OsBSK2 can interact with OsMKKK10 in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. Co-transformation of cGFP-OsBSK3 and nGFP-OsMKKK10, cGFP-OsBSK2 and nGFP-OsMKKK10, leading to the reconstitution of GFP signal, whereas no signal was detected when cGFP-OsBSK1-1 and nGFP-OsMKKK10, or cGFP-OsBSK4 and nGFP-OsMKKK10 were co-expressed, both OsBSK1-1 and OsBSK4 are the orthlog of OsBSK3 and OsBSK2. For each interaction pair, at least 5 independent \u003cem\u003eN. benthamiana\u003c/em\u003e leaves were infiltrated and analyzed.\u003c/p\u003e\n\u003cp\u003e(D) Schematic diagram of OsMKKK10. Red squares represent the kinase domain of OsMKKK10.\u003c/p\u003e\n\u003cp\u003e(E) OsBSK2 interacts with OsMKKK10 in yeast cells. Yeast cells were cultured on SD/-Trp-Leu, SD/-Trp-Leu-His or SD/-Trp-Leu-His-Ade medium. Only the yeast strains that co-transform BD-OsBSK2 and AD-OsMKKK10KD can grow on the SD/-Trp-Leu-His-Ade deficient medium, while other combinations cannot grow. This experiment was repeated more than three times.\u003c/p\u003e\n\u003cp\u003e(F) Co-IP assays indicate that OsBSK2 interacts with OsMKKK10 in planta. FLAG-OsBSK2 and MYC-OsMKKK10KDwere co-expressed in rice protoplasts. FLAG-GFP and MYC-OsMKKK10KD were co-expressed as control. Protein extraction was immunoprecipitated with anti-FLAG antibody, and detected with anti-MYC (top panel) or anti-FLAG (bottom panel) antibodies. This experiment was repeated more than three times.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5897621/v1/7f61400892d166910e3bb2af.png"},{"id":75884805,"identity":"923c2a58-f113-453b-9836-76346b5bf077","added_by":"auto","created_at":"2025-02-10 09:01:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":430301,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBoth OsBSK3 and OsBSK2 can enhance the phosphorylation level of OsMAPK6 in vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blot assays indicate that OsBSK3 and OsBSK2 can enhance the phosphorylation level of OsMAPK6 in rice protoplasts. FLAG-OsBSK3 and MYC-OsMAPK6, FLAG-OsBSK2 and MYC-OsMAPK6 were co-expressed in rice protoplasts. FLAG-GFP and MYC-OsMAPK6 were co-expressed as control. The phosphorylation of OsMAPK6 was detected by anti-pTEpY antibody (top panel), and used anti-MYC (middle panel) or anti-FLAG (bottom panel) antibody to detect MYC-OsMAPK6, FLAG-OsBSK3 and FLAG-OsBSK2. This experiment was repeated more than three times.\u003c/p\u003e\n\u003cp\u003e(B) Western blot assays indicate that both OsBSK3 and OsBSK2 can enhance the phosphorylation level of OsMAPK6 in \u003cem\u003eOsBSK3-OE\u003c/em\u003e and \u003cem\u003eOsBSK2-OE\u003c/em\u003e young spikes. The phosphorylation of OsMAPK6 was detected by anti-pTEpY antibody (top panel), and used anti-Actin (bottom panel) antibody to detect ACTIN, which was used as a loading control. This experiment was repeated more than three times.\u003c/p\u003e\n\u003cp\u003e(C) The relative phosphorylation level of OsMAPK6 in (B). The relative intensity of the phospho-OsMAPK6 bands as determined using Image J software. The ratio of the intensity of the phospho-OsMAPK6 band to that of the ACTIN band in LJ11 was set as 1.0. Data was shown as means±SE (n = 3). P values are comparison between \u003cem\u003eOsBSK3-OE\u003c/em\u003e and LJ11, \u003cem\u003eOsBSK2-OE\u003c/em\u003e and LJ11, which were calculated by Student’s t test. * is P \u0026lt; 0.05, and ** is P\u0026lt;0.01.\u003c/p\u003e\n\u003cp\u003e(D) Western blot assays indicate that both OsBSK3 and OsBSK2 can enhance the the phosphorylation level of OsMAPK6 in \u003cem\u003eosbsk3\u003c/em\u003e and osbsk2 young spikes. The phosphorylation of OsMAPK6 was detected by anti-pTEpY antibody (top panel), and used anti-Actin (bottom panel) antibody to detect ACTIN, which used as a loading control. This experiment was repeated more than three times.\u003c/p\u003e\n\u003cp\u003e(E) The relative phosphorylation level of OsMAPK6 in (D). The relative intensity of the phospho-OsMAPK6 bands as determined using Image J software. The ratio of the intensity of the phospho-OsMAPK6 band to that of the ACTIN band in LJ11 was set as 1.0. Data was shown as means±SE (n = 3). P values are comparison between \u003cem\u003eosbsk3\u003c/em\u003e and LJ11, \u003cem\u003eosbsk2\u003c/em\u003e and LJ11, and were calculated by Student’s t test. * is P \u0026lt; 0.05, and ** is P\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5897621/v1/18ec72128de88f0715f45bd9.png"},{"id":75882818,"identity":"d8a6b7b1-8953-4355-ab95-c9ad7b5d5f7d","added_by":"auto","created_at":"2025-02-10 08:45:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":329894,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe OsMAKKK10-OsMKK4-OsMAPK6 cascade plays an important role in the regulation of rice leaf angle\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Quantification of lamina angles of the flag leaf in\u003cem\u003e smg2-1 \u003c/em\u003eand its control (KYJ) planted in Hainan.\u003c/p\u003e\n\u003cp\u003e(B) Quantification of lamina angles of the flag leaf in \u003cem\u003esmg1-1\u003c/em\u003e and its control (SF43) planted in Hainan.\u003c/p\u003e\n\u003cp\u003e(C) Quantification of lamina angles of the flag leaf in \u003cem\u003edsg1\u003c/em\u003e and its control (ZH11) planted in Hainan.\u003c/p\u003e\n\u003cp\u003e(D) Quantification of lamina angles of the flag leaf in\u003cem\u003e smg2-1 \u003c/em\u003eand its control (KYJ) planted in Harbin.\u003c/p\u003e\n\u003cp\u003e(E) Quantification of lamina angles of the flag leaf in \u003cem\u003esmg1-1\u003c/em\u003e and its control (SF43) planted in Harbin.\u003c/p\u003e\n\u003cp\u003e(F) Quantification of lamina angles of the flag leaf in \u003cem\u003edsg1\u003c/em\u003e and its control (ZH11) planted in Harbin.\u003c/p\u003e\n\u003cp\u003eIn (A)-(F), values are means±SE (n = 30), the asterisks indicate significant differences compared with the control (**P \u0026lt; 0.01, *P\u0026lt; 0.05, Student’s t-test).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5897621/v1/d6d0ab0f460fe23ffa097d98.png"},{"id":75883311,"identity":"3e278b25-24ee-4e5d-beda-b31e9804d792","added_by":"auto","created_at":"2025-02-10 08:53:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1694087,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOsBSK3 acts upstream of OsMKKK10-OsMKK4-OsMAPK6 casade to regulate rice grain size and leaf angle\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The gross morphology of KYJ, \u003cem\u003esmg2-1\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eand \u003cem\u003eOsBSK3-OE smg2-1\u003c/em\u003e at heading stage.\u003c/p\u003e\n\u003cp\u003e(B) The gross morphology of SF43, \u003cem\u003esmg1-1\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eand \u003cem\u003eOsBSK3-OE smg1-1\u003c/em\u003e at heading stage.\u003c/p\u003e\n\u003cp\u003e(C) The gross morphology of ZH11, \u003cem\u003edsg1\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eand \u003cem\u003eOsBSK3-OE dsg1 \u003c/em\u003eat heading stage.\u003c/p\u003e\n\u003cp\u003e(D) Quantification of lamina angles of the flag leaf in KYJ, \u003cem\u003esmg2-1\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eand \u003cem\u003eOsBSK3-OE smg2-1\u003c/em\u003e at heading stage. Data are shown as means±SE (n = 30).\u003c/p\u003e\n\u003cp\u003e(E) Quantification of lamina angles of the flag leaf in SF43, \u003cem\u003esmg1-1\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eand \u003cem\u003eOsBSK3-OE smg1-1\u003c/em\u003e at heading stage. Data are shown as means±SE (n = 30).\u003c/p\u003e\n\u003cp\u003e(F) Quantification of lamina angles of the flag leaf in ZH11, \u003cem\u003edsg1\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eand \u003cem\u003eOsBSK3-OE dsg1 \u003c/em\u003eat heading stage. Data are shown as means±SE (n = 30).\u003c/p\u003e\n\u003cp\u003e(G) Grain morphology in KYJ, \u003cem\u003esmg2-1\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eand \u003cem\u003eOsBSK3-OE smg2-1\u003c/em\u003e plants (Scale bar = 5 mm).\u003c/p\u003e\n\u003cp\u003e(H) Grain morphology in SF43, \u003cem\u003esmg1-1\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eand \u003cem\u003eOsBSK3-OE smg1-1 \u003c/em\u003eplants (Scale bar = 5 mm).\u003c/p\u003e\n\u003cp\u003e(I) Grain morphology in ZH11, \u003cem\u003edsg1\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eand \u003cem\u003eOsBSK3-OE dsg1\u003c/em\u003e plants (Scale bar = 5 mm).\u003c/p\u003e\n\u003cp\u003e(J) and (K) Quantification of grain length (J) and grain width (K) in (G). Data are shown as means±SE (n = 30).\u003c/p\u003e\n\u003cp\u003e(L) and (M) Quantification of grain length (L) and grain width (M) in (H). Data are shown as means±SE (n = 30).\u003c/p\u003e\n\u003cp\u003e(N) and (O) Quantification of grain length (N) and grain width (O) in (I). Data are shown as means±SE (n = 30).\u003c/p\u003e\n\u003cp\u003eEach dot represents the result from one biological replicate, error bars indicate means±SE. Statistically significant differences are indicated by different lowercase letters (P \u0026lt; 0.05, one-way ANOVA with Tukey’s significant difference test).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5897621/v1/a2c1587683129e0cc5561d42.png"},{"id":75884804,"identity":"1500dd9a-8027-4785-aef5-1bd23027c052","added_by":"auto","created_at":"2025-02-10 09:01:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1498188,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOsWRKY53 may act downstream of OsBSK3-OsMKKK10-OsMKK4-OsMAPK6 casade to regulate rice grain size and leaf angle\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The gross morphology of WT, \u003cem\u003eN390\u003c/em\u003e, \u003cem\u003eoswrky53\u003c/em\u003e and \u003cem\u003eN390 oswrky53\u003c/em\u003e at heading stage.\u003c/p\u003e\n\u003cp\u003e(B) Quantification of lamina angles of the flag leaf in WT, \u003cem\u003eN390\u003c/em\u003e, \u003cem\u003eoswrky53\u003c/em\u003e and \u003cem\u003eN390 oswrky53\u003c/em\u003e at heading stage. Data are shown as means±SE (n = 20).\u003c/p\u003e\n\u003cp\u003e(C) Grain morphology in WT, \u003cem\u003eN390\u003c/em\u003e, \u003cem\u003eoswrky53\u003c/em\u003e and \u003cem\u003eN390 oswrky53\u003c/em\u003e plants (Scale bar = 5 mm).\u003c/p\u003e\n\u003cp\u003e(D) and (E) Quantification of grain length (D) and grain width (E) in (C). Data are shown as means±SE (n = 20).\u003c/p\u003e\n\u003cp\u003e(F) The gross morphology of LJ11, \u003cem\u003eosbsk3\u003c/em\u003e, \u003cem\u003eOsWRKY53OE\u003c/em\u003e and o\u003cem\u003esbsk3 OsWRKY53OE\u003c/em\u003e at heading stage.\u003c/p\u003e\n\u003cp\u003e(G) Quantification of lamina angles of the flag leaf in LJ11, \u003cem\u003eosbsk3\u003c/em\u003e, \u003cem\u003eOsWRKY53OE\u003c/em\u003e and o\u003cem\u003esbsk3 OsWRKY53OE\u003c/em\u003e. Data are shown as means±SE (n = 20).\u003c/p\u003e\n\u003cp\u003e(H) Grain morphology in LJ11, \u003cem\u003eosbsk3\u003c/em\u003e, \u003cem\u003eOsWRKY53OE\u003c/em\u003e and o\u003cem\u003esbsk3 OsWRKY53OE\u003c/em\u003e plants (Scale bar = 5 mm).\u003c/p\u003e\n\u003cp\u003e(I) and (J) Quantification of grain length (I) and grain width (J) in (H). Data are shown as means±SE (n = 20).\u003c/p\u003e\n\u003cp\u003eEach dot represents the result from one biological replicate, error bars indicate means±SE. Statistically significant differences are indicated by different lowercase letters (P \u0026lt; 0.05, one-way ANOVA with Tukey’s significant difference test).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5897621/v1/bb1b7f6a14d3039e3b3c4b95.png"},{"id":75882822,"identity":"ccc9ab28-b510-460c-a6d8-993dc80ce185","added_by":"auto","created_at":"2025-02-10 08:45:03","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":197882,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed Working Model of OsBSKs-OsMKKK10-OsMKK4-OsMAPK6-OsWRKY53 in regulating rice grain size and leaf angle\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Both OsBSK3 and OsBSK2 interact with OsMKKK10, and indirectly activate OsMAPK6, postively regulating rice leaf angle and grain size.\u003c/p\u003e\n\u003cp\u003e(B) In \u003cem\u003eosbsk3\u003c/em\u003e and \u003cem\u003eosbsk2\u003c/em\u003e mutants, the loss of function of OsBSK3 and OsBSK2 cannot combine with OsMKKK10, indirectly repress OsMAPK6 activity through the MAPK cascade, thereby reducing the phosphorylation level of OsMAPK6 in the\u003cem\u003e osbsk2\u003c/em\u003e and \u003cem\u003eosbsk3 \u003c/em\u003emutants, ultimately causing a decrease in leaf angle and grain size.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5897621/v1/4d275ddfc2d48f1e6754318d.png"},{"id":81050862,"identity":"09a52a5c-7be6-4f50-9063-bec9da3e29ed","added_by":"auto","created_at":"2025-04-21 16:06:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11959082,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5897621/v1/8cb76a5d-24b2-45e9-8ae8-98adb4b7a57e.pdf"},{"id":75882827,"identity":"83da5d9b-25da-447b-8a8a-cdfa566daa87","added_by":"auto","created_at":"2025-02-10 08:45:03","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":999145,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalfigure.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5897621/v1/382741157cc000427c01d240.pdf"},{"id":75882817,"identity":"ebfe79bd-e291-4540-8897-2f6d52f0e094","added_by":"auto","created_at":"2025-02-10 08:45:03","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14268,"visible":true,"origin":"","legend":"","description":"","filename":"supplementalData1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5897621/v1/81fedca3315ceae7a07de035.xlsx"}],"financialInterests":"","formattedTitle":"OsBSK3 and OsBSK2 regulate grain size and leaf angle via MAPK signaling pathway in rice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRice (\u003cem\u003eOryza sativa\u003c/em\u003e L.) is a staple food crop, providing sustenance to over half of the global population (Wang and Li, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Increasing rice yield is crucial for ensuring global food security. Grain size and leaf angle are the key agronomic traits that determine grain yield in crops. Erect leaves facilitate high-density planting, and grain size is directly correlated with yield per plant (Liu et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). So far, several genes regulating rice grain size and leaf angle have been identified (Fan et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Ishimaru et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Qiao et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the genetic and molecular mechanisms that determine these traits are still largely unknown. Identifying the genetic components that regulate rice grain size and leaf angle holds promise for crop improvement.\u003c/p\u003e \u003cp\u003eRecent advances have identified several signaling pathways that controlling grain size in rice, including the ubiquitin-proteasome pathway, G-protein signaling, mitogen-activated protein kinase (MAPK) signaling, some transcriptional regulators, and phytohormone perception and homeostasis, such as Brassinosteroid (BR) signaling and auxin signaling (Li et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e; Ren et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Leaf angle, the inclination angle between the vertical culm and leaf blade, contributes to rice architecture. Suitably compact plants with erect leaves exhibit enhanced photosynthetic efficiency under dense planting conditions, thereby increasing yields (Liu et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Various plant hormones, such as BRs, auxin, and gibberellic acid, are involved in controlling leaf angle (Luo et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Nolan et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Phytohormones signaling and crosstalk regulate grain size and leaf angle in rice, BRs and auxin are reported to simultaneously regulate these two traits (Luo et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Jang et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Qiao et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eReceptor-like protein kinases (RLKs) constitute an important kinase family in plants. Some RLKs, called cytoplasmic receptor-like kinases (RLCKs), lack extracellular domains (Shubha et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Liang and Zhou, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). BRASSINOSTEROID-SIGNALING KINASES (BSKs) belong to the RLCK XII superfamily, BR induces the BR receptor BR INSENSITIVE1 (BRI1) to phosphorylate and activate BSKs, which then phosphorylate BRI1-SUPPRESSOR1 (BSU1) to transduce the BR signal in Arabidopsis (Tang et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Kim et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The BSKs gene originated in embryophytes and plays a role in various stages of plant development (Li et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e ). Rice contains five OsBSK members, OsBSK1-1 likely serves as a scaffold protein, directly bridging OsBRI1 and OsGSK2 to positively regulate BR signaling (Tian et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). OsBSK1-2 functions as an important regulator in rice plant immunity, and silencing \u003cem\u003eOsBSK1-2\u003c/em\u003e decreased resistance to \u003cem\u003eM. oryzae\u003c/em\u003e (Li et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). OsBSK2, a putative brassinosteroid-signalling kinase, positively controls grain size and can form homodimers or heterodimers with OsBSK3 and OsBSK4 in rice (Yuan et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). OsBSK3 is a positive regulator of BR signaling in rice, OsBRI1 interacts directly with and phosphorylates OsBSK3, disrupting the interaction between its TPR and kinase domains (Zhang et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Additionally, AtBSK3 promotes root elongation by interacting with AtBAK1 under mild nitrogen deficiency conditions in Arabidopsis (Jia et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, although certain functions of OsBSKs have been reported in rice, their overall roles and mechanisms remain poorly understood currently.\u003c/p\u003e \u003cp\u003eThe MAPK cascades consist of protein kinases (MAPKs), MAPK kinases (MAPKKs), and MAPKK kinases (MAPKKKs). In cellular activity, upstream signals recognition activates MAPKKKs, further phosphorylate MAPKs to amplify the signal through the cascade pathway (Liu et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). MAPK cascades play different roles in plant growth, morphogenesis, signal transduction, and responses to biological and abiotic stress by phosphorylating various substrates (Zhang and Zhang, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The single mutants \u003cem\u003eosmkkk10\u003c/em\u003e, \u003cem\u003eosmkk4, osmapk6\u003c/em\u003e exhibit small and light grains. The OsMPKKK10-OsMKK4-OsMAPK6 cascade functions in a common pathway to control grain size and spikelet shape in rice. Additionally, OsWRKY53 acts genetically downstream of this cascade to control leaf angle and seed size (Xu et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Guo et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Tian et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Besides, the OsYDA1/OsYDA2-OsMKK4-OsMPK6 cascade plays a crucial role in tapetal development and male gametophyte fertility (Zeng et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Studies report that AtMKK4/5-AtMPK3/6 negatively regulate freezing tolerance, while AtMEKK1-AtMKK2-AtMAPK4 positively regulate it in Arabidopsis (Teige et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, cold activates OsMAPK3/6, which then phosphorylates and stabilizes OsICE1 to positively regulate cold tolerance at the seedling stage in rice (Zhang et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Moreover, OsMAPK6 also interacts with and phosphorylates OsWRKY78, enhancing its stability to regulate rice panicle exsertion (Mei et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The pathogen-inducible OsMPKK10.2-OsMPK6 cascade phosphorylates the Raf-like kinase OsEDR1, inhibiting its scaffold function to promote rice disease resistance (Ma et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Recent studies have shown that YDA-MKK4/MKK5-MPK3/MPK6 can regulate stomatal development (Li et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Furthermore, rice Salt Intolerance 1 (OsSIT1) is reported to act upstream of MAPK6/3, regulating the accumulation of ROS under salt stress (Li et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Although MAPK cascades have been reported to be involved in regulating various biological functions, but their role of MAPK cascades in regulating leaf angle are still largely unknown. Similarly, the molecular mechanism by how MAPK perceives and transmits upstream signals to regulate rice grain size and leaf angle is poorly understood.\u003c/p\u003e \u003cp\u003eIn this study, we found that OsBSKs regulate rice grain size and leaf angle, with OsBSK3 and OsBSK2 playing more critical roles. The grain size and leaf angle in \u003cem\u003eosbsk3\u003c/em\u003e and \u003cem\u003eosbsk2\u003c/em\u003e mutants are significantly smaller, whereas the \u003cem\u003eOsBSK3\u003c/em\u003e-overexpressing lines (\u003cem\u003eOsBSK3\u003c/em\u003e-OEs) exhibit considerably larger grain size and leaf angle compared to the others. Both OsBSK3 and OsBSK2 can bind to OsMKKK10, indirectly activating OsMAPK6. Furthermore, mutations in MAPK cascade components, such as \u003cem\u003esmg2-1\u003c/em\u003e (\u003cem\u003eosmkkk10\u003c/em\u003e mutant), \u003cem\u003esmg1-1\u003c/em\u003e (\u003cem\u003eosmkk4\u003c/em\u003e mutant), and \u003cem\u003edsg1\u003c/em\u003e (\u003cem\u003eosmapk6\u003c/em\u003e mutant) showed an erect leaf angle. Moreover, these mutations were able to rescue the increased grain size and leaf angle in \u003cem\u003eOsBSK3\u003c/em\u003e overexpression lines. Additionally, OsWRKY53 may a target of the OsBSKs-OsMKKK10-OsMKK4-OsMAPK6 cascade that regulates rice grain size and leaf angle. Taken together, these results suggest that OsBSK3 and OsBSK2 regulate rice grain size and leaf angle via the OsMKKK10-OsMKK4-OsMAPK6-OsWRKY53 cascade, OsBSK3 and OsBSK2 may mediate the crosstalk between BR and MAPK signal.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eOsBSK3 and OsBSK2 play a more critical role in regulating grain size and leaf angle compared to other OsBSKs gene.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe BSKs family genes play a crucial role in regulating plant growth and development, including plant defense and BR signaling responses (Shubha et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Bi et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In our previous study, we generated several mutants of the OsBSK family members. We found that most \u003cem\u003eosbsks\u003c/em\u003e mutants have smaller grain size, while the \u003cem\u003eOsBSKs-OE\u003c/em\u003e (\u003cem\u003eOsBSKs\u003c/em\u003e overexpression lines) have larger grain size (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Surprisingly, compared to other OsBSKs members, \u003cem\u003eosbsk3\u003c/em\u003e and \u003cem\u003eosbsk2\u003c/em\u003e have significantly shorter grain length and width (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). These indicate that OsBSKs members have functional redundancy in regulating grain size, and OsBSK3 and OsBSK2 may play a more critical role. Subsequently, we observed their plant architecture, and found that the leaf angle of \u003cem\u003eosbsk2\u003c/em\u003e and \u003cem\u003eosbsk3\u003c/em\u003e decreased significantly, while the leaf angle of \u003cem\u003eOsBSK3-OE\u003c/em\u003e and \u003cem\u003eOsBSK4-OE\u003c/em\u003e increased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL). This suggests that OsBSKs family genes also play an important role in regulating rice leaf angle. To further verify this result, we generated multiple independent \u003cem\u003eosbsk3\u003c/em\u003e, \u003cem\u003eosbsk2\u003c/em\u003e and \u003cem\u003eOsBSK3-OEs\u003c/em\u003e plants for careful observation. Through years of continuous statistical analysis of the leaf angle of these mutants grown in the fields, we confirmed that \u003cem\u003eosbsk3\u003c/em\u003e and \u003cem\u003eosbsk2\u003c/em\u003e indeed have a decreased leaf angle, while \u003cem\u003eOsBSK3-OEs\u003c/em\u003e have an significantly increased leaf angle (Supplemental Fig.\u0026nbsp;1\u0026ndash;3). Collectively, these findings imply that OsBSKs play an essential role in regulating rice grain size and leaf angle, with OsBSK3 and OsBSK2 potentially playing a more pivotal role. OsBSK3 and OsBSK2 may also be involved in other signaling pathways beyond brassinosteroid (BR) signaling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eOsBSK3 and OsBSK2 interact with OsMKKK10\u003c/h2\u003e \u003cp\u003eTo investigate the molecular mechanism by which OsBSK2 and OsBSK3 regulate rice grain size and leaf angle, we conducted RNA-Seq analysis on 3 cm young spike from \u003cem\u003eosbsk3\u003c/em\u003e mutant and control plants, identifying 1834 differently expressed genes (Supplemental Fig.\u0026nbsp;4A and 4B). KEGG pathway analysis revealed significant enrichment of these genes in the MAPK signaling pathway, suggesting a crucial role for OsBSK3 in this pathway (Supplemental Fig.\u0026nbsp;4C). Given that previous reports have shown multiple members of RLCKs can bind to MKKKs, we performed the LCI (luciferase complementary imaging) assay to examine the interactions between OsBSKs and OsMKKKs. Constructs expressing OsBSK3 and OsBSK2 fused to the C-terminal part of firefly luciferase (cLUC-OsBSK2 and cLUC-OsBSK3) and OsMKKKs fused to the N-terminal part (nLUC-OsMKKKs) were generated. Strong LUC activity was observed when nLUC-OsMKKK10 were co-infiltrated with cLUC-OsBSK3 or cLUC-OsBSK2 into \u003cem\u003eN. benthamiana\u003c/em\u003e leaves, indicating interactions between OsMKKK10 and both OsBSK3 and OsBSK2 in planta (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These interactions were confirmed using BiFC (Bimolecular Fluorescence Complementation) assay, which showed strong GFP fluorescence in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves co-transformed with cGFP-OsOsBSK3 and nGFP-OsMKKK10, as well as cGFP-OsOsBSK2 and nGFP-OsMKKK10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Further validation using a yeast two-hybrid assay demonstrated that OsBSK2 interacts with the kinase domain of OsMKKK10 in vitro (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). The interaction between OsBSK2 and OsMKKK10 was further validated through Co-IP (co-immunoprecipitation) in rice protoplasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Collectively, these results indicate that OsMKKK10 interacts with both OsBSK3 and OsBSK2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eOsBSK3 and OBSK2 activating OsMAPK6\u003c/h3\u003e\n\u003cp\u003eDespite interactions between OsBSK3, OsBSK2 with OsMKKK10, we were unable to show whether OsBSK3 and OsBSK2 can phosohorylate OsMKKK10 in vitro due to difficulties in detecting their kinase activity (Supplemental Fig.\u0026nbsp;5). Given that MAPK cascades function downstream of RLK signaling in Arabidopsis and the OsMKKK10-OsMKK4-OsMAPK6 function in a cascade during control of grain size and weight in rice ((Xu and Zhang, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). We investigated whether OsBSK3 and OsBSK2 can indirectly regulate OsMAPK6 activity. In a rice protoplasts transient expression assay, co-transformed the constructs expressing either Flag-OsBSK3 or Flag-OsBSK2 significantly enhanced the phosphorylation level of OsMAPK6, as detected by an anti-pTEpY (anti phospho-p44/42 MAPK) antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), which specifically recognizes the phosphorylated TEY loop motif of MAPK, detecting phosphorylated OsMAPK6 (Liu et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This suggests that both OsBSK3 and OsBSK2 can activate OsMAPK6 in plant cells. To further verify this result, we assayed the phosphorylation level of OsMAPK6 in the 3 cm young spike of \u003cem\u003eOsBSK3-\u003c/em\u003eOE, \u003cem\u003eOsBSK2-\u003c/em\u003eOE, \u003cem\u003eosbsk3\u003c/em\u003e and \u003cem\u003eosbsk2\u003c/em\u003e plants. We found that gain-of-function of OsBSK3 and OsBSK2 increased the phosphorylation of OsMAPK6, while loss-of-function suppressed it (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Overall, these results indicate that OsBSK3 and OBSK2 can activate OsMAPK6 in vivo.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eThe OsMKKK10-OsMKK4-OsMAPK6 cascade plays a mediating role in the control of rice leaf angle\u003c/h3\u003e\n\u003cp\u003eThe MAPK cascade pathway plays a crucial role in plant growth, development, and the response to both biotic and abiotic stresses (Zhang et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Specifically, the OsMKKK10-OsMKK4-OsMAPK6 cascade regulates rice grain size and weight (Xu et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). additionally, ER1 functions upstream of the OsMKKK10-OsMKK4-OsMAPK6 cascade to regulate spikelet number by modulating cytokinin metabolism (Guo et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). To investigate whether the OsMKKK10-OsMKK4-OsMAPK6 cascade can regulate rice leaf angle, we conducted field trails in Hainan and Harbin using \u003cem\u003esmg2-1\u003c/em\u003e (a small grain 2\u0026thinsp;\u0026minus;\u0026thinsp;1 mutant, which is a \u003cem\u003eosmkkk10\u003c/em\u003e mutant), \u003cem\u003esmg1-1\u003c/em\u003e (a small grain 1\u0026ndash;1 mutant, which is a \u003cem\u003eosmkk4\u003c/em\u003e mutant), and \u003cem\u003edsg1\u003c/em\u003e (a dwarf and small grains 1 mutant, which is a weak allele of \u003cem\u003eOsMAPK6\u003c/em\u003e) (Liu et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Our results showed that \u003cem\u003esmg2-1\u003c/em\u003e, \u003cem\u003esmg1-1\u003c/em\u003e, and \u003cem\u003edsg1\u003c/em\u003e exhibited significantly more erect leaf angle compared to their respective controls, regardless of whether they were planted in Hainan or Harbin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). These findings suggest that OsMKKK10, OsMKK4, and OsMAPK6 regulate leaf angle development in rice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eOsBSK3 and the OsMKKK10-OsMKK4-OsMAPK6 cascade may potentially function within a shared pathway to regulate rice grain size and leaf angle\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs described above, OsBSK3 and OsBSK2 can interact with OsMKKK10, activating the OsMAPK6 in vivo (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Both OsBSK3, OsBSK2 and OsMKKK10-OsMKK4-OsMAPK6 can regulate rice grain size and leaf angle (Xu et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). We investigated whether OsBSKs function genetically in a common signaling cascade with OsMKKK10-OsMKK4-OsMAPK6 to regulate rice grain size and leaf angle. First, we obtained \u003cem\u003eOsBSK3-\u003c/em\u003eOEs under KYJ (genetic background of \u003cem\u003esmg2-1\u003c/em\u003e), SF43 (genetic background of \u003cem\u003esmg1-1\u003c/em\u003e), and ZH11 (genetic background of \u003cem\u003edsg1\u003c/em\u003e) (Supplemental Fig.\u0026nbsp;6A-6C). We found \u003cem\u003eOsBSK3-\u003c/em\u003eOEs in different genetic backgrounds still display increased grain size and leaf angle, consistent with above mentioned (Supplemental Fig.\u0026nbsp;6D -6L). To further explore this, we overexpressed the \u003cem\u003eOsBSK3\u003c/em\u003e in \u003cem\u003esmg2-1\u003c/em\u003e, \u003cem\u003esmg1-1\u003c/em\u003e and \u003cem\u003edsg1\u003c/em\u003e, generating \u003cem\u003eOsBSK3-\u003c/em\u003eOE \u003cem\u003esmg2-1\u003c/em\u003e, \u003cem\u003eOsBSK3-\u003c/em\u003eOE \u003cem\u003esmg1-1\u003c/em\u003e, and \u003cem\u003eOsBSK3-\u003c/em\u003eOE \u003cem\u003edsg1\u003c/em\u003e lines respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC; Supplemental Fig.\u0026nbsp;7A to 7C). It was shown that \u003cem\u003eOsBSK3-\u003c/em\u003eOE \u003cem\u003esmg2-1\u003c/em\u003e, \u003cem\u003eOsBSK3-\u003c/em\u003eOE \u003cem\u003esmg1-1\u003c/em\u003e, and \u003cem\u003eOsBSK3-\u003c/em\u003eOE \u003cem\u003edsg1\u003c/em\u003e lines show similar grain size and leaf angle to s\u003cem\u003emg2-1\u003c/em\u003e, \u003cem\u003esmg1-1\u003c/em\u003e, and \u003cem\u003edsg1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eO). implying that these mutations were able to rescue the increased grain size and leaf angle in \u003cem\u003eOsBSK3\u003c/em\u003e overexpression lines, OsBSK3 may act upstream of the OsMKKK10-OsMKK4-OsMAPK6 cascade in regulating rice grain size and leaf angle.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eOsWRKY53 may a target of the OsBSKs-OsMKKK10-OsMKK4-OsMAPK6 cascade that regulates rice grain size and leaf angle\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOur previously reported data indicates that the OsMKK4-OsMAPK6 cascade phosphorylates and activates OsWRKY53, and the OsMKK4, OsMAPK6, and OsWRKY53 cascade might function in the same pathway to regulate rice grain size (Tian et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Tian et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We then asked whether OsWRKY53 is a downstream component of OsBSKs-regulated signaling in regulating grain size and leaf angle. We therefore Knocking out \u003cem\u003eOsWRKY53\u003c/em\u003e in \u003cem\u003eN390\u003c/em\u003e, which overexpressing the kinase domain (amino acids 1-390) of \u003cem\u003eOsBSK3\u003c/em\u003e (N390), and obtained \u003cem\u003eN390 oswrky53\u003c/em\u003e lines (Zhang et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, plant physiology) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). We found that \u003cem\u003eN390 oswrky53\u003c/em\u003e lines show similar grain size and leaf angle to \u003cem\u003eoswrky53\u003c/em\u003e, \u003cem\u003eoswrky53\u003c/em\u003e can fully rescue the increased grain size and leaf angle in \u003cem\u003eN390\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-E). To further verify this result, we overexpressed \u003cem\u003eOsWRKY53\u003c/em\u003e in the \u003cem\u003eosbsk3\u003c/em\u003e background, generating \u003cem\u003eosbsk3 OsWRKY53-OE\u003c/em\u003e lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Phenotypic analysis demonstrated that \u003cem\u003eOsWRKY53\u003c/em\u003e overexpression fully rescued the reduced leaf angle and grain size in \u003cem\u003eosbsk3\u003c/em\u003e mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG-J). These results implied that OsWRKY53 is a key downstream target of the OsBSKs-OsMKKK10-OsMKK4-OsMAPK6 cascade in regulating seed size and leaf angle in rice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we elucidate the regulatory roles of OsBSK3 and its homolog OsBSK2 in controlling grain size and leaf angle through the OsMKKK10-OsMAPK4-OsMAPK6-OsWRKY53 signaling cascade. First, OsBSKs regulate grain size and leaf angle, with OsBSK2 and OsBSK3 playing more critical roles. The grain size and leaf angle in \u003cem\u003eosbsk3\u003c/em\u003e and \u003cem\u003eosbsk2\u003c/em\u003e are more smaller, while \u003cem\u003eOsBSK3-\u003c/em\u003eoverexpressing lines (\u003cem\u003eOsBSK3-\u003c/em\u003eOEs) have more larger grain size and leaf angle (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplemental Fig.\u0026nbsp;1\u0026ndash;3). Second, both OsBSK3 and OsBSK2 can bind to OsMKKK10, directly activate OsMAPK6 in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Supplemental Fig.\u0026nbsp;4, 5). Third, the leaf angle in \u003cem\u003esmg2-1\u003c/em\u003e (\u003cem\u003eosmkkk10\u003c/em\u003e mutant), \u003cem\u003esmg1-1\u003c/em\u003e (\u003cem\u003eosmkk4\u003c/em\u003e mutant), and \u003cem\u003edsg1\u003c/em\u003e (\u003cem\u003eosmapk6\u003c/em\u003e mutant) showed a significant decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Fourth, mutation in MAPK component (s\u003cem\u003emg2-1\u003c/em\u003e, \u003cem\u003esmg1-1\u003c/em\u003e, and \u003cem\u003edsg1\u003c/em\u003e) can rescue the increased grain size and leaf angle in \u003cem\u003eOsBSK3\u003c/em\u003e overexpression line (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Supplemental Fig.\u0026nbsp;6, 7). Lastly, OsWRKY53 may a target of the OsBSKs-OsMKKK10-OsMKK4-OsMAPK6 cascade in regulating rice grain size and leaf angle. Knockout \u003cem\u003eOsWRKY53\u003c/em\u003e can fully complement the increased leaf angle and grain size in \u003cem\u003eN390\u003c/em\u003e, and \u003cem\u003eOsWRKY53\u003c/em\u003e overexpression can also rescue the decreased leaf angle and grain size in \u003cem\u003eosbsk3\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Supplemental Fig.\u0026nbsp;8, 9). Collectively, these results indicate that OsBSK3 and OsBSK2 regulate rice grain size and leaf angle through the OsBSK3/OsBSK2-OsMKKK10-OsMAPK4-OsMAPK6 cascade, OsBSK2 and OsBSK3 may mediate the crosstalk between BR and MAPK (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eOsBSK3 and OsBSK2 are functional kinases that indirectly activate MAPK6\u003c/h3\u003e\n\u003cp\u003eThe RLCK-XII subfamily in Arabidopsis comprise 12 members, each containing a kinase domain at the N-terminal and tetratricopeptide repeat (TPR) domains at the C terminus (Tang et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). OsBSK3 shares 72% homology with AtBSK3 and process all key features of AtBSK3 (Zhang et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). A protein structure analysis of AtBSK8 indicated that AtBSKs are constitutively inactive protein kinases (Gr\u0026uuml;tter et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). However, AtBSK1 was reported to exhibit weak Mn\u003csup\u003e2+\u003c/sup\u003e-dependent kinase activity (Shi et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). AtBSK1 physically interacts with MPK15 and enhances fungal resistance partly by promoting MPK15 Ser-511 phosphorylation in plant cells (Shi et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). OsBSK3 was reported has a weak autophosphorylation activity, which requiring 1 week exposure of the dired gel under a storage phosphor screen to obtain a clear image, and its kinase activity is crucial for BR signaling function in rice (Zhang et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In this study, although it is difficult to detect the kinase activity of OsBSK3 and OsBSK2 using BTL-104 in vitro, but OsBSK3 and OsBSK2 can activate OsMAPK6 in rice protoplasts and rice young spikes directly (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and supplemental Fig.\u0026nbsp;5). These results suggest that OsBSK3 and OsBSK2 are functional and might promote OsMAPK6 phosphorylation in an OsMKKK10-dependent manner or through an unknown mechanism, which need further investigation.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eOsBSK3 and OsBSK2 may mediate the crosstalk between BR and MAPK signal\u003c/h2\u003e \u003cp\u003eCell surface receptor kinases recognize extracellular signals, such as hormones and environmental cues, thereby regulating multiple processes, and involved in plant growth, development and stress response. Although BSKs have been reported as regulatory factors in the BR signal transduction pathway, leading to activation of the protein phosphatase BSU1, then dephosphorylates and inhibits the GSK3/Shaggy-like kinase BRI1 INSENSITIVE2 (BIN2), thereby activating BR signal response (Tang et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kim et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). However, their role remains incompletely understood in rice, it is currently unclear whether OsBSKs are involved in other signaling pathways, such as MAPK, gibberellin and auxin signaling. In this study, we found that OsBSKs can regulate rice grain size and leaf angle, with OsBSK2 and OBSK3 may play more critical roles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and supplemental Fig.\u0026nbsp;1\u0026ndash;3). These indicated that OsBSK2 and OsBSK3 may also be involved in other signaling pathways besides BR signal. Subsequently, we found that mutation in MAPK component (s\u003cem\u003emg2-1\u003c/em\u003e, \u003cem\u003esmg1-1\u003c/em\u003e, and \u003cem\u003edsg1\u003c/em\u003e) could suppress the enhanced grain size and leaf angle phenotypes caused by \u003cem\u003eOsBSK3\u003c/em\u003e overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Supplemental Fig.\u0026nbsp;6, 7). These above results suggest that OsBSK3 and OsBSK2 regulate multiple biological processes through distinct signaling pathways, and may play a role in mediating the crosstalk between MAPK and BR signaling pathways to control grain size and leaf angle in rice.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOsBSK3 and OsBSK2 may be the upstream messenger of MAPK cascade in regulating leaf angle and grain size\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRLCKs sense external signals and transmit to MAPK cascades, regulating multiple processes of plant growth and development. RLCK185, transmits immune signaling from the PAMP receptor OsCERK1 to an MAPK signaling cascade by interacting with OsMAPKKKs (Wang et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Receptor-like cytoplasmic kinases VII (RLCK VII), which act downstream of PRRs, directly phosphorylate MAPKKK5 at Ser-599, and this phosphorylation is essential for pattern-triggered MPK3/6 activation, defense gene expression, and disease resistance (Bi et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). BSK1 enhances MPK15 phosphorylation at Ser-511, regulating fungal resistance in Arabidopsis (Shi et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). OsBSK1-2 mediates disease resistance in rice through the MAPKKK16/18/19-MAPKK4/5-MAPK3/6 cascade (Li et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In this study, we found that OsBSK3 and OsBSK2 physically interact with OsMKKK10, and directly activate OsMAPK6 in plant cells. This suggests that OsBSK3 and OsBSK2 may be the upstream signal transmitter of the OsMKKK10-OsMKK4-OsMAPK6 cascade in regulating grain size and leaf angle in rice.\u003c/p\u003e \u003c/div\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and growth conditions\u003c/h2\u003e \u003cp\u003eRice (\u003cem\u003eOryza sativa\u003c/em\u003e) cultivar Longjing 11 (\u003cem\u003eOryza sativa\u003c/em\u003e ssp. japonica) was used for generate \u003cem\u003eOsBSK3\u003c/em\u003e and \u003cem\u003eOsBSK2\u003c/em\u003e transgenic plants. Plants were grown in the field (natural long day conditions). Rice cultivar Kuanyejing (KYJ), SF43, and Zhonghua 11 (\u003cem\u003eOryza sativa ssp. japonica\u003c/em\u003e) were used as wild type control to compare with overexpression plants or diverse mutants in relative background. \u003cem\u003eOsMKKK10\u003c/em\u003e mutant \u003cem\u003esmall grain 2\u003c/em\u003e (\u003cem\u003esmg2-1\u003c/em\u003e) (Xu et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), \u003cem\u003eOsMKK4\u003c/em\u003e mutant \u003cem\u003esmall grain 1\u003c/em\u003e (\u003cem\u003esmg1-1\u003c/em\u003e) (Duan et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), \u003cem\u003eOsMAPK6\u003c/em\u003e mutant \u003cem\u003edwarf and small grain\u003c/em\u003e (\u003cem\u003edsg1\u003c/em\u003e) (Liu et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), \u003cem\u003eN390\u003c/em\u003e (which overexpressing the kinase domain (amino acids 1-390) of \u003cem\u003eOsBSK3\u003c/em\u003e) (Zhang et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), \u003cem\u003eoswrky53\u003c/em\u003e (Tian et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and \u003cem\u003eosbsk3\u003c/em\u003e were used to develop double mutant. The \u003cem\u003eUbiquitin\u003c/em\u003e\u003csub\u003e\u003cem\u003epro\u003c/em\u003e\u003c/sub\u003e: \u003cem\u003eOsBSK3\u003c/em\u003e was transformed into \u003cem\u003esmg2-1\u003c/em\u003e, \u003cem\u003esmg1-1\u003c/em\u003e and \u003cem\u003edsg1\u003c/em\u003e to generate \u003cem\u003eOsBSK3-OE smg2-1\u003c/em\u003e, \u003cem\u003eOsBSK3-OE smg1-1\u003c/em\u003e and \u003cem\u003eOsBSK3-OE dsg1\u003c/em\u003e, respectively. \u003cem\u003eUbiquitin\u003c/em\u003e\u003csub\u003e\u003cem\u003epro\u003c/em\u003e\u003c/sub\u003e: \u003cem\u003eOsWRKY53\u003c/em\u003e was transformed into \u003cem\u003eosbsk3\u003c/em\u003e to generate \u003cem\u003eosbsk3 OsWRKY53OE\u003c/em\u003e (Tian et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The \u003cem\u003eN390 oswrky53\u003c/em\u003e line was generated through hybridization between \u003cem\u003eN390\u003c/em\u003e and \u003cem\u003eoswrky53.\u003c/em\u003e Plants were grown in the field, natural long day condition (Harbin) and natural short day condition (Hainan). For all these mutants, the expression level and mutation site of corresponding genes were examined by RT-qPCR, western blot, and DNA sequence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTotal RNA isolation and RT-qPCR analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from leaves using TRIzol (Invitrogen) and then subjected to DNase I digestion. cDNA was synthesized from the extracted total RNA using Superscript II Reverse Transcriptase (Invitrogen). Real-time PCR was performed with a Lightcycler 480 using SYBR Green PCR master mix (Takara). All expressions were standardized for the ubiquitin gene (\u003cem\u003eOs01g0328400\u003c/em\u003e). The primers used are listed in Supplementary Data 1. Three biological replicates were performed for each analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eLUC complementary imaging (LCI) assay\u003c/h2\u003e \u003cp\u003eAs shown in Supplemental Data 1, cLUC-OsBSK3, cLUC-OsBSK2, and nLUC-OsMKKK10 constructs were generated. Agrobacteria harboring different construct combinations infiltrate into \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. Finally, 60 hours after infiltration, the LUC activity of the \u003cem\u003eN. benthamiana\u003c/em\u003e infiltrated leaves was analyzed using Chemiluminescence imaging (Tanon 5200). The assay was conducted in three biological replicates, with three leaves per replicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eBimolecular fluorescence complementation (BiFC) assay\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eFor BiFC assay, OsMKKK10 was fused with partial GFP to generate nGFP-OsMKK10, while OsBSK3 and OsBSK2 were combined with partial GFP to generate cGFP-OsBSK3 and cGFP-OsBSK2, as described in Supplemental Data 1. Transform these vectors into \u003cem\u003eAgrobacterium\u003c/em\u003e strain \u003cem\u003eGV3101\u003c/em\u003e, after different combinations, co-injected into the young leaves of \u003cem\u003eN\u003c/em\u003e. \u003cem\u003ebenthamiana\u003c/em\u003e separately. Subsequently, fluorescence emission was observed under confocal microscopy (Leica) after 60 h. The assay was conducted in three biological replicates, with three leaves per replicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eYeast two-hybrid assay\u003c/h2\u003e \u003cp\u003eCloning the coding sequences of \u003cem\u003eOsBSK3\u003c/em\u003e and \u003cem\u003eOsBSK2\u003c/em\u003e into the \u003cem\u003eEcoRI\u003c/em\u003e and \u003cem\u003ePstI\u003c/em\u003e sites of pGBKT7 vector to generate BD-OsBSK3 and BD-OsBSK2 constructs. And then, the coding sequence of OsMKKK10KD was cloned into the \u003cem\u003eEcoRI\u003c/em\u003e and \u003cem\u003eXhoI\u003c/em\u003e sites of pGADT7 vector to generate the AD-OsMKKK10KD construct. The obtained constructs were transformed into yeast strain Y2H Gold. The presence of plasmids was confirmed by growth on the synthesis limited (SD) medium lacking Trp and Leu (SD-Trp-Leu). Next, the positive yeast clones were suspended in liquid SD-Trp-Leu medium (OD600\u0026thinsp;=\u0026thinsp;1.0) to assess protein interactions. Finally, the suspension cells and their dilutions were plated onto SD-Trp-Leu-His and SD-Trp-Leu-His-Ade medium. Protein interactions were observed after incubation at 30 ℃ for three days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCo-Immunoprecipitation (Co-IP) assays\u003c/h2\u003e \u003cp\u003e \u003cem\u003e35S\u003c/em\u003e \u003csub\u003e \u003cem\u003epro\u003c/em\u003e \u003c/sub\u003e: \u003cem\u003eFLAG-OsBSK2\u003c/em\u003e, \u003cem\u003e35S\u003c/em\u003e\u003csub\u003e\u003cem\u003epro\u003c/em\u003e\u003c/sub\u003e: \u003cem\u003eFLAG-GFP\u003c/em\u003e, and \u003cem\u003e35S\u003c/em\u003e\u003csub\u003e\u003cem\u003epro\u003c/em\u003e\u003c/sub\u003e: \u003cem\u003eMYC-OsMKKK10KD\u003c/em\u003e constructs were made as described in Supplemental Data Set 1 for Co-IP assay, These plasmids were co expressed transiently in rice protoplasts, as shown in the combination. Total protein was extracted using the lysis buffer (50 mM Tris HCl at pH 7.5, 150 mM NaCl, 0.5 mM EDTA at pH 8.0, 10% glycerol, 0.5% Triton X-100) with freshly added protease inhibitor cocktail (Roche, 11873580001), PMSF (phenylmethylsulfonyl fluoride, Roche, 10837091001), and MG132 (Sigma Aldrich, M8699). Before the Co-IP assays, incubating 20 \u0026micro;l of protein-A/G magnetic beads (GenScript, L00277) with 2 \u0026micro;l anti-Flag (Abmart, M20008) for 2 hours, gently rotate at 4\u0026deg;C, then adding an equal amount of total protein extractions and incubating at 4\u0026deg;C for 2 hours. Washing the magnetic beads multiple times with the washing buffer (50 mM Tris HCl at pH 7.5, 100 mM NaCl, 0.5 mM EDTA at pH 8.0, 0.1% Triton X-100). The immunoprecipitates were eluted with SDS sample buffer, separated on SDS-PAGE gel, transferred to PVDF membrane (Millipore 0.45um, IPVH00010), and detected with anti FLAG (Abmart, M20008) and anti MYC (ThermoFisher, 9E10) antibodies respectively. For all Co-IP assays, we conducted at least three independent replications.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eRNA-Seq and data analysis\u003c/h2\u003e \u003cp\u003eFor Illumina sequencing, collecting tiller buds from LJ11 and \u003cem\u003eosbsk3\u003c/em\u003e plants. The extraction and detection of total RNA, library preparation, and Illumina sequencing were performed by Novogene Bioinformatics Technology Co., Ltd., Beijing, China using the Illumina HiSeq 2500 platform. The reference genome and gene information of Rice cultivar Nipponbare can be downloaded from IRGSP-1.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://rapdb.dna.affrc.go.jp/download/irgsp.html\u003c/span\u003e\u003cspan address=\"http://rapdb.dna.affrc.go.jp/download/irgsp.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Using Hisat 2 for mapping quality and saturation analysis. Using DESeq2 to detect differentially expressed genes, with the absolute fold change\u0026thinsp;\u0026gt;\u0026thinsp;1.5, FDR-adjusted P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The GO analysis was carried out by the PANTHER classification system (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.pantherdb.org\u003c/span\u003e\u003cspan class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with FDR\u0026thinsp;\u0026le;\u0026thinsp;0.05, and get the GO annotations based on biological process.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003ekinase assay\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs described in Supplementary Data 1, constructs were prepared for expressing GST-OsBSK3S215E, GST-OsBSK2, and GST-OsMKKK10. Purify the fusion protein according to the manufacturer's instructions. Subsequently, the purified protein was incubated with phosphorylation buffer (25 mM Tris-HCl at pH 7.4, 12 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1 mM DTT, and 1 mM ATP) at 30 ℃ for 45 minutes, and then boiled in SDS loading buffer. And according to the manufacturer's instructions, using Phos-tag Biotin BTL-104 (Wako, Richmond, VI, USA; 301-93531) for detect.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eMAPK activity assay\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe wild-type LJ11, \u003cem\u003eOsBSK3-OE\u003c/em\u003e, \u003cem\u003eOsBSK2-OE\u003c/em\u003e, \u003cem\u003eosbsk3\u003c/em\u003e and \u003cem\u003eosbsk2\u003c/em\u003e plants were grown in a greenhouse, and collect their 3 cm young spikles separately. Then, following the manufacturer's instructions, extract total protein from the plant and the rice protoplast using SDS Lysis solution (Beyotime, P0013G) with added protease inhibitor cocktail (Roche, 11206893001), 50 \u0026micro;M MG132, 1 mM PMSF, and phosphatase inhibitor cocktail (Sigma-Aldrich, P2850) respectively. The proteins were separated by 8% SDS-PAGE and detected by immunoblotting with anti-Phospho-p44/42 MAPK (Cell Signaling Technology, #9101) and anti-ACTIN (Abmart, M20009M) antibodies. In addition, \u003cem\u003e35S\u003c/em\u003e\u003csub\u003e\u003cem\u003epro\u003c/em\u003e\u003c/sub\u003e:\u003cem\u003eFLAG-OsBSK2\u003c/em\u003e, \u003cem\u003e35S\u003c/em\u003e\u003csub\u003e\u003cem\u003epro\u003c/em\u003e\u003c/sub\u003e:\u003cem\u003eFLAG-OsBSK3\u003c/em\u003e, \u003cem\u003e35S\u003c/em\u003e\u003csub\u003e\u003cem\u003epro\u003c/em\u003e\u003c/sub\u003e:\u003cem\u003eGFP-FLAG\u003c/em\u003e, and \u003cem\u003e35S\u003c/em\u003e\u003csub\u003e\u003cem\u003epro\u003c/em\u003e\u003c/sub\u003e:\u003cem\u003eMYC-OsMAPK6\u003c/em\u003e constructs were made as described in Supplemental Data Set 1 for MAPK activity assays, these plasmids were co expressed transiently in rice protoplasts as indicated combination. And perform in vivo MAPK activity assay according to the above method, detecting by immunoblotting with anti-Phospho-p44/42 MAPK (Cell Signaling Technology, #9101), anti FLAG (Abmart, M20008) and anti MYC (ThermoFisher, 9E10) antibodies respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStudent\u0026rsquo;s two-tailed t-test was used to compare the means between two samples/variables, while the one-way ANOVA analyses were used for multiple pairwise comparisons. Tukey\u0026rsquo;s test at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 significance level separated the differences between the means. All the analyses were performed using SPSS software.\u003c/p\u003e \u003cp\u003eStudent's two tailed t-test was used to compare the means between two samples/variables, while the one-way ANOVA analyses was used for multiple pairwise comparisons. Tukey\u0026rsquo;s test at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 significance level separated the differences between the means. All analyses were conducted using SPSS software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eAccession numbers\u003c/h2\u003e \u003cp\u003eDNA sequence data from this article can be found in gramene (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.gramene.org/\u003c/span\u003e\u003cspan address=\"https://www.gramene.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) under the following accession numbers: Ubiquitin (Os01g0328400), OsMAPK6 (Os06g0154500), OsMKKK10 (Os04g0559800), OsBSK3 (Os04g0684200), OsBSK2 (Os10g0571300), OsBSK1-2 (Os10g0542800), OsBSK4 (Os03g0825300).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would thank Prof. Yunhai Li, Prof. Fan Chen and Prof. Wenqiang Tang for providing the relative mutants and vectors. This study was supported by National Key Research and Development Program of China (Grant No. 2024YFD1201001), Youth Innovation Promotion Association CAS (Grant No. 2021229), Young Scientist Group Project of Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences (Grant No. 2023QNXZ02).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX.T., Q.B., and C. L. conceived and supervised the project. X.T., Q.B., and X.J. analyzed the data and wrote the article. X.J. and L.F. performed most of the experiments. J.L., C.C., Y.L., W. Z., and X.L. contributed to diverse experiments. All authors read and approved the contents of this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e\u003cstrong\u003eBi, G.Z., Zhou, Z.Y., Wang, W.B., Li, L., Rao, S.F., Wu, Y., Zhang, X.J., Menke, F.L.H., Chen, S., and Zhou, J.M.\u003c/strong\u003e (2018). Receptor-Like Cytoplasmic Kinases Directly Link Diverse Pattern Recognition Receptors to the Activation of Mitogen-Activated Protein Kinase Cascades in Arabidopsis. The Plant cell \u003cstrong\u003e30, \u003c/strong\u003e1543-1561.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eDong, X.J., Feng, F., Li, Y.J., Li, L., Chen, S., and Zhou, J.M.\u003c/strong\u003e (2023). 14-3-3 proteins facilitate the activation of MAP kinase cascades by upstream immunity-related kinases. 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Developmental cell \u003cstrong\u003e43, \u003c/strong\u003e618.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"theoretical-and-applied-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"taag","sideBox":"Learn more about [Theoretical and Applied Genetics](https://www.springer.com/journal/122)","snPcode":"122","submissionUrl":"https://submission.nature.com/new-submission/122/3","title":"Theoretical and Applied Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"MAPK cascades, OsBSK3, OsBSK2, Grain size, Leaf angle","lastPublishedDoi":"10.21203/rs.3.rs-5897621/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5897621/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGrain size and leaf angle are key agronomic traits that determine the final yield. OsBSKs (BRASSINOSTEROID-SIGNALING KINASES) and OsMAPKs (MITOGEN ACTIVATED PROTEIN KINASE) are known to play essential roles in plant growth, development, and stress responses. However, the potential crosstalk between these pathways and their specific roles in regulating grain size and leaf angle remain largely unexplored in rice. Here, we characterized OsBSKs regulate grain size and leaf angle in rice, and among these, OsBSK2 and OsBSK3 may play more critical roles. The grain size and leaf angle in \u003cem\u003eosbsk3\u003c/em\u003e and \u003cem\u003eosbsk2\u003c/em\u003e mutants are significantly smaller, whereas the \u003cem\u003eOsBSK3\u003c/em\u003e-overexpressing lines (\u003cem\u003eOsBSK3\u003c/em\u003e-OEs) exhibit considerably larger grain size and leaf angle compared to the others. Furthermore, both OsBSK3 and OsBSK2 interact with OsMKKK10, indirectly activating OsMAPK6 in plant cells. Notably, mutations in MAPK cascade components, such as \u003cem\u003esmg2-1\u003c/em\u003e (\u003cem\u003eosmkkk10\u003c/em\u003e mutant), \u003cem\u003esmg1-1\u003c/em\u003e (\u003cem\u003eosmkk4\u003c/em\u003e mutant), and \u003cem\u003edsg1\u003c/em\u003e (\u003cem\u003eosmapk6\u003c/em\u003e mutant), resulted in significantly reduced leaf angles. Moreover, these mutations were able to rescue the increased grain size and leaf angle in \u003cem\u003eOsBSK3\u003c/em\u003e overexpression lines. Additionally, we also identified OsWRKY53 as a potential downstream target of the OsBSKs-OsMKKK10-OsMKK4-OsMAPK6 cascade in regulation of grain size and leaf angle. Taken together, the above results not only highlight the essential and specific roles of OsBSK3 and OsBSK2 in regulating rice grain size and leaf angle, but also reveal the mechanism which OsBSK3/OsBSK2 mediating MAPK cascade to regulate grain size and leaf angle, OsBSK3 and OsBSK2 may act as key mediator of crosstalk between BR and MAPK signaling.\u003c/p\u003e","manuscriptTitle":"OsBSK3 and OsBSK2 regulate grain size and leaf angle via MAPK signaling pathway in rice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-10 08:44:58","doi":"10.21203/rs.3.rs-5897621/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2025-02-26T02:19:52+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-02-07T10:52:55+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-02-07T09:32:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-02-03T06:22:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Theoretical and Applied Genetics","date":"2025-02-01T10:14:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"theoretical-and-applied-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"taag","sideBox":"Learn more about [Theoretical and Applied Genetics](https://www.springer.com/journal/122)","snPcode":"122","submissionUrl":"https://submission.nature.com/new-submission/122/3","title":"Theoretical and Applied Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c08bf4da-bc04-4ca8-9f56-5321038cb1db","owner":[],"postedDate":"February 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-21T16:00:54+00:00","versionOfRecord":{"articleIdentity":"rs-5897621","link":"https://doi.org/10.1007/s00122-025-04889-w","journal":{"identity":"theoretical-and-applied-genetics","isVorOnly":false,"title":"Theoretical and Applied Genetics"},"publishedOn":"2025-04-20 15:57:36","publishedOnDateReadable":"April 20th, 2025"},"versionCreatedAt":"2025-02-10 08:44:58","video":"","vorDoi":"10.1007/s00122-025-04889-w","vorDoiUrl":"https://doi.org/10.1007/s00122-025-04889-w","workflowStages":[]},"version":"v1","identity":"rs-5897621","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5897621","identity":"rs-5897621","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}