MAPK signaling modulates the partition of DCP1 between processing bodies and stress granules in plant cells

MAPK signaling modulates the partition of DCP1 between processing bodies and stress granules in plant cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article MAPK signaling modulates the partition of DCP1 between processing bodies and stress granules in plant cells Siou-Luan He, Ying Wang, Libo Shan, Ping He, Jyan-Chyun Jang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7292794/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Feb, 2026 Read the published version in Discover Life → Version 1 posted 11 You are reading this latest preprint version Abstract Processing bodies (PBs) and stress granules (SGs) are membrane-less cellular compartments consisting of ribonucleoprotein complexes. Whereas PBs are more ubiquitous, SGs are assembled mainly in response to stress. PBs and SGs are known to physically interact and molecules exchange between the two have been documented in mammals. However, the molecular mechanisms underpinning these processes are unknown in plants. We recently reported that tandem CCCH zinc finger 1 (TZF1) protein can recruit mitogen-activated protein kinase (MAPK) signaling components to SGs. Here, we show that TZF1-MPK3/6-MKK4/5 form a protein-protein interacting network in SGs. The mRNA decapping factor 1 (DCP1) is a core component of PBs. MAPK signaling mediated phosphorylation triggers a rapid reduction of DCP1 partition into PBs, concomitantly associated with an increase of DCP1 assembly into SGs. Furthermore, we found that the plant SG marker protein, oligouridylate binding protein 1b (UBP1b), plays a role in maintaining DCP1 in PBs by suppressing the accumulation of MAPK signaling components. Together, we propose that MAPK signaling and UBP1b mediate the dynamics of PBs and SGs in plants. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Introduction Processing bodies (PBs) and stress granules (SGs) are two types of cytoplasmic biomolecular condensates that dynamically assembled in response to environmental stresses. Dysregulation of PBs and SGs has been implicated in various human diseases such as neuro- and muscular-degenerative diseases, neuro-developmental diseases, and cancers (1,2). Although previous studies have suggested that PBs and SGs perform distinct functions, as each contains a unique set of proteins, multiple observations indicate that SGs interact with PBs and likely exchange messenger ribonucleoproteins (mRNPs) between each other (3). Under specific stress conditions, PBs often dock with SGs, and the overexpression of certain proteins that localize to both structures can lead to the fusion of PBs and SGs (4). For example, the overexpression of tristetraprolin (TTP) or butyrate response factor-1 (BRF-1), two RNA-binding proteins that target ARE-containing mRNAs to PBs for degradation (5) or cytoplasmic polyadenylation element binding protein (CPEB1), another dual SG/PB protein (6), lead to the tight clustering of PBs around and within SGs (5-7). Overexpression of ubiquitin-associated protein 2-like (UBAP2L), and the interaction between UBAP2L and SG and PB nucleating protein such as stress granule assembly factor (G3BP) and DEAD-box helicase 6 (DDX6), respectively, induces hybrid granules containing SG and PB components in the cells (8). Furthermore, PBs can play a role in promoting SG assembly by providing untranslated mRNAs, shared proteins, and translational repressors that are necessary for nucleating SGs during cellular stress conditions (9). On the other hand, SGs could still form in the absence of PBs. DDX6 (Rck/p54) is an evolutionarily conserved member of the DEAD-box RNA helicase family involved in the inhibition of translation and storage and the degradation of cellular mRNAs in PBs. When DDX6 expression is reduced, PB formation is strongly impaired, whereas DDX6 knockdown causes the PB-specific protein DCP1 to relocate to SGs (10,11), suggesting that some proteins involved in PB assembly may switch roles and contribute to co-aggregate with SG proteins under specific stress conditions. In plants, RNA-binding protein 47b (Rbp47b) and Tudor Staphylococcal Nuclease (TSN2) are two core components of plant SGs (12). Rbp47b and TSN2 interactome analysis in Arabidopsis revealed the presence of mitogen-activated protein kinase (MAPK) signaling components MPK3, MKK4, and MKK5 (13), suggesting that MAPK signaling could mediate SG dynamics, perhaps by phosphorylating key SG components. MAPK cascades (including MPK3 and MPK6 and their upstream kinases MKK4 and MKK5) play a crucial role in plant lifecycle, where they regulate a wide range of physiological processes, including growth and development as well as in responses to environmental cues such as cold, heat, drought, and especially pathogen attack (14). Some other kinases can also be recruited to SGs and PBs (15,16). The SG component Ras-GAP SH3-binding protein (G3BP1) recruits casein kinase 2 (CK2) to SGs and phosphorylation of G3BP1 by CK2 promotes dissociation of G3BP from SGs and triggers SG disassembly (17). The yeast kinase Sky1 is recruited to heat-induced SGs, where it can phosphorylate substrates Npl3 (a nucleocytoplasmic mRNA shuttling protein) to promote SG dissolution (18). SGs also play a role in sequestering signaling molecules as a protective mechanism. For example, high-heat stress stimulates MAPK activation, which causes fission yeast protein kinase C (Pck2) translocation from the plasma membrane into SGs to suppresses MAPK hyperactivation and cell death (19). In plants, the molecular mechanisms underpinning the interaction and equilibrium between PBs and SGs are unclear. Here we present multiple lines of evidence to propose that a model in which MAPK signaling pathway is involved in triggering a major PB component DCP1 to relocate to SGs. We previously showed that tandem CCCH zinc finger 1 (TZF1) recruits MAPK signaling components to SGs (20). Using a high throughput and high-fidelity Arabidopsis protoplast transient expression system, we have found that MPK3, MPK6, MKK4, and MKK5 form homo- and hetero-dimers with each other in SGs. DCP1 is phosphorylated by MPK3 and MPK6 and the kinase activity of MKK5 is required for DCP1 to localize to SGs. In support of this notion, the phospho-dead form of DCP1 S237A is mainly localized in PBs and phosphor-mimetic form of DCP1 S237D is mainly localized in SGs. Oligouridylate binding protein 1b (UBP1b) is an RNA-binding protein and it has been used widely as an SG marker. Surprisingly, MAPK signaling-mediated reduction of DCP1 association with PBs is antagonized by the SG marker UBP1b, as UBP1b suppresses the accumulation of MPK3/6 and MKK4/5. Consistent with this idea, the abundance of DCP1 association with PBs is enhanced by the co-expression of UBP1b, as well as an additional mechanism independent of DCP1’s phosphorylation status. Together, our results indicate that MAPK phosphorylation could serve as a switch for DCP1 sequestration from PBs to SGs. This pathway is counteracted by an SG marker UBP1b that could diminish the level of MAPK signaling components. Results TZF1-MPK3/6-MKK4/5 interacting network We have shown previously that TZF1 could interact with MPK3/6 and MKK4/5 in SGs (20). Here, we found that MPK3 and MPK6 could interact with itself and each other in bimolecular fluorescence complementation (BiFC) analysis. MKK4 and MKK5 could also interact with itself and each other (Fig. 1A). Furthermore, MPK3 or MPK6 could also interact with MKK4 or MKK5 in BiFC analysis (Fig. 1B). Remarkably, all the interacting BiFC signals were co-localized with an SG marker TZF1 (21) in cytoplasmic granules, suggesting that MPK3/6 and MKK4/5 interact in SGs. To validate protein-protein interactions identified in BiFC analyses, co-immunoprecipitation (Co-IP) assays were conducted. Pair-wise protein components with various tags were co-expressed in an Arabidopsis protoplasts transient expression system and IP was carried out using GFP-antibody. Results indicated that MKK4 and MKK5 (Fig. 1C) as well as MPK3 and MPK6 (Fig. 1D) could self- and cross-interact. MKK4 or MKK5 could cross-interact with MPK3 or MPK6 as well (Fig. 1E). No Co-IP signals were found when the MKK construct was co-expressed with the empty construct with free GFP (Fig. 1F), indicating the specificity of the Co-IP results. Interestingly, when the BiFC analysis was conducted in the presence of nuclear marker NLS-RFP, the interaction between MPK3 or MPK6 with MKK4 (not shown) or MKK5 was mainly in the nucleus (Fig. 2), as opposed to in the cytoplasmic granules when co-expressed with TZF1 (Fig. 1A and B). However, BiFC signals of MKK4 and MKK5 self- and cross-interactions remained distinctively in cytoplasmic granules when co-expressed with NLS-RFP (Fig. 2). These results suggest that TZF1 recruits cross-interactions between MPK3/6 and MKK4/5 to SGs. To determine if protein-protein interactions were taken place in PBs or SGs, additional markers were co-expressed in the BiFC analyses. Results indicated that most of the self- and cross-interactions among MPK3/6 and cross interactions between MPK3/6 and MKK4/5 were not colocalized with a major PB component DCP1 (Fig. 3A), whereas almost completely co-localized with SG marker UBP1b (Fig. 3B). Note that BiFC signals of MPK3-MKK5 were occasionally overlapped with DCP1-mCherry signals (indicated by an arrow in Fig. 3A). Furthermore, UBP1b could be localized to both SGs and the nucleus. It appeared that most of the aforementioned interactions were co-localized with UBP1b in the nucleus (Fig. 3B), suggesting a dominant role of protein-protein interaction in affecting UBP1b subcellular localization. Intriguingly, the above-mentioned interactions appeared to be suppressed by the co-expression of UBP1b in some cells, as evidenced by their very weak or missing BiFC signals and strong UBP1b-mcherry signals in SGs (Fig. 3C). This raised a possibility that MPK3-MPK3 and MPK3-MPK6 interactions were more stable in the nucleus than in SGs (with strongly co-expressed UBP1b). For MKK4 and MKK5, the BiFC signals of both self- and cross-interactions were predominately localized in cytoplasmic granules and only partially (indicated by arrows) co-localized with DCP1, but almost completely co-localized with SG marker UBP1b (Fig. 4). Based on the results of BiFC (Fig. 1A-B and 2-4), Co-IP (Fig. 1C-F), and previous report (20), we proposed a model in which TZF1, MPK3/6, and MKK4/5 formed an interactome in SGs (Fig. S1). DCP1 granule assembly affected by MPK3/6 and MKK4/5 In the course of conducting BiFC analyses, it was noted that the signal levels of PB component DCP1-mCherry varied, depending on a specific co-expressed pair of BiFC constructs. For example, the DCP1 granule number was abundant when co-expressed with MKK4-nYFP+MKK4-cYFP, but much lower when co-expressed with MKK4-nYFP+MKK5-cYFP, MKK5-nYFP+MKK4-cYFP, and MKK5-nYFP+MKK5-cYFP, respectively (Fig. S2A and B). Likewise, DCP1 granule number was abundant when co-expressed with MPK3-nYFP+MPK3-cYFP, but low when co expressed with MPK3-nYFP+MKK4-cYFP, MPK3-nYFP+MKK5-cYFP, and MPK3-nYFP+MPK6-cYFP, respectively (Fig. S3A and B). It was also noted that both the number and the size of DCP1 granules were varied among samples. To verify if differential signal levels of DCP1 granules were due to co-expressed individual proteins, additional co-expression analyses were conducted using full-length GFP fusion proteins. The number of DCP1 granules was slightly reduced by co-expression of MPK3, moderately by MKK4, but severely reduced by MPK6 and MKK5. By contrast, the size of DCP1 granules appeared to be strongly enhanced by MPK3 and slightly enhanced by MPK6, whereas reduced by MKK4 and MKK5, as compared to the sample co-expressed with free GFP (Fig. 5). Given MPK6 appeared to have a stronger effect than MPK3, additional family members of MPKs were tested using available BiFC constructs. Results showed that the BiFC constructs were as effective as the GFP-fusion constructs, because MPK6-cYFP could suppress DCP1 granule accumulation, as compared to GFP-MPK6. In additional to MPK6, MPK1, 4, and 11 had similar effects in reducing DCP1 granule accumulation (Fig. S4). From the cellular images in (Fig. S4), it appeared that the protein expression levels of MPKs, MKKs, and DCP1 varied in different samples. As protein level might affect biomolecular condensate assembly (22), immunoblot analyses were conducted. To further delineate the effects of MPKs/MKKs on DCP1 accumulation, two dozes of plasmid DNA (20 and 40 mg) were used in the protoplast transient expression analysis. Consistent with cellular images in (Fig. S4), the levels of protein expression displayed large differences ranging from weak to strong in the order of MPK3, MPK6, MKK4, and MKK5. By contrast, DCP1 level varied just slightly between samples co-expressed with MPK3/6 and MKK4/5, with only negligible differences between the two samples transformed with different doses of DCP1-mCherry plasmid DNA (Fig. 5C). Similar analysis was carried out for various MPK-cYFP and DCP1-mCherry samples as shown in (Fig. S5). Results showed whereas the levels of MPKs varied, with MPK11-cYFP at the lowest abundance, the level of DCP1-mCherry remained at similar levels across different samples. Of note, MPK3-cYFP and MPK6-cYFP appeared to be much more stable than their counterparts fused with GFP. However, the co-expressed DCP1-mC was accumulated at a remarkably consistent level across all samples. Together, these results suggest that post-translational regulation plays a major role on DCP1 granule dynamics, although DCP1 protein accumulation might also play a secondary role. Phosphorylation activity of MKK5 modulates the partition of DCP1 between PBs and SGs We showed previously that plant SG assembly is affected by the phosphorylation status of a core SG protein TZF1 (20). Co-expression analysis was then carried out to determine if the phosphorylation status of MKK5 could affect DCP1 granule assembly. Results showed that co-expression of MKK5 WT reduced the number of DCP1 granules to about 50%, whereas the MKK5 DD , a constitutively active form of MKK5 T215D/S221D (23,24), exerted even stronger suppression. By contrast, the MKK5 KR , a loss-of-function mutation of the conserved K to R change in the kinase ATP-binding loop of MKK5 K99R (23), enhanced the accumulation of DCP1 granules (Fig. 6A and B). Noticeably, the decrease of DCP1 granules was associated with an increased number of cells containing a dominant large DCP1 granule. We showed previously that flg22-induced MAPK signaling cascade could trigger DCP1 phosphorylation and rapid disassembly of DCP1 granules, whereas the phospho-dead DCP1 S237A was resistant to the effect of flg22 (25). Here, by expressing DCP1 phosphorylation mutant constructs alone, it was found that the accumulation of granules from DCP1 S237A was far greater, whereas DCP1 S237D (phospho-mimetic) was far less than that from DCP1 WT (Fig. 6). When the individual DCP1 constructs were co-expressed with phosphorylation mutants of MKK5, the DCP1 S237A granules remained highly abundant across each combination (Fig. 6C and D), whereas the accumulation of DCP1 S237D granules was low and dominated by the single large granule pattern across each combination of DCP1+MKK5 (Fig. 6E and F). These results suggest that MKK5 acts upstream of DCP1 and phosphorylation status of DCP1 is a determinant for the accumulation/assembly and form/size of DCP1 granules. To determine if MKK5-mediated DCP1 granule dynamics was related to protein accumulation, immunoblot analysis was conducted. Interestingly, compared to the MKK5 WT , MKK5 DD was accumulated at a higher level, whereas MKK5 KR was accumulated at a lower level, across various samples. By contrast, the accumulation of DCP1 WT , DCP1 S237A , and DCP1 S237D showed a remarkably consistent level across various samples (Fig. 7). These results indicate that DCP1 granule dynamics is likely orchestrated by post-translational regulation but not protein accumulation. As DCP2 granule dynamics was similarly regulated by MKK5 kinase activity, same assay was also conducted (results not shown). Similar results were obtained as those from DCP1 (Fig. 7, right panel). Phosphorylation triggers the sequestration of DCP1 from PBs to SGs Because a high number of cells contained coalesced large DCP1 S237D granules in (Fig. 6E), we sought to determine the identity of the large DCP1 granules. Intriguingly, albeit differing in number, both large and small granules were present in all three types of DCP1, independent of its phosphorylation status (Fig. 6). We first examined the relationship between small granules and various cellular markers. Results showed that small granules of all three types of DCP1 were independent of nuclear marker NLS-RFP (Fig. 8A). The small granules of DCP1 WT and DCP1 S237A were not co-localized with the SG marker UBP1b either, supporting their identity as PBs. By contrast, the DCP1 S237D granules were generally larger and they completely co-localized with UBP1b, suggesting that phosphorylation of DCP1 is a trigger to sequester DCP1 from PBs to SGs (Fig. 8B). Paradoxically, the large ‘nucleus-like’ DCP1 granules were not co-localized with the nuclear marker NLS-RFP (Fig. 9A), but instead partially or completely co-localized with the SG marker UBP1b (Fig. 9B). These results suggest that phosphorylated DCP1 is mainly sequestered to SGs, but the phosphorylation is not the only prerequisite for SG localization, as small percentage of cells with large granules were also found in DCP1 S237A samples and they could also co-localize with UBP1b. We speculate that there might be redundant post-translational modification events that could trigger the sequestration of DCP1 S237A to SGs. Together, these results suggest that protein phosphorylation and additional post-translational modification mechanisms likely play a role in the sequestration of DCP1 between PBs and SGs. UBP1b suppresses MAPK signaling to maintain DCP1 in PBs In contrast to the relationship between DCP1 and MPK3/6 and MKK4/5, co-expression of UBP1b appeared to suppress the BiFC signals generated from interactions within and between MPK3/6 (Fig. 3C). To decipher if UBP1b suppressed the signals of protein-protein interactions or suppressed the individual protein accumulation, co-expression analyses were conducted using single gene-GFP fusion constructs. Results showed that the signals from MPK3, MPK6, and MKK4 were dampened by the co-expression of UBP1b (Fig. 10A). Consistent with the cellular imaging results, immunoblot analysis revealed that co-expression of two different doses of UBP1b resulted in a reduction of the accumulation of MPK6 and MKK4, with a lesser extent on MKK5. MPK3 level was too low to be determined (Fig. 10B). Given UBP1b appeared to cause a general reduction of MPKs/MKKs protein accumulation, the variation of MPKs/MKKs granule intensity/dynamics could have been contributed by the protein abundance, although it was not as obvious for MPK6 and MKK4 from cellular images (Fig. 10A). Given DCP1 granule assembly could be modulated by MAPK signaling components whose accumulation appeared to be affected by UBP1b, the relationship between DCP1 phosphorylation status and UBP1b was investigated. Compared to the DCP1 WT , DCP1 S237A had increased and DCP1 S237D had decreased number of granules (Fig. 11A). When UBP1b was co-expressed, DCP1 granule abundance was enhanced and there was no significant difference between the three types of DCP1 (Fig. 11B and C). Because none of the DCP1-mCherry granules were co-localized with the SG marker UBP1b-GFP, the small and distinct DCP1-mCherry granules were likely PBs (Fig. 11B). Immunoblot analysis was conducted to determine if increased DCP1 granule abundance was due to higher level of protein accumulation. Compared to the samples co-expressed with free GFP, a general reduction of the accumulation of all three types of DCP1, independent of their phosphorylation status, was found when co-expressed with UBP1b-GFP. More importantly, the three DCP1 proteins accumulated at nearly the same level, ruling out the possibility of increased DCP1 granule abundance due to elevated protein accumulation (Fig. 11D). Together, these results suggest that UBP1b suppresses MAPK signaling components that act as negative regulators of DCP1 large granule assembly. We propose a model in which flg22 activates MAPK signaling mediated post-translational modification of DCP1 that results in a decrease of DCP1 localization to PBs, whereas an increase of DCP1 sequestration to SGs. UBP1b, on the other hand, acts as a negative regulator of certain MPKs and MKKs, hence counteracting with the MAPK signaling effects and resulting in the maintenance of DCP1 to be associated with PBs (Fig. 12). Because UBP1b-mediated increase of DCP1 sequestration to PBs appeared to override the phosphorylation status of DCP1, it is likely that an additional unknown mechanism exerted by UBP1b is also operating in this process (Fig. 12). We also speculate that UBP1b, a key modulator of SG assembly, and MAPK signaling play a role in the homeostasis control of PBs and SGs in response to stresses other than flg22, such as heat, salt, and ABA (26-28). Discussion PBs and SGs share similarities in composition and function in various cellular processes. Although distinct core components of PB and SG have been redefined by using sophisticated proximity mapping (29), recent reports have also found an extensive overlap of composition across PBs and SGs, such as poorly translated mRNAs and low complexity RNA-binding proteins (30). In mammals, double knockout of RNA-binding protein G3BP1 and G3BP2 prevents SG assembly induced by eukaryotic initiation factor 2a phosphorylation. In this double mutant background, phosphor-mimetic mutant G3BP S149E failed to rescue SG assembly, highlighting the importance of the role of PTM on G3BP. In another scenario, Caprin and USP10 bind G3BP in a mutually exclusive way, whereas G3BP-Caprin complex promotes SG assembly, G3BP-USP10 complex promotes SG disassembly (31,32). The plant ubiquitin-specific protease family members are homologs of USP10, including UBP24, a negative regulator of ABA signaling (33). It is not known if UBP24 is involved in PB-SG interaction. Confusing in nomenclature, although UBP1b is not an UBP family member, it is a key modulator of SG assembly in plants (26). On the other hand, the mammalian DDX6 (Rck/p54), a major PB scaffold component, plays a key role in PB-SG interaction. DDX6 limits itself and other RNPs to be assembled into SGs. In the absence of DDX6, more RNPs are partitioned into SGs. Loss PB scaffold proteins such as DCP1 and DDX6 also causes reduction in PB growth and enhances incompletely assembled PBs docking with SGs to form hybrid granules with irregular shapes (11,34). SG assembly provides a means for the temporal and spatial compartmentalization of signaling components critical for cell growth and defense response (35). In fission yeast, while it is not demonstrated that MAPK signaling components are sequestered to SGs, the high heat stress induced MAPK activation triggers the sequestration of upstream regulator PKC into SGs thereby deactivating MAPK hyperactivation induced cell death (19,36). While some limited information is available from studies using non-plant models, the molecular mechanisms mediating PB-SG dynamics in plants are virtually unknown. MPK-MKK-TZF1 interactome in SGs In this report, we have found that MAPK signaling components, including MPK3, MPK6, MKK4, and MKK5, can self- and cross-interact with each other (Fig. 1-4). These protein-protein interactions take place mainly in SGs, with a slight chance in PBs. MPK3 and MPK6 self- and cross-interactions, as well as cross-interactions between MPK3/6 and MKK4/5 often occur in the nucleus where the SG marker UBP1b can also be localized when co-expressed with the MAPK BiFC constructs (Fig. 3). In contrast, MKK4 and MKK5 self- and cross-interactions are primarily taken place in cytoplasmic granules with complete co-localization with SG marker UBP1b and limited co-localization with the core PB component DCP1 (Fig. 4). More importantly, all interactions are co-localized with TZF1 cytoplasmic granules, including MPK3 and MPK6 self- and cross-interactions (Fig. 1). As we have demonstrated in an earlier report that TZF1 interacts with MPK3, MPK6, MKK4, and MKK5 in vivo and in vitro (20), we propose that TZF1 forms an interacting network with MAPK components in SGs (Fig. S1). MAPK signaling modulates DCP1 granule assembly In the process of conducting BiFC analyses, it was noted that the proteins generated from BiFC constructs had significant interactions with co-expressed PB (DCP1) or SG (UBP1b) marker protein. For example, DCP1-mCherry granules were suppressed by all combinations except MKK4-nYFP+MKK4-cYFP (Fig. S2A and B) and MPK3-nYFP+MPK3-cYFP (Fig. S3A and B). Further analysis indicated that DCP1-mCherry granules were differentially affected by MAPK signaling components: (1) DCP1 granule size was enhanced by the co-expression of MPK3 and MPK6; (2) DCP1 granule number was progressively reduced by co-expression of MAPK components in the order of MPK6, MKK5, MKK4, and MPK3 (Fig. 5B). Additional analysis revealed that MPK1, MPK4, and MPK11 could also reduce the number of DCP1-mCherry granules (Fig. S4). This is potentially important because the two MKK4/5-MPK3/6 and MEKK1-MKK2-MPK4 MAPK signaling cascades play opposing roles in plant cold response (24). Our preliminary results indicated that MEKK1 could enhance DCP1 granule assembly (Fig. S6A) in a DCP1 phosphorylation-independent manner (Fig. S6B). Moving forward, it would be interesting to clarify how components in the MEKK1-MKK2-MPK4 cascade affects DCP1 granule assembly. In fact, it would be more important to compare how MKK4/5-MPK3/6 and MEKK1-MKK2-MPK4 cascades affect plant PB/SG assembly in general. In an earlier stage of this study, we speculated that DCP1 granule assembly might be affected by differential protein accumulation of co-expressed MAPK signaling components. However, a series of protein immunoblot analyses revealed that although the level of protein accumulation of MPKs and MKKs varied, the co-expressed DCP1 was accumulated in a remarkable consistent level across different samples (Fig. 5C, 7, and S5). These results suggest that the size and number of DCP1 granules are modulated by MAPK signaling post-translationally. To potentiate the idea that MAPK signaling can modulate DCP1 granule assembly, the kinase activity of MKK5 was examined, as MKK5 exerted a strong effect on DCP1 granule assembly. Results showed that kinase activity was required for MKK5 to reduce the number of DCP1 granules (Fig. 6A and B). It was also found that MKK5 kinase activity could trigger the assembly of unusually large DCP1 granules, which was validated to be SGs by co-localization with UBP1b (Fig. 9B). On the basis of these findings, we hypothesized that MAPK signaling induced DCP1 phosphorylation could reduce the sequestration of DCP1 into PBs but enhance it into SGs. To test this hypothesis directly, we used phospho-dead form of DCP1 S237A and phospho-mimetic form of DCP1 S237D . The results indicated that neither DCP1 S237A nor DCP1 S237D were affected by the kinase activity of MKK5 (Fig. 6C-F). Furthermore, compared to the DCP1 WT , there was a significant increase of small DCP1 S237A granules (PBs) (Fig. 6C and D) and decrease of small but increase of large DCP1 S237D granules (SGs) (Fig. 6E and F). More importantly, the level of protein accumulation across DCP1 WT , DCP1 S237A , and DCP1 S237D co-expressed with MKKs of different phosphorylation capacity showed a remarkable consistent level (Fig. 7), again supporting the notion that the dynamics of DCP1 granule assembly is orchestrated by MAPK signaling mediated post-translational modification. On the other hand, we showed previously that flag22-induced MPK3/6 phosphorylation of DCP1 is required for the positive effects of DCP1-DCP2 complex on plant microbe associated molecular patterns (MAMPs)-triggered responses and immunity against pathogenic bacteria (25). Given the new findings presented here, it would be informative to determine if phosphorylated DCP1 localization in SGs is a pre-requisite for plant immunity. In a separate study, we reported recently that Arabidopsis TZF1 recruits MPK3 and MPK6 to SGs and TZF1 is phosphorylated by MPK3/6. Interestingly, TZF1 is differentially phosphorylated and de-phosphorylated on various residues by flg22-activated MAPK signaling cascade. Mutations of different TZF1 phosphorylation sites could either enhance or reduce TZF1 granule assembly. Remarkably, some of the phosphorylation mutations could similarly trigger the assembly of unusually large TZF1 granules (20). As TZF1 is mainly localized in SGs and only partially co-localizes with PB components such as DCP1 and DCP2 (37). As in the case of DDX6 (Rck/p54) in mammals (11), it would be interesting in the future to further delineate how reversible phosphorylation of different residues plays a distinctive role in shuttling TZF1 between PBs and SGs or simply enhance or reduce the size of TZF1 SGs. In contrast to the effects of MAPK signaling on the sequestration of DCP1 from PBs to SGs, here we have found that the SG marker UBP1b appeared to play a positive role in maintaining DCP1 in PBs (Fig. 11). UBP1b is an RNA-binding protein and a plant SG marker. UBP1b is a homolog of mammalian TIA-1 and TIAR that can promote the sequestration of untranslated mRNAs into SGs (38). The UBP1b SGs are induced by heat in the UBP1b overexpression (OX) plants that are heat stress tolerant (28). It was proposed that UBP1b sequesters mRNAs encoding DnaJ heat shock protein and other stress-related proteins into SGs to achieve heat-tolerance. UBP1b OX plants are also ABA hypersensitive. Curiously, MPK3 , MKK4 , and MKK9 were up-regulated, but their half-lives were unaltered in UBP1b OX plants, indicating that these mRNAs were not the direct targets of UBP1b (27). The subcellular localization of MAPK signaling components and their relationship with UBP1b remains unclear. In this report, we have found that UBP1b could moderately reduce the protein accumulation of MPK6 and MKK4 (Fig. 10B). Given that MAPK signaling negatively regulates DCP1 sequestration into PBs, we hypothesized that UBP1b could counteract this effect by suppressing MAPK signaling. Our hypothesis was supported by the results in which DCP1 was highly sequestered into PBs independent of its phosphorylation status when UBP1b was co-expressed (Fig. 11). Interestingly, the enhancement of DCP1 PB assembly was not due to elevated DCP1 protein accumulation, suggesting that the dynamics of DCP1 shuttling between PBs and SGs are controlled by MAPK signaling mediated post-translational modifications (Fig. 12). Conclusion In summary, although the interaction between PBs and SGs and molecules exchange between the two have been well-documented in mammals (1), the molecular details of these processes are unknown in plants. Our findings have revealed a molecular mechanism mediating PB-SG dynamics in plants. We have shown recently that TZF1 with two intrinsically disordered domains is able to recruit MAPK signaling components to SGs (20). Here, we have found that TZF1-MPK3/6-MKK4/5 forms a protein-protein interacting network (Fig. S1). DCP1, a core component of plant PBs, is phosphorylated by MPK3/6 and the phosphorylation triggers a rapid reduction of DCP1 partition into PBs (25). We found that this reduction is concomitantly associated with the increase of DCP1 partition into SGs, hence establishing a role for MAPK signaling in mediating PB-SG dynamics in plants. Furthermore, we have found that plant SG marker protein UBP1b plays a role in maintaining DCP1 in PBs by suppressing the accumulation of MAPK signaling components. Together, we propose that MAPK signaling and UBP1b control the balance of DCP1 partition into PBs and SGs in plants. Materials and Methods Plant materials Wild-type Arabidopsis ( Arabidopsis thaliana ) ecotype Columbia-0 (Col-0) was used in this study. Seeds were obtained from the Arabidopsis Biological Resource Center (ABRC). Protoplast transient expression analysis Arabidopsis protoplasts transient expression analyses were conducted mainly as described (39), with additional modifications as described (20). BiFC analysis The CDS of MKK4, MKK5, MPK3, and MPK6 were cloned into pA7-YN (containing N-terminal half of YFP) and pA7-YC (containing C-terminal half of YFP) vector (40), respectively. Each pair of BiFC construct and an additional cellular localization marker were co-transformed into Arabidopsis protoplasts. Co-IP assay Total proteins from Arabidopsis protoplasts co-expressing plasmid pairs were lysed with lysis buffer (100 mM Tris pH 8.0, 150 mM NaCl, 5 mM EDTA, 10 mM DTT, 0.1% NP-40, proteases inhibitor cocktail). Extracted proteins were then incubated with equilibrated GFP-trap beads (Chromotek) at 4°C for 2 hr under gentle agitation, followed by 3 times of washing with wash buffer (100 mM Tris pH 8.0, 150 mM NaCl, 5 mM EDTA, 10 mM DTT, 0.1% NP-40, proteases inhibitor cocktail). Immunoblots were performed using α-GFP, α-FLAG or α-myc antibodies. Accession numbers The accession numbers used are as follows: TZF1 (At2g25900), DCP1 (At1g08370), UBP1b (At1g17370), MEKK1 (At4g08500), MKK4 (At1g51660), MKK5 (At3g21220), MPK1 (At1g10210), MPK3 (At3g45640), MPK4 (At4g01370), MPK6 (At2g43790), MPK7 (At2g18170), and MPK11 (At1g01560). Declarations Acknowledgements We would like to thank undergraduate researchers Hannah Furness, Gianni Giarrano, Alex Tarter, and Yu Wang for assistance in various experiments. Author contributions Siou-Luan He and Jyan-Chyun Jang conceived and designed the experiments; Siou-Luan He performed most of the experiments; Siou-Luan He and Jyan-Chyun Jang wrote the manuscript; Ping He, Libo Shan, and Ying Wang provided comments, tools, and reagents for the project. Funding This work was supported by the grants from National Science Foundation MCB-1906060 to Jyan-Chyun Jang, Ping He, Libo Shan, and Ying Wang; Ohio State University President’s Research Excellence Accelerator award, Ohio Agricultural Research and Development Center SEEDS Program #2018007, Ohio State University College of Food, Agricultural, and Environmental Sciences Internal Grant Program #2022014, and Ohio State University Center for Applied Plant Sciences Research Enhancement Grant to Jyan-Chyun Jang. Data availability All data supporting the findings of this study are provided in the paper and its supplementary information file. Ethics approval and consent to participate The authors confirm that all experimental protocols were conducted in accordance with the relevant guidelines and regulations of The Ohio State University. The use of plants in this study complied with the university’s research ethics guidelines and required no further approval. Consent for publication Not applicable. Disclosure and competing interests statement The authors declare no competing interests. References Riggs, C.L., Kedersha, N., Ivanov, P. and Anderson, P. (2020) Mammalian stress granules and P bodies at a glance. J Cell Sci , 133 . Ripin, N. and Parker, R. (2023) Formation, function, and pathology of RNP granules. Cell , 186 , 4737-4756. Buchan, J.R. and Parker, R. (2009) Eukaryotic stress granules: the ins and outs of translation. Mol Cell , 36 , 932-941. Stoecklin, G. and Kedersha, N. (2013) Relationship of GW/P-bodies with stress granules. Adv Exp Med Biol , 768 , 197-211. Franks, T.M. and Lykke-Andersen, J. 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(2024) Modulation of stress granule dynamics by phosphorylation and ubiquitination in plants. iScience , 27 , 111162. He, S.L., Li, B., Zahurancik, W.J., Arthur, H.C., Sidharthan, V., Gopalan, V., Wang, L. and Jang, J.C. (2024) Overexpression of stress granule protein TZF1 enhances salt stress tolerance by targeting ACA11 mRNA for degradation in Arabidopsis. Front Plant Sci , 15 , 1375478. Liu, D., Riggi, M., Lee, H.O., Currie, S.L., Goodsell, D.S., Iwasa, J.H. and Rog, O. (2023) Depicting a cellular space occupied by condensates. Mol Biol Cell , 34 , tp2. Ren, D., Yang, H. and Zhang, S. (2002) Cell death mediated by MAPK is associated with hydrogen peroxide production in Arabidopsis. J Biol Chem , 277 , 559-565. Zhao, C., Wang, P., Si, T., Hsu, C.C., Wang, L., Zayed, O., Yu, Z., Zhu, Y., Dong, J., Tao, W.A. et al. (2017) MAP Kinase Cascades Regulate the Cold Response by Modulating ICE1 Protein Stability. Dev Cell , 43 , 618-629 e615. Yu, X., Li, B., Jang, G.J., Jiang, S., Jiang, D., Jang, J.C., Wu, S.H., Shan, L. and He, P. (2019) Orchestration of Processing Body Dynamics and mRNA Decay in Arabidopsis Immunity. Cell Rep , 28 , 2194-2205 e2196. Yan, Y., Gan, J., Tao, Y., Okita, T.W. and Tian, L. (2022) RNA-Binding Proteins: The Key Modulator in Stress Granule Formation and Abiotic Stress Response. Front Plant Sci , 13 , 882596. Nguyen, C.C., Nakaminami, K., Matsui, A., Watanabe, S., Kanno, Y., Seo, M. and Seki, M. (2017) Overexpression of oligouridylate binding protein 1b results in ABA hypersensitivity. Plant Signal Behav , 12 , e1282591. Nguyen, C.C., Nakaminami, K., Matsui, A., Kobayashi, S., Kurihara, Y., Toyooka, K., Tanaka, M. and Seki, M. (2016) Oligouridylate Binding Protein 1b Plays an Integral Role in Plant Heat Stress Tolerance. Front Plant Sci , 7 , 853. Youn, J.Y., Dunham, W.H., Hong, S.J., Knight, J.D.R., Bashkurov, M., Chen, G.I., Bagci, H., Rathod, B., MacLeod, G., Eng, S.W.M. et al. (2018) High-Density Proximity Mapping Reveals the Subcellular Organization of mRNA-Associated Granules and Bodies. Mol Cell , 69 , 517-532 e511. Kershaw, C.J., Nelson, M.G., Lui, J., Bates, C.P., Jennings, M.D., Hubbard, S.J., Ashe, M.P. and Grant, C.M. (2021) Integrated multi-omics reveals common properties underlying stress granule and P-body formation. RNA Biol , 18 , 655-673. Kedersha, N., Panas, M.D., Achorn, C.A., Lyons, S., Tisdale, S., Hickman, T., Thomas, M., Lieberman, J., McInerney, G.M., Ivanov, P. et al. (2016) G3BP-Caprin1-USP10 complexes mediate stress granule condensation and associate with 40S subunits. J Cell Biol , 212 , 845-860. Krapp, S., Greiner, E., Amin, B., Sonnewald, U. and Krenz, B. (2017) The stress granule component G3BP is a novel interaction partner for the nuclear shuttle proteins of the nanovirus pea necrotic yellow dwarf virus and geminivirus abutilon mosaic virus. Virus Res , 227 , 6-14. Zhao, J., Zhou, H., Zhang, M., Gao, Y., Li, L., Gao, Y., Li, M., Yang, Y., Guo, Y. and Li, X. (2016) Ubiquitin-specific protease 24 negatively regulates abscisic acid signalling in Arabidopsis thaliana. Plant Cell Environ , 39 , 427-440. Majerciak, V., Zhou, T., Kruhlak, M.J. and Zheng, Z.M. (2023) RNA helicase DDX6 and scaffold protein GW182 in P-bodies promote biogenesis of stress granules. Nucleic Acids Res , 51 , 9337-9355. Kedersha, N., Ivanov, P. and Anderson, P. (2013) Stress granules and cell signaling: more than just a passing phase? Trends Biochem Sci , 38 , 494-506. Kanda, Y., Satoh, R., Takasaki, T., Tomimoto, N., Tsuchiya, K., Tsai, C.A., Tanaka, T., Kyomoto, S., Hamada, K., Fujiwara, T. et al. (2021) Sequestration of the PKC ortholog Pck2 in stress granules as a feedback mechanism of MAPK signaling in fission yeast. J Cell Sci , 134 . Pomeranz, M.C., Hah, C., Lin, P.C., Kang, S.G., Finer, J.J., Blackshear, P.J. and Jang, J.C. (2010) The Arabidopsis tandem zinc finger protein AtTZF1 traffics between the nucleus and cytoplasmic foci and binds both DNA and RNA. Plant Physiol , 152 , 151-165. Kedersha, N.L., Gupta, M., Li, W., Miller, I. and Anderson, P. (1999) RNA-binding proteins TIA-1 and TIAR link the phosphorylation of eIF-2 alpha to the assembly of mammalian stress granules. J Cell Biol , 147 , 1431-1442. Yoo, S.D., Cho, Y.H. and Sheen, J. (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc , 2 , 1565-1572. Chen, S., Tao, L., Zeng, L., Vega-Sanchez, M.E., Umemura, K. and Wang, G.-L. (2006) A highly efficient transient protoplast system for analyzing defense gene expression and protein-protein interactions in rice. Mol Plant Path , 7 , 417-427. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFiguresMPKDCP1PBSGSLH.pptx Supplementaryfileuncroppedimages.pptx Cite Share Download PDF Status: Published Journal Publication published 07 Feb, 2026 Read the published version in Discover Life → Version 1 posted Editorial decision: Revision requested 18 Nov, 2025 Reviews received at journal 24 Oct, 2025 Reviewers agreed at journal 20 Oct, 2025 Reviewers agreed at journal 15 Oct, 2025 Reviews received at journal 30 Sep, 2025 Reviewers agreed at journal 22 Sep, 2025 Reviewers agreed at journal 21 Sep, 2025 Reviewers invited by journal 02 Sep, 2025 Editor assigned by journal 22 Aug, 2025 Submission checks completed at journal 21 Aug, 2025 First submitted to journal 21 Aug, 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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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7292794","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":509007847,"identity":"ebea08bb-3ba7-4473-8a8f-aec8ffbf43fc","order_by":0,"name":"Siou-Luan He","email":"","orcid":"","institution":"The Ohio State University","correspondingAuthor":false,"prefix":"","firstName":"Siou-Luan","middleName":"","lastName":"He","suffix":""},{"id":509007848,"identity":"b3f78856-e788-42cb-9b28-020f9d7b7280","order_by":1,"name":"Ying Wang","email":"","orcid":"","institution":"University of 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16:50:56","extension":"pptx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":28054592,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFiguresMPKDCP1PBSGSLH.pptx","url":"https://assets-eu.researchsquare.com/files/rs-7292794/v1/4773d22d721b6825dc764ac2.pptx"},{"id":90933858,"identity":"8035012f-b547-4fa3-bfab-beedf45e306c","added_by":"auto","created_at":"2025-09-09 16:50:57","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":80279768,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfileuncroppedimages.pptx","url":"https://assets-eu.researchsquare.com/files/rs-7292794/v1/692fe272c3df6cb9db14370c.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"MAPK signaling modulates the partition of DCP1 between processing bodies and stress granules in plant cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eProcessing bodies (PBs) and stress granules (SGs) are two types of cytoplasmic biomolecular condensates that dynamically assembled in response to environmental stresses. Dysregulation of PBs and SGs has been implicated in various human diseases such as neuro- and muscular-degenerative diseases, neuro-developmental diseases, and cancers (1,2). Although previous studies have suggested that PBs and SGs perform distinct functions, as each contains a unique set of proteins, multiple observations indicate that SGs interact with PBs and likely exchange messenger ribonucleoproteins (mRNPs) between each other (3). Under specific stress conditions, PBs often dock with SGs, and the overexpression of certain proteins that localize to both structures can lead to the fusion of PBs and SGs (4). For example, the overexpression of tristetraprolin (TTP) or butyrate response factor-1 (BRF-1), two RNA-binding proteins that target ARE-containing mRNAs to PBs for degradation (5) or cytoplasmic polyadenylation element binding protein (CPEB1), another dual SG/PB protein (6), lead to the tight clustering of PBs around and within SGs (5-7). Overexpression of ubiquitin-associated protein 2-like (UBAP2L), and the interaction between UBAP2L and SG and PB nucleating protein such as stress granule assembly factor (G3BP) and DEAD-box helicase 6 (DDX6), respectively, induces hybrid granules containing SG and PB components in the cells (8). Furthermore, PBs can play a role in promoting SG assembly by providing untranslated mRNAs, shared proteins, and translational repressors that are necessary for nucleating SGs during cellular stress conditions (9). On the other hand, SGs could still form in the absence of PBs. DDX6 (Rck/p54) is an evolutionarily conserved member of the DEAD-box RNA helicase family involved in the inhibition of translation and storage and the degradation of cellular mRNAs in PBs. When DDX6 expression is reduced, PB formation is strongly impaired, whereas DDX6 knockdown causes the PB-specific protein DCP1 to relocate to SGs (10,11), suggesting that\u0026nbsp;some proteins involved in PB assembly may switch roles and contribute to co-aggregate with SG proteins under specific stress conditions.\u003c/p\u003e\n\u003cp\u003eIn plants, RNA-binding protein 47b (Rbp47b) and Tudor Staphylococcal Nuclease (TSN2) are two core components of plant SGs (12). Rbp47b and TSN2 interactome analysis in Arabidopsis revealed the presence of mitogen-activated protein kinase (MAPK) signaling components MPK3, MKK4, and MKK5 (13), suggesting that MAPK signaling could mediate SG dynamics, perhaps by phosphorylating key SG components. MAPK cascades (including MPK3 and MPK6 and their upstream kinases MKK4 and MKK5) play a crucial role in plant lifecycle, where they regulate a wide range of physiological processes, including growth and development as well as in responses to environmental cues such as cold, heat, drought, and especially pathogen attack (14). Some other kinases can also be recruited to SGs and PBs (15,16). The SG component Ras-GAP SH3-binding protein (G3BP1) recruits casein kinase 2 (CK2) to SGs and phosphorylation of G3BP1 by CK2 promotes dissociation of G3BP from SGs and triggers SG disassembly (17). The yeast kinase Sky1 is recruited to heat-induced SGs, where it can phosphorylate substrates Npl3 (a nucleocytoplasmic mRNA shuttling protein) to promote SG dissolution (18). SGs also play a role in sequestering\u0026nbsp;signaling molecules as a protective mechanism. For example, high-heat stress stimulates MAPK activation, which causes fission yeast protein kinase C (Pck2) translocation\u0026nbsp;from the plasma membrane into SGs to suppresses MAPK hyperactivation and cell death\u0026nbsp;(19).\u003c/p\u003e\n\u003cp\u003eIn plants, the molecular mechanisms underpinning the interaction and equilibrium between PBs and SGs are unclear. Here we present multiple lines of evidence to propose that a model in which MAPK signaling pathway is involved in triggering a major PB component DCP1 to relocate to SGs. We previously showed that tandem CCCH zinc finger 1 (TZF1) recruits MAPK signaling components to SGs (20). Using a high throughput and high-fidelity Arabidopsis protoplast transient expression system, we have found that MPK3, MPK6, MKK4, and MKK5 form homo- and hetero-dimers with each other in SGs. DCP1 is phosphorylated by MPK3 and MPK6 and the kinase activity of MKK5 is required for DCP1 to localize to SGs. In support of this notion, the phospho-dead form of DCP1\u003csup\u003eS237A\u003c/sup\u003e is mainly localized in PBs and phosphor-mimetic form of DCP1\u003csup\u003eS237D\u003c/sup\u003e is mainly localized in SGs. Oligouridylate binding protein 1b (UBP1b) is an RNA-binding protein and it has been used widely as an SG marker. Surprisingly, MAPK signaling-mediated reduction of DCP1 association with PBs is antagonized by the SG marker UBP1b, as UBP1b suppresses the accumulation of MPK3/6 and MKK4/5. Consistent with this idea, the abundance of DCP1 association with PBs is enhanced by the co-expression of UBP1b, as well as an additional mechanism independent of DCP1’s phosphorylation status. Together, our results indicate that MAPK phosphorylation could serve as a switch for DCP1 sequestration from PBs to SGs. This pathway is counteracted by an SG marker UBP1b that could diminish the level of MAPK signaling components.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eTZF1-MPK3/6-MKK4/5 interacting network\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe have shown previously that TZF1 could interact with MPK3/6 and MKK4/5 in SGs (20). Here, we found that MPK3 and MPK6 could interact with itself and each other in bimolecular fluorescence complementation (BiFC) analysis. MKK4 and MKK5 could also interact with itself and each other (Fig.\u0026nbsp;1A).\u0026nbsp;Furthermore, MPK3 or MPK6 could also interact with MKK4 or MKK5 in BiFC analysis (Fig.\u0026nbsp;1B). Remarkably, all the interacting BiFC signals were co-localized with an SG marker TZF1\u0026nbsp;(21)\u0026nbsp;in cytoplasmic granules, suggesting that MPK3/6 and MKK4/5 interact in SGs. To validate protein-protein interactions identified in BiFC analyses, co-immunoprecipitation (Co-IP) assays were conducted. Pair-wise protein components with various tags were co-expressed in an Arabidopsis protoplasts transient expression system and\u0026nbsp;IP\u0026nbsp;was carried out using GFP-antibody. Results indicated that MKK4 and MKK5 (Fig.\u0026nbsp;1C) as well as MPK3 and MPK6 (Fig.\u0026nbsp;1D) could self- and cross-interact. MKK4 or MKK5 could cross-interact with MPK3 or MPK6 as well (Fig.\u0026nbsp;1E). No\u0026nbsp;Co-IP signals were found when the MKK construct was co-expressed with the empty construct with free GFP (Fig.\u0026nbsp;1F), indicating the specificity of the\u0026nbsp;Co-IP results.\u0026nbsp;Interestingly, when the BiFC analysis was conducted in the presence of nuclear marker NLS-RFP, the interaction between MPK3 or MPK6 with MKK4 (not shown) or MKK5 was mainly in the nucleus (Fig.\u0026nbsp;2), as opposed to in the cytoplasmic granules when co-expressed with TZF1 (Fig.\u0026nbsp;1A and\u0026nbsp;B). However, BiFC signals of MKK4 and MKK5 self- and cross-interactions remained distinctively in cytoplasmic granules when co-expressed with NLS-RFP (Fig.\u0026nbsp;2). These results suggest that TZF1 recruits cross-interactions between MPK3/6 and MKK4/5 to SGs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo determine if protein-protein interactions were taken place in PBs or SGs, additional markers were co-expressed in the BiFC analyses. Results indicated that most of the self- and cross-interactions among MPK3/6 and cross interactions between MPK3/6 and MKK4/5 were not colocalized with a major PB component DCP1 (Fig. 3A), whereas almost completely co-localized with SG marker UBP1b (Fig. 3B). Note that BiFC signals of MPK3-MKK5 were occasionally overlapped with DCP1-mCherry signals (indicated by an arrow in Fig. 3A). Furthermore, UBP1b could be localized to both SGs and the nucleus. It appeared that most of the aforementioned interactions were co-localized with UBP1b in the nucleus (Fig. 3B), suggesting a dominant role of protein-protein interaction in affecting UBP1b subcellular localization. Intriguingly, the above-mentioned interactions appeared to be suppressed by the co-expression of UBP1b in some cells, as evidenced by their very weak or missing BiFC signals and strong UBP1b-mcherry signals in SGs (Fig. 3C). This raised a possibility that MPK3-MPK3 and MPK3-MPK6 interactions were more stable in the nucleus than in SGs (with strongly co-expressed UBP1b). For MKK4 and MKK5, the BiFC signals of both self- and cross-interactions were predominately localized in cytoplasmic granules and only partially (indicated by arrows) co-localized with DCP1, but almost completely co-localized with SG marker UBP1b (Fig. 4).\u003c/p\u003e\n\u003cp\u003eBased on the results of BiFC (Fig. 1A-B and 2-4), Co-IP (Fig. 1C-F), and previous report (20), we proposed a model in which TZF1, MPK3/6, and MKK4/5 formed an interactome in SGs (Fig. S1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDCP1 granule assembly affected by MPK3/6 and MKK4/5\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the course of conducting BiFC analyses, it was noted that the signal levels of PB component DCP1-mCherry varied, depending on a specific co-expressed pair of BiFC constructs. For example, the DCP1 granule number was abundant when co-expressed with MKK4-nYFP+MKK4-cYFP, but much lower when co-expressed with MKK4-nYFP+MKK5-cYFP, MKK5-nYFP+MKK4-cYFP, and MKK5-nYFP+MKK5-cYFP, respectively (Fig. S2A and B). Likewise, DCP1 granule number was abundant when co-expressed with MPK3-nYFP+MPK3-cYFP, but low when co expressed with MPK3-nYFP+MKK4-cYFP, MPK3-nYFP+MKK5-cYFP, and MPK3-nYFP+MPK6-cYFP, respectively (Fig. S3A and B). It was also noted that both the number and the size of DCP1 granules were varied among samples. To verify if differential signal levels of DCP1 granules were due to co-expressed individual proteins, additional co-expression analyses were conducted using full-length GFP fusion proteins. The number of DCP1 granules was slightly reduced by co-expression of MPK3, moderately by MKK4, but severely reduced by MPK6 and MKK5. By contrast, the size of DCP1 granules appeared to be strongly enhanced by MPK3 and slightly enhanced by MPK6, whereas reduced by MKK4 and MKK5, as compared to the sample co-expressed with free GFP (Fig. 5). Given MPK6 appeared to have a stronger effect than MPK3, additional family members of MPKs were tested using available BiFC constructs. Results showed that the BiFC constructs were as effective as the GFP-fusion constructs, because MPK6-cYFP could suppress DCP1 granule accumulation, as compared to GFP-MPK6. In additional to MPK6, MPK1, 4, and 11 had similar effects in reducing DCP1 granule accumulation (Fig. S4).\u003c/p\u003e\n\u003cp\u003eFrom the cellular images in (Fig. S4), it appeared that the protein expression levels of MPKs, MKKs, and DCP1 varied in different samples. As protein level might affect biomolecular condensate assembly (22), immunoblot analyses were conducted. To further delineate the effects of MPKs/MKKs on DCP1 accumulation, two dozes of plasmid DNA (20 and 40\u0026nbsp;mg) were used in the protoplast transient expression analysis. Consistent with cellular images in\u0026nbsp;(Fig.\u0026nbsp;S4), the levels of protein expression displayed large differences ranging from weak to strong in the order of MPK3, MPK6, MKK4, and MKK5. By contrast, DCP1 level varied just slightly between samples co-expressed with MPK3/6 and MKK4/5, with only negligible differences between the two samples transformed with different doses of DCP1-mCherry plasmid DNA (Fig.\u0026nbsp;5C). Similar analysis was carried out for various MPK-cYFP and DCP1-mCherry samples as shown in\u0026nbsp;(Fig.\u0026nbsp;S5). Results showed whereas the levels of MPKs varied, with MPK11-cYFP at the lowest abundance, the level of DCP1-mCherry remained at similar levels across different samples. Of note, MPK3-cYFP and MPK6-cYFP appeared to be much more stable than their counterparts fused with GFP. However, the co-expressed DCP1-mC was accumulated at a remarkably consistent level across all samples. Together, these results suggest that post-translational regulation plays a major role on DCP1 granule dynamics, although DCP1 protein accumulation might also play a secondary role.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhosphorylation activity of MKK5 modulates the partition of DCP1 between PBs and SGs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe showed previously that plant SG assembly is affected by the phosphorylation status of a core SG protein TZF1 (20). Co-expression analysis was then carried out to determine if the phosphorylation status of MKK5 could affect DCP1 granule assembly. Results showed that co-expression of MKK5\u003csup\u003eWT\u003c/sup\u003e reduced the number of DCP1 granules to about 50%, whereas the MKK5\u003csup\u003eDD\u003c/sup\u003e, a constitutively active form of MKK5\u003csup\u003eT215D/S221D\u003c/sup\u003e (23,24), exerted even stronger suppression. By contrast, the MKK5\u003csup\u003eKR\u003c/sup\u003e, a loss-of-function mutation of the conserved K to R change in the kinase ATP-binding loop of MKK5\u003csup\u003eK99R\u003c/sup\u003e (23), enhanced the accumulation of DCP1 granules (Fig. 6A and B). Noticeably, the decrease of DCP1 granules was associated with an increased number of cells containing a dominant large DCP1 granule. We showed previously that flg22-induced MAPK signaling cascade could trigger DCP1 phosphorylation and rapid disassembly of DCP1 granules, whereas the phospho-dead DCP1\u003csup\u003eS237A\u003c/sup\u003e was resistant to the effect of flg22 (25). Here, by expressing DCP1 phosphorylation mutant constructs alone, it was found that the accumulation of granules from DCP1\u003csup\u003eS237A\u003c/sup\u003e was far greater, whereas DCP1\u003csup\u003eS237D\u003c/sup\u003e (phospho-mimetic) was far less than that from DCP1\u003csup\u003eWT\u003c/sup\u003e (Fig. 6). When the individual DCP1 constructs were co-expressed with phosphorylation mutants of MKK5, the DCP1\u003csup\u003eS237A\u003c/sup\u003e granules remained highly abundant across each combination (Fig. 6C and D), whereas the accumulation of DCP1\u003csup\u003eS237D\u003c/sup\u003e granules was low and dominated by the single large granule pattern across each combination of DCP1+MKK5 (Fig. 6E and F). These results suggest that MKK5 acts upstream of DCP1 and phosphorylation status of DCP1 is a determinant for the accumulation/assembly and form/size of DCP1 granules.\u003c/p\u003e\n\u003cp\u003eTo determine if MKK5-mediated DCP1 granule dynamics was related to protein accumulation, immunoblot analysis was conducted. Interestingly, compared to the MKK5\u003csup\u003eWT\u003c/sup\u003e, MKK5\u003csup\u003eDD\u003c/sup\u003e was accumulated at a higher level, whereas MKK5\u003csup\u003eKR\u003c/sup\u003e was accumulated at a lower level, across various samples. By contrast, the accumulation of DCP1\u003csup\u003eWT\u003c/sup\u003e, DCP1\u003csup\u003eS237A\u003c/sup\u003e, and DCP1\u003csup\u003eS237D\u003c/sup\u003e showed a remarkably consistent level across various samples (Fig. 7). These results indicate that DCP1 granule dynamics is likely orchestrated by post-translational regulation but not protein accumulation. As DCP2 granule dynamics was similarly regulated by MKK5 kinase activity, same assay was also conducted (results not shown). Similar results were obtained as those from DCP1 (Fig. 7, right panel).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhosphorylation triggers the sequestration of DCP1 from PBs to SGs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBecause a high number of cells contained coalesced large DCP1\u003csup\u003eS237D\u003c/sup\u003e granules in (Fig. 6E), we sought to determine the identity of the large DCP1 granules. Intriguingly, albeit differing in number, both large and small granules were present in all three types of DCP1, independent of its phosphorylation status (Fig. 6). We first examined the relationship between small granules and various cellular markers. Results showed that small granules of all three types of DCP1 were independent of nuclear marker NLS-RFP (Fig. 8A). The small granules of DCP1\u003csup\u003eWT\u003c/sup\u003e and DCP1\u003csup\u003eS237A\u003c/sup\u003e were not co-localized with the SG marker UBP1b either, supporting their identity as PBs. By contrast, the DCP1\u003csup\u003eS237D\u003c/sup\u003e granules were generally larger and they completely co-localized with UBP1b, suggesting that phosphorylation of DCP1 is a trigger to sequester DCP1 from PBs to SGs (Fig. 8B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eParadoxically, the large ‘nucleus-like’ DCP1 granules were not co-localized with the nuclear marker NLS-RFP (Fig. 9A), but instead partially or completely co-localized with the SG marker UBP1b (Fig. 9B). These results suggest that phosphorylated DCP1 is mainly sequestered to SGs, but the phosphorylation is not the only prerequisite for SG localization, as small percentage of cells with large granules were also found in DCP1\u003csup\u003eS237A\u003c/sup\u003e samples and they could also co-localize with UBP1b. We speculate that there might be redundant post-translational modification events that could trigger the sequestration of DCP1\u003csup\u003eS237A\u003c/sup\u003e to SGs. Together, these results suggest that protein phosphorylation and additional post-translational modification mechanisms likely play a role in the sequestration of DCP1 between PBs and SGs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUBP1b suppresses MAPK signaling to maintain DCP1 in PBs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn contrast to the relationship between DCP1 and MPK3/6 and MKK4/5, co-expression of UBP1b appeared to suppress the BiFC signals generated from interactions within and between MPK3/6 (Fig. 3C). To decipher if UBP1b suppressed the signals of protein-protein interactions or suppressed the individual protein accumulation, co-expression analyses were conducted using single gene-GFP fusion constructs. Results showed that the signals from MPK3, MPK6, and MKK4 were dampened by the co-expression of UBP1b (Fig. 10A).\u0026nbsp;Consistent with the cellular imaging results, immunoblot analysis revealed that co-expression of two different doses of UBP1b resulted in a reduction of the accumulation of MPK6 and MKK4, with a lesser extent on MKK5. MPK3 level was too low to be determined (Fig. 10B). Given UBP1b appeared to cause a general reduction of MPKs/MKKs protein accumulation, the variation of MPKs/MKKs granule intensity/dynamics could have been contributed by the protein abundance, although it was not as obvious for MPK6 and MKK4 from cellular images (Fig. 10A). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGiven DCP1 granule assembly could be modulated by MAPK signaling components whose accumulation appeared to be affected by UBP1b, the relationship between DCP1 phosphorylation status and UBP1b was investigated. Compared to the DCP1\u003csup\u003eWT\u003c/sup\u003e, DCP1\u003csup\u003eS237A\u003c/sup\u003e had increased and DCP1\u003csup\u003eS237D\u003c/sup\u003e had decreased number of granules (Fig. 11A). When UBP1b was co-expressed, DCP1 granule abundance was enhanced and there was no significant difference between the three types of DCP1 (Fig. 11B and C). Because none of the DCP1-mCherry granules were co-localized with the SG marker UBP1b-GFP, the small and distinct DCP1-mCherry granules were likely PBs (Fig. 11B). Immunoblot analysis was conducted to determine if increased DCP1 granule abundance was due to higher level of protein accumulation. Compared to the samples co-expressed with free GFP, a general reduction of the accumulation of all three types of DCP1, independent of their phosphorylation status, was found when co-expressed with UBP1b-GFP. More importantly, the three DCP1 proteins accumulated at nearly the same level, ruling out the possibility of increased DCP1 granule abundance due to elevated protein accumulation (Fig. 11D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTogether, these results suggest that UBP1b suppresses MAPK signaling components that act as negative regulators of DCP1 large granule assembly.\u0026nbsp;We propose a model in which flg22 activates MAPK signaling mediated post-translational modification of DCP1 that results in a decrease of DCP1 localization to PBs, whereas an increase of DCP1 sequestration to SGs. UBP1b, on the other hand, acts as a negative regulator of certain MPKs and MKKs, hence counteracting with the MAPK signaling effects and resulting in the maintenance of DCP1 to be associated with PBs (Fig.\u0026nbsp;12). Because UBP1b-mediated increase of DCP1 sequestration to PBs appeared to override the phosphorylation status of DCP1, it is likely that an additional unknown mechanism exerted by UBP1b is also operating in this process (Fig.\u0026nbsp;12). We also speculate that UBP1b, a key modulator of SG assembly, and MAPK signaling play a role in the homeostasis control of PBs and SGs in response to stresses other than flg22, such as heat, salt, and ABA\u0026nbsp;(26-28).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePBs and SGs share similarities in composition and function in various cellular processes. Although distinct core components of PB and SG have been redefined by using sophisticated proximity mapping (29), recent reports have also found an extensive overlap of composition across PBs and SGs, such as poorly translated mRNAs and low complexity RNA-binding proteins (30). In mammals, double knockout of RNA-binding protein G3BP1 and G3BP2 prevents SG assembly induced by eukaryotic initiation factor 2a\u0026nbsp;phosphorylation. In this double mutant background, phosphor-mimetic mutant G3BP\u003csup\u003eS149E\u003c/sup\u003e failed to rescue SG assembly, highlighting the importance of the role of PTM on G3BP. In another scenario, Caprin and USP10 bind G3BP in a mutually exclusive way, whereas G3BP-Caprin complex promotes SG assembly, G3BP-USP10 complex promotes SG disassembly\u0026nbsp;(31,32). The plant ubiquitin-specific protease family members are homologs of USP10, including UBP24, a negative regulator of ABA signaling\u0026nbsp;(33). It is not known if UBP24 is involved in PB-SG interaction. Confusing in nomenclature, although UBP1b is not an UBP family member, it is a key modulator of SG assembly in plants\u0026nbsp;(26). On the other hand, the mammalian DDX6 (Rck/p54), a major PB scaffold component, plays a key role in PB-SG interaction. DDX6 limits itself and other RNPs to be assembled into SGs. In the absence of DDX6, more RNPs are partitioned into SGs. Loss PB scaffold proteins such as DCP1 and DDX6 also causes reduction in PB growth and enhances incompletely assembled PBs docking with SGs to form hybrid granules with irregular shapes\u0026nbsp;(11,34). SG assembly provides a means for the temporal and spatial compartmentalization of signaling components critical for cell growth and defense response\u0026nbsp;(35). In fission yeast, while it is not demonstrated that MAPK signaling components are sequestered to SGs, the high heat stress induced MAPK activation triggers the sequestration of upstream regulator PKC into SGs thereby deactivating MAPK hyperactivation induced cell death\u0026nbsp;(19,36). While some limited information is available from studies using non-plant models, the molecular mechanisms mediating PB-SG dynamics in plants are virtually unknown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMPK-MKK-TZF1 interactome in SGs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this report, we have found that MAPK signaling components, including MPK3, MPK6, MKK4, and MKK5, can self- and cross-interact with each other (Fig. 1-4). These protein-protein interactions take place mainly in SGs, with a slight chance in PBs. MPK3 and MPK6 self- and cross-interactions, as well as cross-interactions between MPK3/6 and MKK4/5 often occur in the nucleus where the SG marker UBP1b can also be localized when co-expressed with the MAPK BiFC constructs (Fig. 3). In contrast, MKK4 and MKK5 self- and cross-interactions are primarily taken place in cytoplasmic granules with complete co-localization with SG marker UBP1b and limited co-localization with the core PB component DCP1 (Fig. 4). More importantly, all interactions are co-localized with TZF1 cytoplasmic granules, including MPK3 and MPK6 self- and cross-interactions (Fig. 1). As we have demonstrated in an earlier report that TZF1 interacts with MPK3, MPK6, MKK4, and MKK5 \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e (20), we propose that TZF1 forms an interacting network with MAPK components in SGs (Fig. S1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMAPK signaling modulates DCP1 granule assembly\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the process of conducting BiFC analyses, it was noted that the proteins generated from BiFC constructs had significant interactions with co-expressed PB (DCP1) or SG (UBP1b) marker protein. For example, DCP1-mCherry granules were suppressed by all combinations except MKK4-nYFP+MKK4-cYFP (Fig. S2A and B) and MPK3-nYFP+MPK3-cYFP (Fig. S3A and B). Further analysis indicated that DCP1-mCherry granules were differentially affected by MAPK signaling components: (1) DCP1 granule size was enhanced by the co-expression of MPK3 and MPK6; (2) DCP1 granule number was progressively reduced by co-expression of MAPK components in the order of MPK6, MKK5, MKK4, and MPK3 (Fig. 5B). Additional analysis revealed that MPK1, MPK4, and MPK11 could also reduce the number of DCP1-mCherry granules (Fig. S4). This is potentially important because the two MKK4/5-MPK3/6 and MEKK1-MKK2-MPK4 MAPK signaling cascades play opposing roles in plant cold response (24). Our preliminary results indicated that MEKK1 could enhance DCP1 granule assembly (Fig. S6A) in a DCP1 phosphorylation-independent manner (Fig. S6B). Moving forward, it would be interesting to clarify how components in the MEKK1-MKK2-MPK4 cascade affects DCP1 granule assembly. In fact, it would be more important to compare how MKK4/5-MPK3/6 and MEKK1-MKK2-MPK4 cascades affect plant PB/SG assembly in general. In an earlier stage of this study, we speculated that DCP1 granule assembly might be affected by differential protein accumulation of co-expressed MAPK signaling components. However, a series of protein immunoblot analyses revealed that although the level of protein accumulation of MPKs and MKKs varied, the co-expressed DCP1 was accumulated in a remarkable consistent level across different samples (Fig. 5C, 7, and S5). These results suggest that the size and number of DCP1 granules are modulated by MAPK signaling post-translationally.\u003c/p\u003e\n\u003cp\u003eTo potentiate the idea that MAPK signaling can modulate DCP1 granule assembly, the kinase activity of MKK5 was examined, as MKK5 exerted a strong effect on DCP1 granule assembly. Results showed that kinase activity was required for MKK5 to reduce the number of DCP1 granules (Fig. 6A and B). It was also found that MKK5 kinase activity could trigger the assembly of unusually large DCP1 granules, which was validated to be SGs by co-localization with UBP1b (Fig. 9B). On the basis of these findings, we hypothesized that MAPK signaling induced DCP1 phosphorylation could reduce the sequestration of DCP1 into PBs but enhance it into SGs. To test this hypothesis directly, we used phospho-dead form of DCP1\u003csup\u003eS237A\u003c/sup\u003e and phospho-mimetic form of DCP1\u003csup\u003eS237D\u003c/sup\u003e. The results indicated that neither DCP1\u003csup\u003eS237A\u003c/sup\u003e nor DCP1\u003csup\u003eS237D\u003c/sup\u003e were affected by the kinase activity of MKK5 (Fig. 6C-F). Furthermore, compared to the DCP1\u003csup\u003eWT\u003c/sup\u003e, there was a significant increase of small DCP1\u003csup\u003eS237A\u003c/sup\u003e granules (PBs) (Fig. 6C and D) and decrease of small but increase of large DCP1\u003csup\u003eS237D\u003c/sup\u003e granules (SGs) (Fig. 6E and F). More importantly, the level of protein accumulation across DCP1\u003csup\u003eWT\u003c/sup\u003e, DCP1\u003csup\u003eS237A\u003c/sup\u003e, and DCP1\u003csup\u003eS237D\u003c/sup\u003e co-expressed with MKKs of different phosphorylation capacity showed a remarkable consistent level (Fig. 7), again supporting the notion that the dynamics of DCP1 granule assembly is orchestrated by MAPK signaling mediated post-translational modification. On the other hand, we showed previously that flag22-induced MPK3/6 phosphorylation of DCP1 is required for the positive effects of DCP1-DCP2 complex on plant microbe associated molecular patterns (MAMPs)-triggered responses and immunity against pathogenic bacteria (25). Given the new findings presented here, it would be informative to determine if phosphorylated DCP1 localization in SGs is a pre-requisite for plant immunity.\u003c/p\u003e\n\u003cp\u003eIn a separate study, we reported recently that Arabidopsis TZF1 recruits MPK3 and MPK6 to SGs and TZF1 is phosphorylated by MPK3/6. Interestingly, TZF1 is differentially phosphorylated and de-phosphorylated on various residues by flg22-activated MAPK signaling cascade. Mutations of different TZF1 phosphorylation sites could either enhance or reduce TZF1 granule assembly. Remarkably, some of the phosphorylation mutations could similarly trigger the assembly of unusually large TZF1 granules (20). As TZF1 is mainly localized in SGs and only partially co-localizes with PB components such as DCP1 and DCP2 (37). As in the case of DDX6 (Rck/p54) in mammals (11), it would be interesting in the future to further delineate how reversible phosphorylation of different residues plays a distinctive role in shuttling TZF1 between PBs and SGs or simply enhance or reduce the size of TZF1 SGs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn contrast to the effects of MAPK signaling on the sequestration of DCP1 from PBs to SGs, here we have found that the SG marker UBP1b appeared to play a positive role in maintaining DCP1 in PBs (Fig. 11). UBP1b is an RNA-binding protein and a plant SG marker. UBP1b is a homolog of mammalian TIA-1 and TIAR that can promote the sequestration of untranslated mRNAs into SGs (38). The UBP1b SGs are induced by heat in the\u003cem\u003e\u0026nbsp;UBP1b\u003c/em\u003e overexpression (OX) plants that are heat stress tolerant (28). It was proposed that UBP1b sequesters mRNAs encoding DnaJ heat shock protein and other stress-related proteins into SGs to achieve heat-tolerance. \u003cem\u003eUBP1b OX\u003c/em\u003e plants are also ABA hypersensitive. Curiously, \u003cem\u003eMPK3\u003c/em\u003e, \u003cem\u003eMKK4\u003c/em\u003e, and \u003cem\u003eMKK9\u003c/em\u003e were up-regulated, but their half-lives were unaltered in \u003cem\u003eUBP1b OX\u003c/em\u003e plants, indicating that these mRNAs were not the direct targets of UBP1b (27). The subcellular localization of MAPK signaling components and their relationship with UBP1b remains unclear. In this report, we have found that UBP1b could moderately reduce the protein accumulation of MPK6 and MKK4 (Fig. 10B). Given that MAPK signaling negatively regulates DCP1 sequestration into PBs, we hypothesized that UBP1b could counteract this effect by suppressing MAPK signaling. Our hypothesis was supported by the results in which DCP1 was highly sequestered into PBs independent of its phosphorylation status when UBP1b was co-expressed (Fig. 11). Interestingly, the enhancement of DCP1 PB assembly was not due to elevated DCP1 protein accumulation, suggesting that the dynamics of DCP1 shuttling between PBs and SGs\u0026nbsp;are controlled by MAPK signaling mediated post-translational modifications (Fig. 12).\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, although the interaction between PBs and SGs and molecules exchange between the two have been well-documented in mammals (1), the molecular details of these processes are unknown in plants. Our findings have revealed a molecular mechanism mediating PB-SG dynamics in plants. We have shown recently that TZF1 with two intrinsically disordered domains is able to recruit MAPK signaling components to SGs (20). Here, we have found that TZF1-MPK3/6-MKK4/5 forms a protein-protein interacting network (Fig. S1). DCP1, a core component of plant PBs, is phosphorylated by MPK3/6 and the phosphorylation triggers a rapid reduction of DCP1 partition into PBs (25). We found that this reduction is concomitantly associated with the increase of DCP1 partition into SGs, hence establishing a role for MAPK signaling in mediating PB-SG dynamics in plants. Furthermore, we have found that plant SG marker protein UBP1b plays a role in maintaining DCP1 in PBs by suppressing the accumulation of MAPK signaling components. Together, we propose that MAPK signaling and UBP1b control the balance of DCP1 partition into PBs and SGs in plants. \u0026nbsp; \u0026nbsp;\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003ePlant materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWild-type Arabidopsis (\u003cem\u003eArabidopsis thaliana\u003c/em\u003e) ecotype Columbia-0 (Col-0) was used in this study. Seeds were obtained from the Arabidopsis Biological Resource Center (ABRC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtoplast transient expression analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eArabidopsis protoplasts transient expression analyses were conducted mainly as described (39), with additional modifications as described (20).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiFC analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe CDS of MKK4, MKK5, MPK3, and MPK6 were cloned into pA7-YN (containing N-terminal half of YFP) and pA7-YC (containing C-terminal half of YFP) vector (40), respectively. Each pair of BiFC construct and an additional cellular localization marker were co-transformed into Arabidopsis protoplasts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-IP assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal proteins from Arabidopsis protoplasts co-expressing plasmid pairs were lysed with lysis buffer (100 mM Tris pH 8.0, 150 mM NaCl, 5 mM EDTA, 10 mM DTT, 0.1% NP-40, proteases inhibitor cocktail). Extracted proteins were\u0026nbsp;then incubated with equilibrated GFP-trap beads (Chromotek) at 4°C for 2 hr under gentle agitation, followed by 3 times of washing with wash buffer (100 mM Tris pH 8.0, 150 mM NaCl, 5 mM EDTA, 10 mM DTT, 0.1% NP-40, proteases inhibitor cocktail). Immunoblots were performed using α-GFP, α-FLAG or α-myc antibodies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAccession numbers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe accession numbers used are as follows:\u0026nbsp;TZF1 (At2g25900), DCP1 (At1g08370), UBP1b (At1g17370), MEKK1 (At4g08500), MKK4 (At1g51660), MKK5 (At3g21220), MPK1 (At1g10210), MPK3 (At3g45640), MPK4 (At4g01370), MPK6 (At2g43790), MPK7 (At2g18170), and MPK11 (At1g01560).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank undergraduate researchers Hannah Furness, Gianni Giarrano, Alex Tarter, and Yu Wang for assistance in various experiments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSiou-Luan He and Jyan-Chyun Jang conceived and designed the experiments; Siou-Luan He performed most of the experiments; Siou-Luan He and Jyan-Chyun Jang wrote the manuscript; Ping He, Libo Shan, and Ying Wang provided comments, tools, and reagents for the project.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the grants from National Science Foundation MCB-1906060 to Jyan-Chyun Jang, Ping He, Libo Shan, and Ying Wang; Ohio State University President\u0026rsquo;s Research Excellence Accelerator award, Ohio Agricultural Research and Development Center SEEDS Program #2018007, Ohio State University College of Food, Agricultural, and Environmental Sciences Internal Grant Program #2022014, and Ohio State University Center for Applied Plant Sciences Research Enhancement Grant to Jyan-Chyun Jang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are provided in the paper and its supplementary information file.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors confirm that all experimental protocols were conducted in accordance with the relevant guidelines and regulations of The Ohio State University. The use of plants in this study complied with the university\u0026rsquo;s research ethics guidelines and required no further approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure and competing interests statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRiggs, C.L., Kedersha, N., Ivanov, P. and Anderson, P. (2020) Mammalian stress granules and P bodies at a glance. \u003cem\u003eJ Cell Sci\u003c/em\u003e, \u003cstrong\u003e133\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eRipin, N. and Parker, R. (2023) Formation, function, and pathology of RNP granules. \u003cem\u003eCell\u003c/em\u003e, \u003cstrong\u003e186\u003c/strong\u003e, 4737-4756.\u003c/li\u003e\n\u003cli\u003eBuchan, J.R. and Parker, R. 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(2006) A highly efficient transient protoplast system for analyzing defense gene expression and protein-protein interactions in rice. \u003cem\u003eMol Plant Path\u003c/em\u003e, \u003cstrong\u003e7\u003c/strong\u003e, 417-427.\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-life","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Life](https://link.springer.com/journal/11084)","snPcode":"11084","submissionUrl":"https://submission.springernature.com/new-submission/11084/3","title":"Discover Life","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7292794/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7292794/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Processing bodies (PBs) and stress granules (SGs) are membrane-less cellular compartments consisting of ribonucleoprotein complexes. Whereas PBs are more ubiquitous, SGs are assembled mainly in response to stress. PBs and SGs are known to physically interact and molecules exchange between the two have been documented in mammals. However, the molecular mechanisms underpinning these processes are unknown in plants. We recently reported that tandem CCCH zinc finger 1 (TZF1) protein can recruit mitogen-activated protein kinase (MAPK) signaling components to SGs. Here, we show that TZF1-MPK3/6-MKK4/5 form a protein-protein interacting network in SGs. The mRNA decapping factor 1 (DCP1) is a core component of PBs. MAPK signaling mediated phosphorylation triggers a rapid reduction of DCP1 partition into PBs, concomitantly associated with an increase of DCP1 assembly into SGs. Furthermore, we found that the plant SG marker protein, oligouridylate binding protein 1b (UBP1b), plays a role in maintaining DCP1 in PBs by suppressing the accumulation of MAPK signaling components. Together, we propose that MAPK signaling and UBP1b mediate the dynamics of PBs and SGs in plants.","manuscriptTitle":"MAPK signaling modulates the partition of DCP1 between processing bodies and stress granules in plant cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-09 16:50:50","doi":"10.21203/rs.3.rs-7292794/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-18T12:50:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-24T06:11:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"38507146878861448795152157921443098838","date":"2025-10-20T15:01:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"339957975161323078513624522283622951271","date":"2025-10-16T02:59:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-30T06:49:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"336184950450367729361108292980882474975","date":"2025-09-22T10:11:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"124464796537156556083300486319472443633","date":"2025-09-21T06:31:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-02T09:49:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-22T08:52:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-21T18:28:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Life","date":"2025-08-21T18:24:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-life","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Life](https://link.springer.com/journal/11084)","snPcode":"11084","submissionUrl":"https://submission.springernature.com/new-submission/11084/3","title":"Discover Life","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a77dad77-c89a-41dd-91d7-d2c3bbfc42d2","owner":[],"postedDate":"September 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-09T16:05:08+00:00","versionOfRecord":{"articleIdentity":"rs-7292794","link":"https://doi.org/10.1007/s11084-026-09721-z","journal":{"identity":"discover-life","isVorOnly":false,"title":"Discover Life"},"publishedOn":"2026-02-07 15:59:37","publishedOnDateReadable":"February 7th, 2026"},"versionCreatedAt":"2025-09-09 16:50:50","video":"","vorDoi":"10.1007/s11084-026-09721-z","vorDoiUrl":"https://doi.org/10.1007/s11084-026-09721-z","workflowStages":[]},"version":"v1","identity":"rs-7292794","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7292794","identity":"rs-7292794","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}