OsNAC016 regulates plant architecture and drought tolerance by interacting with the kinases GSK2 and SAPK8

Abstract

Ideal plant architecture and drought tolerance are important determinants of yield potential in rice (Oryza sativa). Here, we found that OsNAC016, a rice NAC (NAM, ATAF, and CUC) transcription factor, functions as a regulator in the crosslink between brassinosteroid (BR)-mediated plant architecture and abscisic acid (ABA)-regulated drought responses. The loss-of-function mutant osnac016 exhibited erect leaves and shortened internodes, but OsNAC016-overexpressing plants had opposite phenotypes. Further investigation revealed that OsNAC016 regulated the expression of the BR biosynthesis gene D2 by binding to its promoter. Moreover, OsNAC016 interacted with and was phosphorylated by GSK3/SHAGGY-LIKE KINASE2 (GSK2), a negative regulator in the BR pathway. Meanwhile, the mutant osnac016 had improved drought stress tolerance, supported by a decreased water loss rate and enhanced stomatal closure in response to exogenous ABA, but OsNAC016-overexpressing plants showed attenuated drought tolerance and reduced ABA sensitivity. Further, OSMOTIC STRESS/ABA-ACTIVATED PROTEIN KINASE8 (SAPK8) phosphorylated OsNAC016 and reduced its stability. The ubiquitin/26S proteasome system is an important degradation pathway of OsNAC016 via the interaction with PLANT U-BOX PROTEIN43 (OsPUB43) that mediates the ubiquitination of OsNAC016. Notably, RNA-sequencing analysis revealed global roles of OsNAC016 in promoting BR-mediated gene expression and repressing ABA-dependent drought-responsive gene expression, which was confirmed by chromatin immunoprecipitation quantitative PCR analysis. Our findings establish that OsNAC016 is positively involved in BR-regulated rice architecture, negatively modulates ABA-mediated drought tolerance, and is regulated by GSK2, SAPK8, and OsPUB43 through posttranslational modification. Our data provide insights into how plants balance growth and survival by coordinately regulating the growth-promoting signaling pathway and response under abiotic stresses.

The rice transcription factor OsNAC016 regulates plant architecture and drought tolerance by mediating the crosslink between brassinosteroid and abscisic acid signaling.

Introduction

As one of the most important staple crops, rice (Oryza sativa L.) feeds more than half of the population around the world. The plant architecture of rice contributes greatly to grain yield such as leaf inclination, plant height, and tiller number. Plant steroid hormone brassinosteroids (BRs) play crucial roles in modulating plant architecture and yield potential (Yamamuro et al., 2000; Hong et al., 2003; Li et al., 2009; Qiao et al., 2017; Tian et al., 2017; Liu et al., 2018). The BR-biosynthetic deficient or BR signaling-defective rice mutants showed erect leaves and dwarfism, such as osdwarf4-1, d11, d2, and d61-7 (Hong et al., 2003; Tanabe et al., 2005; Morinaka et al., 2006; Sakamoto et al., 2006). In rice, BRs are perceived through the receptor kinase BRASSINOSTEROID INSENSITIVE 1 (OsBRI1), which inactivates GSK3/SHAGGY-LIKE KINASE 2 (GSK2) and thus activates key transcription factor BRASSINAZOLE RESISTANT 1 (OsBZR1; Yamamuro et al., 2000; Bai et al., 2007; Tong et al., 2012). Activated OsBZR1 regulates BR-responsive gene expression and modulates plant growth in rice (Qiao et al., 2017; Fang et al., 2020; Wang et al., 2020a).

BRs have been shown to regulate various stress responses, such as drought, salinity, and cold responses (Chen et al., 2017; Ye et al., 2019; Nolan et al., 2020). Although several studies support the positive roles for BRs or BR signaling in drought tolerance in plants (Fabregas et al., 2018; Cui et al., 2019), it has been recognized that BR biosynthesis and signaling loss-of-function mutants display increased survival under drought conditions (Chen et al., 2017; Ye et al., 2017; Castorina et al., 2018; Xie et al., 2019). For instance, BR biosynthesis loss-of-function mutant lil1-1 in maize has increased drought tolerance (Castorina et al., 2018). On the contrary, BR signaling gain-of-function mutant bes1-D was more hypersensitive to drought than wild-type (Ye et al., 2017).

The antagonism between BR signaling and drought responses has been revealed to be closely associated with abscisic acid (ABA; Ryu et al., 2014; Wang et al., 2020b). The “PYR/PYL/RCAR-PP2C-SnRK2” cascade model for ABA signaling was well known in Arabidopsis (Park et al., 2009; Fuchs et al., 2014). In this model, ABA binding to PYR/PYL/RCAR receptors initiates binding of PP2Cs, thus releasing and activating SNF1-related kinases 2 (SnRK2s). Activated SnRK2s further pass the signals to downstream transcription factors through protein phosphorylation (Umezawa et al., 2009; Moreno-Alvero et al., 2017). One critical junction point between the BR and ABA pathways is BRASSINOSTEROID-INSENSITIVE 2 (BIN2) that is the main repressor of BR pathway and activated by ABA (Wang and Wang, 2018). Without BRs, BIN2 promotes ABA signaling outputs by stabilizing SnRK2.2/2.3 as well as downstream transcription factor ABSCISIC ACID INSENSITIVE 5 (ABI5) by phosphorylation (Cai et al., 2014; Hu and Yu, 2014). In turn, in the absence of ABA, PP2Cs dephosphorylate and inhibit BIN2 activity to enhance BR signaling (Wang et al., 2018). In addition to BIN2, BRI1-EMS-SUPPRESSOR 1 (BES1), the core transcription factor involved in BR signaling, was found to interact with ABI5 and attenuate ABA signaling through preventing ABI5 from binding to the promoters of downstream ABA-responsive genes (Zhao et al., 2019).

The ubiquitin/26S proteasome system (UPS) plays an important role in developmental process and physiological responses (Moon et al., 2004; Smalle and Vierstra, 2004; Vierstra, 2009). During the ubiquitination process, E3 ligases play crucial roles in defining specific substrate (Smalle and Vierstra, 2004). Plants have several types of ubiquitin E3 ligases including HECT, RING, or U-Box domains containing proteins (Moon et al., 2004). The plant U-box (PUB) ubiquitin E3 ligases are involved in plant-specific cellular processes and biotic/abiotic stress responses, and hormone pathways (Cho et al., 2008; Zhou et al., 2018; Wang et al., 2019). In Arabidopsis, PUB12/13 mediate the degradation of ABA co-receptor ABI1 and thus promotes ABA response, while PUB40 induces the degradation of BZR1, a positive component in BR signaling (Kong et al., 2015; Kim et al., 2019). In rice, phosphorylated OsPUB24 by OsSK22/GSK2 mediates OsBZR1 turnover, which is crucial for fine-tuning the BR response (Min et al., 2019).

In this study, we characterized a dwarf BR-insensitive rice mutant osnac016 with erect leaves. OsNAC016 promotes the BR response and plays a negative role in drought tolerance. We demonstrated that GSK2 in BR signaling and SAPK8 in ABA signaling can phosphorylate OsNAC016. Further, we identified the E3 ubiquitin ligase OsPUB43 that mediates OsNAC016 degradation through 26S proteasome system. Our study explored the mechanism by which OsNAC016 regulate the BR-mediated plant architecture and ABA-regulated drought tolerance.

Results

OsNAC016 regulates plant architecture by promoting BR signaling

To gain further insight into the role of rice NAC transcription factors in plant growth and development, we screened a set of T-DNA insertion mutants of OsNACs and identified an erect-leaf mutant in which T-DNA was inserted in the second exon of OsNAC016 (Figure 1, A–C; Supplemental Figure S1, A–F), which was similar to that of the BR signaling-defective mutants d61-1 and dlt (Yamamuro et al., 2000; Tong et al., 2009). Knockout of OsNAC016 reduced shoot length and inhibited the root growth severely, and the cell length in the primary root (PR) of osnac016 mutant was significantly reduced (Supplemental Figure S1, G and H; Supplemental Method S1). The spatio-temporal expression profile of OsNAC016 was analyzed using the PlaNet Browser (Mutwil et al., 2011) and real time-quantitative PCR (RT-qPCR) analysis. OsNAC016 is preferentially expressed in the root, and low levels of mRNA were detected in other organs (Supplemental Figure S2). To confirm the erect leaves and growth inhibition in osnac016 mutant is caused by OsNAC016 disruption, we performed the backcross and generated OsNAC016 knockout mutants by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Multiple CRISPR/Cas9 mutants of OsNAC016 (OsNAC016^ko) exhibited a typical BR-deficient phenotype with erect leaves and extreme dwarfism (Figure 1, C–F; Supplemental Figure S3), which strongly supported that OsNAC016 is indispensable for plant growth in rice. Consistently, OsNAC016^ko mutants are sterile more severely than osnac016 mutant. We further confirm the roles of OsNAC016 in plant growth by constructing rice transgenic plants overexpressing OsNAC016. Contrary to osnac016 mutants, the plants overexpressing OsNAC016 had a phenotype of enlarged leaf inclination (Figure 1, C and F), which is similar to the osbzr1-D, a gain-of-function mutant in BR pathway (Qiao et al., 2017). We examined the response of OsNAC016 to exogenous brassinolide (BL) and found that its expression was repressed in wild-type after BL treatment (Figure 1G). We further assessed the BR sensitivity of wild-type, osnac016 mutant and OE2 by lamina inclination assay. With exogenous BL, the lamina joint inclination was enhanced significantly in wild-type, but not altered too much in osnac016 mutant, indicating that osnac016 mutant was less sensitive to BL, but OE2 was more sensitive to BL than wild-type (Figure 1, H–J). To investigate the regulatory relationship between OsNAC016 and BR signaling, we tested the transcript levels of BR biosynthetic genes such as D2, D11, and DWARF (Hong et al., 2003; Tanabe et al., 2005) in wild-type, osnac016 mutant, and OE2, with or without BL treatment. We found that the transcription of these BR biosynthetic genes was substantially reduced in different genotypes after BL treatment, but still higher in osnac016 mutant and lower in OE2 than that in wild-type (Figure 1K). The inhibition of BR biosynthetic gene as well as OsNAC016 expression by BL treatment may be caused by a negative feedback regulation in the BR signaling pathway (Wang et al., 2002; Qiao et al., 2017). These results demonstrate BR response is inhibited in osnac016 mutant, while enhanced in OE2, and that OsNAC016 promotes BR-regulated plant architecture by positively regulating BR biosynthetic gene expression.

Figure 1.

OsNAC016 regulates plant architecture by promoting BR signaling. A, Schematic diagram indicating the T-DNA insertion site in a coding region in osnac016 mutant. Black boxes represent exons, and the line between black boxes is the intron, and white boxes represent the 5′- and 3′-untranslated regions. The line arrow indicates the transcription orientation. B, Molecular identification of the T-DNA insertion mutant osnac016 by PCR analysis. C, Morphological phenotypes of 4-month-old wild-type (DJ), osnac016, OsNAC016 overexpression line (OE2) and OsNAC016 knockout mutants (OsNAC016^ko). Bars = 15 cm. D, Schematic diagram indicating the gRNA locations in the genomic regions in OsNAC016^ko mutants generated by the CRISPR/Cas9 system. E, Statistical analysis of plant height of indicated plants in (C). Error bars indicate se (n = 10). F, Statistical data of the lamina angle of indicated plants in (C). Error bars indicate se (n = 10). G, Expression analysis of OsNAC016 in the roots of 2-week-old wild-type plants treated with or without 10 μM BL (24-epiBL) for 4 h. Error bars indicate se (n = 3). H and I, Lamina joint bending response to BL. J, Statistical data for lamina joint bending angle assay described in (H and I). Error bars indicate se (n = 10). K, Expression analysis of BR biosynthetic genes in the roots of 2-week-old plants with or without 10-μM BL treatment for 4 h. Error bars indicate se (n = 3). In (E, F, and J), the significant difference was determined by Student’s t test. ^*P < 0.05, ^**P < 0.01, or ^***P < 0.001, NS, not significant.

GSK2 phosphorylates OsNAC016 and reduces its stability

As a transcription factor, OsNAC016 was localized in the nucleus, but the full-length protein did not exhibit transactivation activity in yeast (Figure 2, A and B). Partial fragments of OsNAC016 with a series of deletions based on the functional domains were then evaluated in yeast, and the result showed that the transactivation domain of OsNAC016 is located in the C-terminal part of the protein (Figure 2B). It is notable that the transactivation activity was detected only when domain D was absent in truncated OsNAC016 protein, suggesting that domain D may play a prominent role in the regulation of the transactivation activity of OsNAC016 (Figure 2B;Supplemental Figure S4).

Figure 2.

GSK2 phosphorylates OsNAC016 and reduces its stability. A, Subcellular localization of OsNAC016 in the epidermal cells of N. benthamiana leaves. Bars = 40 μm. B, Sketches of OsNAC016 with functional domains and their autoactivation assays in yeast. QDO: SD/-Ade-His-Leu-Trp. (A–E) represent subdomains (A) to (E) of NAC-binding domain. P represents (PEST) sequence. PEST sequence is a region, in which P (proline), E (glutamate), S (serine), and T (threonine) are rich. C, Y2H assays of OsNAC016 and GSK2. DDO: SD/-Leu-Trp. D, The interaction of OsNAC016 with GSK2 by BiFC in the epidermal cells of N. benthamiana leaves. Bars = 40 μm. E, The interaction of OsNAC016 with GSK2 by split luciferase complementation assays in N. benthamiana leaves. Bar = 0.5 cm. F, Semi-endogenous Co-IP analysis showing the interaction of GST-GSK2 with endogenous OsNAC016-GFP. Briefly, the total protein lysates extracted from DJ or OsNAC016-GFP plants were incubated with GST-GSK2 for 2 h, and then the immunoprecipitation was performed with anti-GFP antibody. The immunoprecipitated proteins were detected with anti-GST and anti-GFP antibodies, respectively. The immunoblot analysis of Histone H3.1 (H3.1) was used as a control. G, OsNAC016 interacts with GSK2 in the in vitro pull-down assays. H, Sketches of various deletions of OsNAC016 used for pull-down assays. I, In vitro pull-down assays showing the interactions of GSK2 with OsNAC016 containing functional domains. J, The in vitro kinase assays for phosphorylation of His-OsNAC016 by GST-GSK2, using a Phos-tag gel. K, The kinase assay for phosphorylation of His-OsNAC016 by GST-GSK2 kinase in E. coli co-expressing GST-GSK2 and His-OsNAC016, using a Phos-tag gel. L, OsNAC016 phosphorylation mediated by GSK2, using the Phosbind biotin. M, BL or bikinin induced the accumulation of OsNAC016-GFP in OsNAC016-GFP plants. H3.1 was used as a control. N, Cell-free degradation assays of His-OsNAC016 or His-OsNAC016^S/T-D in the root protein extracts from wild-type. Recombinant proteins were detected by anti-His antibody. The equal amount of protein stained by Coomassie Brilliant Blue (CBB) was used as a loading control. OsNAC016^S/T-D, partial conserved Ser and Thr sites in the motifs recognized by GSK2 were substituted with Asp in OsNAC016. In (J, K, and L), upward arrows indicate phosphorylated proteins.

OsNAC016 contained multiple potential phosphorylation sites (S/TXXXS/T, where X is any amino acid, S is serine, and T is threonine) recognized by BIN2 (Zhao et al., 2002; Supplemental Figure S5), suggesting that OsNAC016 might be a substrate of GSK2. To test our hypothesis, yeast two-hybrid (Y2H) assays was used and OsNAC016 was found to interact with GSK2 (Figure 2C). The direct interaction between OsNAC016 and GSK2 was confirmed by semi-endogenous coimmunoprecipitation (Co-IP), in vitro pull-down and in vivo bimolecular fluorescence complementation (BiFC) assays (Figure 2, D–G). Full-length and truncated His-OsNAC016 proteins were used in pull-down assays to map the regions of OsNAC016 that mediate its interaction with GSK2 (Figure 2H). As shown in Figure 2I, the interaction of the domain (200–340 amino acid [aa]) of OsNAC016 with GSK2 is drastically reduced, compared to the full length of OsNAC016, indicating that deletion of NAC domains (1–200 aa) attenuated the interaction.

Given the kinase feature of GSK2, the GSK2–OsNAC016 interaction promoted us to check whether GSK2 phosphorylates OsNAC016. The kinase assays in vitro by using the Phos-tag-based approach showed that GST-GSK2, but not GST alone, phosphorylates His-OsNAC016 (Figure 2J). The kinase–substrate relationship between GSK2 and OsNAC016 was also confirmed by kinase assays in Escherichia coli co-expressing GST-GSK2 and His-OsNAC016 (Figure 2K, left lane); meanwhile, the mutated OsNAC016 (OsNAC016^S/T-A) in which multiple conserved Ser and Thr sites in the motifs recognized by GSK2 were substituted with Ala clearly had reduced phosphorylation mediated by GSK2 (right lane). Moreover, phosphorylation of OsNAC016 mediated by GSK2 was attenuated by adding Lambda protein phosphatase, further confirming the kinase–substrate relationship (Figure 2L).

Previous reports indicated that BIN2/GSK2 phosphorylation leads to protein destabilization in plants (Youn and Kim, 2015). We first investigated the effect of BL and bikinin (the GSK3-like kinase inhibitor) on OsNAC016 stability in plants. The result showed that the accumulation of OsNAC016-GFP was markedly increased in OsNAC016-GFP plants after BL or bikinin treatment (Figure 2M), suggesting BRs promote OsNAC016 accumulation through the inhibition of GSK2 activity in part. To further determine the effects of phosphorylation mediated by GSK2 on OsNAC016 stability, we then generated the mimicked phosphorylated form of OsNAC016 (His-OsNAC016^S/T-D), in which multiple conserved Ser and Thr sites in the motifs recognized by GSK2 were substituted with Asp. As shown in Figure 2N, when incubated with protein extracts from wild-type seedlings root, the degradation rate of His-OsNAC016^S/T-D is much quicker than that of the native His-OsNAC016, indicating that phosphorylation form promoted OsNAC016 degradation. The result showed that OsNAC016 phosphorylation mediated by GSK2 is critical for its stability. Taken together, these results illustrate that OsNAC016 functions in the BR pathway and is regulated by GSK2 by protein phosphorylation.

OsNAC016 plays a negative role in the drought response via ABA signaling

The expression of OsNAC016 was induced by ABA treatment in roots (Figure 3A), suggesting that it might have a role in rice response to water-deficit stress. As shown in Figure 3, B and C, after withdrawing water and rewatering, the survival rate of osnac016 mutant was significantly higher than that of wild-type (DJ), but OE2 exhibited markedly reduced survival. In parallel water-withholding assays, compared to wild-type plants that almost perished, ∼40% plants of osnac016 mutant still survived, as it lost water more slowly than wild-type under hydropenic conditions (Supplemental Figure S6; Supplemental Method S2). ABA regulation of stomatal movement is a well-recognized model system for the study of plant response to drought stress. Thus, we examined the ABA-induced stomatal closure in plants of different genotypes. Without ABA, there was little difference in the proportions of completely open stomata among different genotypic plants; however, after ABA treatment, the proportions of the completely open stomata were significantly lower in osnac016 mutant, while higher in OE2 than that in wild-type (Figure 3D), suggesting OsNAC016 is involved in ABA-induced stomatal closure.

Figure 3.

OsNAC016 negatively regulates the drought response via ABA signaling. A, Expression of OsNAC016 in root after treatment with 50 μM for different times. Error bars indicate se (n = 3). B, Images showing the phenotypes of OsNAC016-overexpressing line (OE2), wild-type (DJ) and osnac016 mutant under drought stress. Three independent experiments were performed. Bars = 5 cm. C, The survival rates of plants after drought stress and then rewatering. Error bars indicate se (n = 30). D, Percentages of completely open (CO), partially open (PO), and completely closed (CC) stomata in the different genotypes under normal and ABA treatment conditions. Error bars indicate SE (n = 200). Bars = 10 μm. E and G, Responses of OE2, wild-type and osnac016 mutant to ABA. Three independent experiments were performed. Bars = 2 cm. F and H, Statistical analysis of primary root (PR) length of indicated plants in (E and G), respectively. Error bars indicate se (n = 10). I and J, Relative transcription levels of ABA early-response genes in the leaves of 2-week-old plants under air drought for 30 min. Error bars indicate se (n = 3). In (C, F, and H), the significant difference was determined by Student’s t test. ^*P < 0.05, ^**P < 0.01, or ^***P < 0.001. In (D), Two-way analysis of variance followed by Bonferroni’s post-hoc test was performed. Different letters with the same superscript mark indicate significant differences (P < 0.05) between plants under the same conditions.

We then examined the sensitivity of different genotypic plants to ABA in PR elongation. The result showed that OE2 plants were more resistant to inhibition of PR growth by exogenous ABA than wild-type (Figure 3, E and F). On the contrary, osnac016 mutant had markedly enhanced ABA sensitivity and ABA response (Figure 3, G and H). The osnac016 mutant also shows reduced seed germination (Supplemental Figure S7, A and B). ABA signaling plays an essential role in promotes root hair elongation (Li et al., 2015; Wang et al., 2017). Interestingly, in the presence of ABA, the root hair growth in osnac016 mutant was greatly increased, compared to that in wild-type (Supplemental Figure S7C). Furthermore, we found that, after drought treatment, the expression of early-response genes in ABA signaling such as OsPP2C61 and OsPP2C68 in osnac016 mutant was much higher than that in wild-type (Figure 3, I and J). Together, these results clearly showed that OsNAC016 negatively regulates plant drought response by repressing ABA response.

SAPK8 phosphorylates OsNAC016 and reduces its stability

Rice SnRK2 kinases (SAPK8, SAPK9, and SAPK10) were indicated to play essential roles in transduction of ABA signaling (Kobayashi et al., 2005; Wang et al., 2020c). We found that there were two potential SnRK2s phosphorylation sites (RXXS/T) in OsNAC016 protein (Supplemental Figure S5), suggesting that OsNAC016 might be a substrate of SnRK2s. We first tested the interaction between OsNAC016 and a subset of SAPKs by Y2H and found that OsNAC016 interact with SAPK8, SAPK9, and SAPK10, respectively (Figure 4A), implying OsNAC016 might function in ABA signaling. Considering the stronger interaction of OsNAC016 with SAPK8, we chose SAPK8 for further mechanistic investigation. The direct interaction between OsNAC016 and SAPK8 was confirmed by in vitro pull-down, in vivo BiFC, and Co-IP assays (Figure 4, B–E). Further interaction of SAPK8 with multiple truncated OsNAC016 proteins containing different functional domains showed that N-terminal parts of OsNAC016 is responsible for the interaction (Figure 4F). We conducted the kinase assays in BL21 (DE3), and the result showed that SAPK8 was able to phosphorylate OsNAC016 (Figure 4G). We then generated the mimicked dephosphorylated form of OsNAC016, in which two conserved SnRK2s-recognized sites (Ser-244 and Thr-283) were substituted with Ala. Compared to the native OsNAC016, the phosphorylation of OsNAC016^{S244A T283A} is greatly reduced (Figure 4G), suggesting that S244 and T283 are major sites of the SAPK8-mediated phosphorylation. Recent research showed that SAPK10 exhibits autophosphorylation activity on Ser177 (Wang et al., 2020c), which corresponds to Ser187 in SAPK8, based on the sequence similarity analysis. To further confirm that OsNAC016 was the substrate of SAPK8 and could be phosphorylated by SAPK8, SAPK8^S187A (Ser187 to Ala) was used to perform in vitro kinase assays. We found that GST-SAPK8^S187A could hardly phosphorylate His-OsNAC016 (Figure 4H), supporting that OsNAC016 is a substrate for SAPK8. Considering OsNAC016 plays a negative role in ABA signaling (Figure 3), we then examined the effect of exogenous ABA on OsNAC016 stability by using the cell-free protein degradation assays. The result indicated that ABA treatment resulted in His-OsNAC016 destabilization, and its degradation rate depends on ABA treatment time (Figure 4I). To explore whether ABA promoting His-OsNAC016 degradation is mediated via the phosphorylation by SAPK8, the mimicked phosphorylated version of His-OsNAC016 (His-OsNAC016^{S244D T283D}) was used for cell-free degradation assays. As shown in Figure 4J, in comparison with the native His-OsNAC016, its mimicked phosphorylation substantially destabilized the protein, suggesting SAPK8-mediated phosphorylation is negatively involved in modulating the stability of OsNAC016.

Figure 4.

SAPK8 phosphorylates OsNAC016 and reduces its stability. A, The interaction of OsNAC016 with a subset of SAPK members in an Y2H assay. DDO: SD/-Leu-Trp. QDO: SD/–Ade–His–Leu–Trp. B, The interaction of OsNAC016 with SAPK8 in the pull-down assays. C and D, OsNAC016–SAPK8 interaction indicated by BiFC in rice protoplasts and the epidermal cells of N. benthamiana leaves. Bars in (C) = 5 μm. Bars in (D) = 40 μm. E, OsNAC016–SAPK8 interaction by Co-IP assay. F, In vitro pull-down assay showing the interactions of SAPK8 with the functional domains of OsNAC016. G, The kinase assay for OsNAC016 phosphorylation mediated by SAPK8 in E. coli, using a Phos-tag gel. H, Ser187 in SAPK8 is essential for OsNAC016 phosphorylation indicated by the kinase assay in E. coli. I, Cell-free degradation assays of His-OsNAC016 in the root protein extracts from 2-week-old wild-type (DJ) treated with or without 50-μM ABA for different times. J, Cell-free degradation assays for His-OsNAC016 or His-OsNAC016^{S244D T283D} (Ser-244 and Thr-283 to Asp) in the root protein extracts from 2-week-old wild-type. K, Degradation of OsNAC016-GFP is under both the 26S proteasome and autophagy pathways in plants. Tubulin was used as a control. L, ABA induces the degradation of the phosphorylated OsNAC016-GFP (OsNAC016-GFP-P). Histone H3.1 was used as a control. In (G, H), upward arrows indicate phosphorylated proteins. In (I and J), Coomassie Brilliant Blue (CBB) was used as a loading control.

Our results showed that OsNAC016 degradation in plants is under both the 26S proteasome as well as autophagy pathway (Figure 4K; Supplemental Figure S8), suggested by the result of 26S proteasome inhibitor MG132 and autophagy inhibitor E64d treatment (Zhang et al., 2015; Nolan et al., 2017). Then we examined the phosphorylation status of OsNAC016-GFP in plants by the phos-tag-based technique. We found that, in OsNAC016-GFP plants treated by MG132/E64d, phosphorylated OsNAC016-GFP (OsNAC016-GFP-P) accumulated; however, when ABA was added, the band of OsNAC016-GFP-P faded, and only less dephosphorylated OsNAC016-GFP was detected (Figure 4L, top). Notably, in the presence of E64d and MG132, total OsNAC016-GFP protein (phosphorylated and dephosphorylated) greatly accumulated; however, the abundance of total OsNAC016-GFP protein clearly decreased after 2 h of treatment of ABA (Figure 4L, middle). The ABA can accelerate OsNAC016 protein degradation whereas induce OsNAC016 RNA transcription (Figures 3A and 4L), suggesting that OsNAC016 may play a prominent role in the repression and termination of ABA signaling. Together, these observations indicate that ABA triggers OsNAC016 phosphorylation and therefore promotes its degradation in plants.

OsNAC016 degradation is mediated by interacting with the E3 ubiquitin ligase OsPUB43

ABA promoting the degradation of OsNAC016 urges us to explore the relationship between ABA-induced phosphorylation and ubiquitination of OsNAC016. As shown in Figure 5A, ABA markedly promoted the phosphorylation and ubiquitination of OsNAC016-GFP, compared to the mock. These results indicated that SAPK8-mediated OsNAC016 phosphorylation facilitates its degradation by promoting the ubiquitination of OsNAC016. Thus, identifying the E3 ubiquitin ligase specific for OsNAC016 was crucial, which can explain the pathway of OsNAC016 turnover under drought stress in rice.

Figure 5.

OsPUB43 interacts with OsNAC016 and mediates its degradation in plants. A, ABA induces the phosphorylation as well as ubiquitination of OsNAC016-GFP in plants. B, The interaction of OsNAC016 with a subset of rice U-box/ARM proteins in an Y2H assay. DDO: SD/-Leu–Trp. QDO: SD/–Ade–His–Leu–Trp. C, OsPUB43–OsNAC016 interaction by BiFC in the epidermal cells of N. benthamiana leaves. Bars = 40 μm. D, In vitro pull-down assays for OsPUB43-OsNAC016 interaction. E, Semi-endogenous Co-IP analysis showing the interaction of His-OsPUB43 with endogenous OsNAC016-GFP. Briefly, the total protein lysates extracted from DJ or OsNAC016-GFP plants were incubated with His-OsPUB43 for 2 h, and then the immunoprecipitation was performed with anti-GFP antibody. The immunoprecipitated proteins were detected with anti-His and anti-GFP antibodies. The immunoblot analysis of Histone H3.1 (H3.1) was used as a control. F, OsPUB43–OsNAC016 interaction by split luciferase complementation assays in N. benthamiana leaves. Bar = 0.5 cm. G, In vitro pull-down assay showing the interactions of OsPUB43 with the functional domains of OsNAC016. H, Cell-free protein degradation assays for His-OsNAC016 with different functional domains in the root extracts from wild-type (DJ). I, Cell-free protein degradation assays for His-OsNAC016 in the shoot and root extracts from wild-type (DJ). J, OsPUB43 ubiquitinates OsNAC016 in N. benthamiana leaves transiently co-expressing OsNAC016-3×FLAG with OsPUB43-GFP. K, OsPUB43 induces OsNAC016 degradation in vivo. Proteins were extracted from N. benthamiana leaves transiently expressing OsNAC016-3×FLAG, OsPUB43-GFP, or GFP alone. Extracts containing OsNAC016-3×FLAG were incubated with OsPUB43-GFP or GFP extracts at different times. Degradation of OsNAC016-3×FLAG was detected by anti-FLAG antibody. An equal amount of OsPUB43-GFP or GFP was detected by anti-GFP antibody. The equal amount of protein stained by CBB was used as a loading control.

We chose seven PUB E3 ligases (OsPUB3, OsPUB16, OsPUB24, OsPUB28, OsPUB33, OsPUB43, and OsPUB75) that belong to five clusters of Arabidopsis PUB groups based on the alignment (Wiborg et al., 2008; Tamura et al., 2013; Supplemental Figure S9A; Supplemental Method S3), to test their interaction with OsNAC016. The result showed that OsNAC016 interacts with OsPUB16 and OsPUB43 in Y2H yeast (Figure 5B;Supplemental Figure S9B). We selected the stronger interaction of OsPUB43-OsNAC016 for further validation. Semi-endogenous Co-IP, Pull-down and BiFC assays confirmed OsPUB43 physically interacts with OsNAC016 (Figure 5, C–F). The interaction of OsPUB43 with OsNAC016 with functional domains was performed by pull-down assays. Interestingly, similar to GSK2 and SAPK8, OsPUB43 also had stronger interaction with the N-terminal (1–200 aa) than full length of OsNAC016 but did not interact with the C-terminal (200–340 aa) (Figure 5G).

Further, we analyzed the stability of different functional domains of His-OsNAC016 by using cell-free degradation assay. As shown in Figure 5H, the N-terminal (1–200 aa) degraded more quickly, whereas C-terminal (200–340 aa) exhibited a much slower degradation rate, suggesting that the domain (1–200 aa) is responsible for OsNAC016 degradation. It is worth noting that His-OsNAC016 is more unstable in the root protein extracts than that in the shoot protein extracts (Figure 5I), which might be due to high expression of OsNAC016 and OsPUB43 in the root (Supplemental Figure S2). We then examined whether OsNAC016 is ubiquitinated by OsPUB43 in plants by using N. benthamiana leaves transiently co-expressing OsNAC016-3×FLAG with OsPUB43-GFP. We found that OsNAC016 ubiquitination was enhanced in the presence of OsPUB43 (Figure 5J). Then we examined OsPUB43-mediated OsNAC016 degradation in plants. As shown in Figure 5K, the degradation of OsNAC016-3×FLAG was much quicker when co-incubated with OsPUB43-GFP than with GFP alone, illustrating that OsPUB43 targets OsNAC016 for degradation. These results indicated that destabilization of OsNAC016 in plants was at least partially mediated by OsPUB43.

OsNAC016 plays an important role in balancing BR and ABA signaling pathways

To further understand the functions of OsNAC016 in the crosslink between BR and ABA signaling, we performed whole transcriptome RNA sequencing (RNA-seq) using root samples from wild-type, osnac016 mutant, and OE2, and identified differentially expressed genes (DEGs) in osnac016 and OE2, in comparisons with wild-type (Supplemental Table S1). OsNAC016 promotes BR-induced plant growth, implying that OsNAC016-regulated genes might be regulated by OsBZR1. To test this hypothesis, we compared DEGs in OE2 with OsBZR1-regulated genes that was identified in osbzr1-D by RNA-seq recently (Ren et al., 2020). The result showed that OsNAC016-regulated genes were largely modulated by OsBZR1. Specifically, 107 (14.4%) of 742 OsNAC016-promoted genes were upregulated in osbzr1-D, and 118 (5.3%) of 2,241 OsNAC016-repressed genes were downregulated in osbzr1-D (Figure 6A). The expression of OsNAC016 was greatly reduced in osbzr1 mutant (Supplemental Figure S10), suggesting OsNAC016 is likely to be a target of OsBZR1. It is notable that DEGs in OE2 had multiple downregulated genes involved in BR biosynthesis and BR signaling pathway (Supplemental Table S2), confirming that OsNAC016 promotes BR signaling. On the other hand, we compared the DEGs in osnac016 mutant with previously published drought-responsive genes regulated by OsbZIP23 (Zong et al., 2016), and found that 41 (17.4%) of 235 OsNAC016-repressed genes and 40 (19.9%) of 201 OsNAC016-activated genes were found in drought-induced and drought-repressed genes, respectively (Figure 6B). Additionally, we found multiple upregulated genes responsible for reactive oxygen species (ROS) scavenging in DEGs in osnac016 mutant, whereas high frequency of downregulated genes encoding ROS-scavenging enzymes in DGEs in OE2 (Supplemental Table S3), which supports the fact that osnac016 mutant has increased drought tolerance, but OE2 acts in the opposite way. The antagonism of ABA to BRs is further confirmed by the fact that ABA repressed the expression of BR biosynthetic genes such as D2 and D11 but promoted mRNA levels of ABA-responsive genes OsPP2C68 and OsbZIP46 (Figure 6C). These results provided convincing evidence that OsNAC016 acts a negative regulator in ABA signaling but functions positively in BR signaling, suggesting that OsNAC016 is important in the antagonism between BR and ABA signaling pathways.

Figure 6.

OsNAC016 promotes BR biosynthetic genes and represses ABA/drought-responsive genes. A, Venn diagrams showing overlap among OsBZR1-regulated genes and DEGs from RNA-seq analysis in OsNAC016-overexpressing plants (OE2). Circles indicate genes commonly regulated by OsNAC016 and OsBZR1. B, Venn diagrams showing overlap among drought-regulated genes and DEGs from RNA-seq analysis in osnac016 mutant. Circles indicate genes commonly regulated by OsNAC016 and drought. C, ABA represses the expression of BR biosynthetic genes, but promotes ABA-responsive genes in plants, especially in osnac016 mutant. Error bars indicate se (n = 3). D, Schematic diagram of D2 promoter region (2-kb upstream from the transcription start site) showing the positions of key motifs and P1–P4 fragments amplified by ChIP-qPCR analysis. BRRE (CGTGT/CG) and N-box motif (CACG(A/C)G). E, Transactivation activity of OsNAC016 indicated by the dual-LUC reporter assay. Error bars indicate se (n = 3). F, ChIP-qPCR analysis of D2 promoter fragments enriched by OsNAC016-GFP in OsNAC016-GFP plants. P1–P4 represent the regions shown in (D). G, Schematic diagram of OsPP2C68 promoter region (2-kb upstream from the transcription start site) showing the positions of ABRE motifs and P1–P5 fragments amplified by ChIP-qPCR analysis. ABRE (ACGTG). H, ChIP-qPCR analysis of OsPP2C68 promoter fragments enriched by OsNAC016-GFP in OsNAC016-GFP plants. P1–P5 represent the regions shown in (G). In (F and H), the enrichment values were normalized to input. Error bars indicate se with biological triplicates. In (E, F, and H), the significant difference was determined by Student’s t test. ^*P < 0.05, ^**P < 0.01, or ^***P < 0.001.

We then next tested whether OsNAC016 directly regulates the BR/ABA biosynthetic or responsive genes. We selected the BR biosynthetic gene D2 and ABA early-responsive gene OsPP2C68 as potential targets of OsNAC016. NAC family transcription factors were previously reported to bind conserved DNA-binding elements such as BR-response element (BRRE) (CGTGT/CG), ABA-response element (ABRE) (ACGTG), and N-box [CACG(A/C)G] (Lu et al., 2007; Balazadeh et al., 2011; Cordeiro et al., 2016; Peres et al., 2019), which contain the core sequence of CACG. Multiple BRRE motifs were found in the D2 promoter sequence (Figure 6D). Then we performed transient transactivation assays in N. benthamiana leaves and found that OsNAC016 significantly induced the expression of LUC driven by D2 promoter (Figure 6E), suggesting that D2 is regulated by OsNAC016. To further determine whether OsNAC016 recognizes the D2 promoter in vivo, we performed chromatin immunoprecipitation (ChIP)-qPCR assays in OsNAC016-GFP plants. Up to three-fold enrichment was observed in the D2 promoter region containing BRRE, while no enrichment was found in other regions (Figure 6, D and F). These data clearly indicated that OsNAC016 regulates the expression of D2 in vivo. Expectedly, OsNAC016 binds to the OsPP2C68 promoter regions containing ABRE, indicated by ChIP-qPCR analysis (Figure 6, G and H).

Discussion

OsNAC016 plays crucial roles in BR-mediated plant growth

Leaf inclination is a critical determinant of plant architecture and crop yield. Exploring the molecular mechanisms of rice plant architecture will provide theoretical guidance and valuable gene resources for breeding elite rice varieties with ideal plant architecture. Despite the recent progress, our understanding of the molecular mechanisms that control rice leaf inclination is still limited. NAC transcription factors have been characterized in plant growth and stress response; however, to our knowledge, their positive roles in BR signaling in rice have not been previously reported. In this study, we demonstrated that OsNAC016 plays important role in BR-mediated plant architecture but inhibits drought tolerance. Under normal conditions, OsNAC016 promotes BR-mediated plant growth by regulating the expression of BR biosynthetic genes. Our study provided a series of convincing evidence to support the hypothesis that OsNAC016 is an essential component positively regulating BR signaling in rice. First, osnac016 and OsNAC016^ko mutants exhibited BR-deficient phenotypes similar to BR-insensitive mutants such as d61-1 and dlt (Yamamuro et al., 2000; Tong et al., 2009), while the plant architecture of OE2 resembled that of GSK2-Ri or the gain-of-function mutant Osbzr1-D (Qiao et al., 2017; Figure 1C;Supplemental Figures S1 and S3). Second, osnac016 mutant is less sensitive, whereas OE2 is more sensitive to BL than wild-type (Figure 1, H–J). Third, biochemical evidence demonstrated that OsNAC016 interacted with and was phosphorylated by GKS2 (Figure 2), illustrating that OsNAC016 is regulated by GSK2 through posttranslational modification. Fourth, we proved the reduced transcription of OsNAC016 in osbzr1 mutant (Supplemental Figure S10). Finally, the observation implied that the BL and bikinin treatments increase OsNAC016 accumulation, but BL inhibit the transcription of OsNAC016 in rice (Figures 1, G and 2, M), which may be caused by negative feedback in the BR signaling and is generally considered as a central mechanism to maintain BRs homeostasis in plants (Wang et al., 2002; Tanaka et al., 2005; Qiao et al., 2017). However, there is no direct interaction between OsNAC016 and OsBZR1 (Supplemental Figure S11A). Thus, we conclude that OsNAC016 plays positive roles in BR-mediated plant growth.

OsNAC016 mediates the antagonism between BR and ABA pathways

Recent studies have demonstrated that BR-abiotic stress signaling is closely associated with ABA, with BIN2 mediating the crosstalk between ABA/abiotic stress and BR/developmental signaling pathways (Jiang et al., 2019; Wang et al., 2020b). There are other critical components for the crosslink of ABA and BR, such as BES1 and ABI5 (Hu and Yu, 2014; Wang et al., 2018; Zhao et al., 2019). In our study, OsNAC016 promotes plant architecture and reduces drought tolerance by functioning in both BR and ABA signaling pathways (Figures 1–4). Recently, Arabidopsis WKRY, AP2/ERF, and RD26 have been reported to mediate the crosstalk between BR signaling and drought stress (Chen et al., 2017; Ye et al., 2017; Xie et al., 2019). In contrast to Arabidopsis RD26 playing a negative role in the BR signaling pathway but promoting ABA signaling output (Jiang et al., 2019), OsNAC016 positively regulates plant growth through the BR pathway but reduces ABA-mediated drought tolerance in rice (Figures 1–4). Compared to wild-type, osnac016 mutant displayed high tolerance to drought stress (Figure 3), which is in agreement with the general notion that stress tolerance in plants is often associated with growth inhibition (Bechtold and Field, 2018). At present, a series of evidence showed that SnRKs interact with BIN2 (Cai et al., 2014; Wang et al., 2018). However, in our study, there is no direct interaction between GSK2 and SAPK8 (Supplemental Figure S11B), both of which mediate the phosphorylation and promote degradation of OsNAC016. Our study begins to fill the void of knowledge of OsNAC function in rice, thus OsNAC016 is identified as a molecular link that coordinates BR and ABA pathways.

Phosphorylation of OsNAC016 facilitates its ubiquitination

Protein phosphorylation represents an important mechanism of the signal transduction of ABA and BR (Yang et al., 2017). Our result showed that GSK2 in BR signaling and SAPK8 in ABA signaling can interact with and phosphorylate OsNAC016, respectively (Figures 2, C–L and 4, A–H). Protein phosphorylation has been linked to targeting protein turnover and stability (Chen et al., 2015; Kong et al., 2015; Kim et al., 2019; Ruan et al., 2019). In our study, the mimicked phosphorylated version of His-OsNAC016 mediated by GSK2 or SPAK8 was degraded much quicker than its native version (Figures 2, N and 4, J), suggesting that GSK2 and SAPK8-mediated phosphorylation of OsNAC016 facilitates the degradation of OsNAC016. Consistent with this, the phosphorylation of OsNAC016 in planta treated with ABA was higher than without ABA, which was accompanied by the high ubiquitination of OsNAC016 (Figure 5A).

To acquire deeper insight into the degradation mechanism of OsNAC016 mediated by phosphorylation in rice, we identified an E3 ligase, OsPUB43, which physically interacts with OsNAC016 (Figure 5, B–F). More importantly, GSK2, SAPK8, or OsPUB43 interacts with the same functional domain of OsNAC016 (the N-terminal, 1–200 aa; Figures 2, I, 4, F, and 5, G), suggesting that this functional domain might be critical for the conformational maintenance of the protein or for interaction with its partner(s). This idea is confirmed by the much faster degradation rate of this domain (1–200 aa) in the cell-free degradation assays (Figure 5H). As to the reasons, we supposed that, under drought stress conditions or in the presence of ABA, activated protein kinases including GSK2 and SAPK8 phosphorylate OsNAC016, and phosphorylated OsNAC016 might be preferentially ubiquitinated by OsPUB43, resulting in OsNAC016 degradation. As a transcription factor, in addition to activation/deactivation, its phosphorylation/dephosphorylation and stabilization/destabilization also posttranslationally fine-tune its functions. At present, the effects of phosphorylation on OsNAC016 turnover or ubiquitination are somehow elusive, which is worth investigating in the future.

Based on our findings, we proposed a model to elucidate how OsNAC016 acts with its partner(s) to balance BR-mediated plant growth development and ABA-regulated drought response in rice (Figure 7). Under normal conditions with high BR/low ABA levels, the activity of GSK2 as well as SAPK8 is inhibited, resulting in the dephosphorylation and accumulation of OsNAC016, which then positively regulates BR-responsive gene expression but negatively controls ABA-responsive drought-related genes to promote plant growth (Figure 7, left). When water is withheld, ABA accumulation activates SAPK8 and GSK2, both of which phosphorylate OsNAC016. Phosphorylated OsNAC016 preferentially interacts with OsPUB43, leading to the degradation of OsNAC016 via UPS, which leads to repression of growth-related genes but alleviation of OsNAC016 inhibitory effect on drought-related genes, and finally resulting in reduced growth and increased drought tolerance (Figure 7, right). Thus, the regulatory networks of OsNAC016, GSK2, SAPK8, and OsPUB43 are crucial for fine-tuning the BR-mediated plant architecture and ABA-regulated drought responses in rice.

Figure 7.

A proposed working model for OsNAC016 in BR-regulated plant architecture and ABA-mediated drought response. OsNAC016 promotes plant growth but inhibits drought response. Under normal conditions, BRs repress GSK2 activity, which releases and stabilizes OsNAC016. OsNAC016 positively regulates the expression of BR responsive genes at the mRNA level by binding to the BRRE sites, resulting in promoting BR-regulated plant growth; meanwhile, OsNAC016 inhibits drought responses by repressing ABA/drought-responsive genes (left). Under drought stress condition, ABA is produced and activates SAPK8, which results in OsNAC016 phosphorylation. Phosphorylated OsNAC016 facilitates its ubiquitination by interacting with ubiquitin E3 Ligase OsPUB43, and therefore is destabilized, which leads to repression of growth-related genes and alleviation of the OsNAC016 inhibitory effect on drought-related genes, resulting in reduced growth and increased drought tolerance (right).

Materials and methods

Plant materials and growth conditions

Rice (O. sativa ssp. japonica) cv Dongjin (DJ) and cv Zhonghua 11 (ZH11) were used in this study. For constructing OsNAC016-GFP transgenic plants, the coding sequence (CDS) of OsNAC016 was cloned into pCAMBIA1301-eGFP, driven by 35S promoter. Mutants of OsNAC016 were generated by CRISPR/Cas9 system as described previously (Ma et al., 2015). These constructs were introduced into DJ by Agrobacterium tumefaciens-mediated transformation, respectively. Loss-of-function mutant osnac016 (PFG_1B-15010) was obtained from the T-DNA insertional population in DJ (Jeon et al., 2000, 2006), and osbzr1 (RMD_04Z11PM21) was identified from RMD mutant database of ZH11 (Zhang et al., 2006). The plants were grown in the field from April to October under natural conditions in Chongqing, China, or in the greenhouse under a 14 h: 10 h, light: dark photoperiod with 60% humidity in winter. Primers were given in Supplemental Table S4.

Rice stomata imaging

Full expanded young leaves of 2-week-old plants were detached and treated with 30-μM ABA in MES-KCl buffer (50-mM KCl, 10-mM MES-KOH, pH 6.15) for 2 h. Stomatal closure was detected by Hitachi SU3500 scanning electron microscope with a −40°C cool stage. Two hundred stomata from 10 plants of each genotype were observed.

RNA-seq and RNA analysis

Root samples of 2-week-old seedlings were collected for RNA extraction. RNA-Seq libraries were prepared following the manufacturer’s instructions and sequenced on an Illumina Hiseq4000 (Illumina Inc., San Diego, CA, USA) by Majorbio. Inc. (Shanghai, China). The clean reads were mapped to the genome of O. sativa L. Nip using Tophat (Trapnell et al., 2012). Gene expression levels were quantified with  AQ4FPKM (fragments per kilobase of exon model per million mapped reads) calculation method using Cufflinks and Cuffdiff (Trapnell et al., 2012). DEGs between the two groups were determined using the edger program (Robinson et al., 2010; Trapnell et al., 2012). Rice OsActin1 was used as the internal control, and relative changes in gene expression levels were quantified based on three biological replicates via the 2^−ΔΔCt method (Livak and Schmittgen, 2001). The primers were listed in Supplemental Table S4.

Y2H assay

For Y2H assays, the open reading frames (ORFs) of OsNAC016, GSK2, SAPK4, SAPK8, SAPK9, SAPK10, OsPUB3, OsPUB16, OsPUB24, OsPUB28, OsPUB33, OsPUB43, and OsPUB75 were cloned into the pGBKT7 or pGADT7 vectors, respectively. These resulted constructs or the corresponding empty vectors were cotransformed into the yeast strain Y2HGold, and colonies of the corresponding transformants were incubated at 30°C on synthetic defined (SD)/-Leu/-Trp and SD/-Ade/-His/-Leu/-Trp medium for 2–3 d. The lack of Trp and Leu in the selective media is required for maintaining the bait plasmid and the prey plasmid, and the lack of His and Ade in the selective media is crucial for selecting two-hybrid interaction. Primers for these constructs were listed in Supplemental Table S4.

Pull-down assay

The CDS of OsNAC016 was cloned into pET-28a or pET-32a, and GSK2, SAPK8, or OsPUB43 into pGEX-4T-1. These resulted constructs were transformed into E. coli BL21 (DE3) to produce recombinant proteins. For pull-down assay, GST-OsNAC016 or GST was incubated with GST Bind Resin at 4°C for 2 h, and then 0.5 mg of purified recombinant protein with His tag was added. The incubation continued for another 6 h, and the beads were washed with pull-down buffer for 3 times. The bounded proteins were finally eluted, and the pulled down proteins were analyzed by western blot with the anti-His antibody (Proteintech, Rosemont, IL, USA; 66005-1-Ig) and anti-GST antibody (Proteintech; 66001-2-Ig).

BiFC assay

Rice protoplasts were prepared from 2-week-old seedlings of rice as described previously (Zhang et al., 2011). BiFC vectors pFGC-nYFP and pFGC-cYFP were used (Kim et al., 2008). The CDS of OsNAC016 was cloned into pFGC-cYFP, and GSK2, SAPK8, or OsPUB43 into pFGC-nYFP. The generated cYFP and nYFP vectors were co-transformed into rice protoplasts by PEG-mediated transformation method (Zhang et al., 2011), or co-transformed into 4-week-old Nicotiana benthamiana leaves with an efficient agroinfiltration expression system (Liu et al., 2010). The nYFP and cYFP empty vectors were used as the negative controls for the assay. Yellow fluorescent protein fluorescence signals were visualized using a Leica SP8 confocal microscope with the indicted parameters (Zoom 4.86; HyD Gain 100 for fluorescence signals; PMT Trans Gain 180 for bright signals; Frame Average 1; Line Average 1).

Split luciferase complementation assays

The JW771 and JW772 vectors were used for split luciferase (LUC) complementation assay (SLCAs) based on firefly LUC (Gou et al., 2011; Wang et al., 2021). The ORFs of OsNAC016, GSK2, SAPK8, and OsPUB43 were cloned into these vectors, respectively. Agrobacterium (GV3101) cells harboring the constructed plasmids were resuspended (optical density at 600 nm [OD600] of 1.5) in infiltration buffer (10-mM MgCl₂ and 200-μM acetosyringone). Equal volumes of the LUCn and LUCc suspensions were mixed and infiltrated into N. benthamiana leaves. At 36 h after infiltration, the leaves were sprayed with 0.32-mg/mL D-Luciferin potassium salt in 0.1% (v/v) Triton X-100 and fluorescence was detected by a CCD camera (Uvitec Alliance Q9).

Co-IP assay

Co-IP assays were performed as previous description (Liu et al., 2010), with minor modification. Briefly, these constructs were transiently expressed in 4-week-old N. benthamiana leaves, respectively, with the agroinfiltration expression system. Proteins were extracted with the NP40 lysis buffer (P0013F; Beyotime Biotechnology, Jiangsu, China). Corresponding antibodies were added to the cell extracts, and protease inhibitor cocktail and MG132 were also added to prevent protein degradation. The mixtures were kept shaking gently at 4°C for 3 h. The immunocomplex was captured by adding 50-μL ml⁻¹ protein A/G agarose resin (Yeasen) and shaking for another 3 h. The agarose beads were recovered by centrifugation at 14,000g for 5 s and washed with cold PBS for three times. The precipitated samples were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and detected with anti-GFP antibody (Abmart, Shanghai, China; M20004) and anti-FLAG antibody (Abmart; M20008), respectively.

Kinase assay in vitro

In vitro kinase assays were performed according to the previous description (Qiao et al., 2017), with minor modification. Each kinase assay used 0.5 mg His-OsNAC016 or mutated fusion protein and 0.1-mg GST-GSK2. Kinase reaction buffer was composed of 25 mM Tris (pH 7.4), 12-mM MgCl₂, 1-mM DTT, and 1-mM ATP. The reactions were incubated at 37°C for 5 h and boiled with 3× SDS loading buffer, and then separated by SDS–PAGE with or without 50-mM Phos-tag. The signals were detected with anti-His antibody (Proteintech; 66005-1-Ig).

Kinase assays in E. coli were performed as previously described (Wang et al., 2020c). Briefly, the native or mutated His-OsNAC016 was co-expressed with GST-SAPK8 in BL21 (DE3), purified using the His60 Ni Superflow Resin (Clontech, Mountain View, CA, USA), and then separated by SDS–PAGE with or without 50 mM Phos-tag (APE×BIO). The signals were detected with anti-His antibody (Proteintech; 66005-1-Ig).

Plant hormone treatment

For in vivo lamina joint assays, the micro-drop method was performed as described previously (Hong et al., 2003). The lamina joints of the second leaf of 4-d-old seedlings were spotted with 1,000 ng of BL (24-epibrassinolide, an active analogs of BRs) in 1 μL ethanol by micropipette. The angles between the leaf lamina of the second leaf blade and sheath were measured after treatment for 3 d by analyzing digital images using ImageJ software.

Ubiquitination assay in vivo

Nicotiana benthamiana leaves transiently co-expressing OsNAC016-3×FLAG with OsPUB43-GFP or GFP alone for 2–3 d were collected, and 50 μM MG132 dissolved in 10-mM MgCl₂ is infiltrated into the previously infiltrated region 12 h before sample collection. Total proteins were extracted with NP40 lysis buffer and immunoprecipitated with anti-FLAG beads. The poly-ubiquitination of OsNAC016-FLAG was detected with anti-FLAG antibody (Abmart; M20008) and anti-Ub antibody (Santa Cruz Biotechnology, Dallas, TX, USA; sc-8017), respectively.

Cell-free protein degradation assay

The cell-free degradation assays were performed as previously described (Qiao et al., 2017). Two-week-old seedlings were used to extract protein in the extraction buffer (25-mM Tris–HCl, pH 7.5, 10-mM NaCl, 10-mM MgCl₂, 5-mM DTT, and 1-mM PMSF). The same amount of extracts was added to the tubes containing equal amounts of recombinant proteins and incubated at 30°C for different times, in the presence of 10-mM ATP.

ChIP-qPCR assay

Chromatin was isolated from 2g crosslinked leaves of 2-week-old OsNAC016-GFP transgenic plants. Isolated chromatin was sonicated for DNA fragmentation ranging from 200 to 500 bp. Subsequently, the DNA–protein complex was immunoprecipitated with anti-GFP antibody or IgG. Amounts of the DNA–protein complex that was immunoprecipitated were calculated relative to 10% of the total DNA–protein complexes before the immunoprecipitation experiment. The immunoprecipitated DNA fragments were detected by qPCR with gene-specific primers, respectively. Primers used were listed in Supplemental Table S4.

Transient transactivation assay

For transient transactivation assay, pGreenII cloning vectors were used (Hellens et al., 2005). The CDS of OsNAC016 was cloned into pGreenII 62-SK to function as the effector. For the reporter, the promoter region (−22 to −2,055 bp) of D2 was cloned into pGreenII 0800-LUC. These generated constructs were cotransformed into 4-week-old N. benthamiana leave with the agroinfiltration expression system described above. After incubation for 2–3 d, the activities of firefly LUC and renilla LUC were measured with a dual-LUC reporter assay kit (Promega, Madison, WI, USA; E1910). Primers used were listed in Supplemental Table S4.

Statistical analysis

Statistical differences between samples were analyzed by Student’s t test (*P < 0.05, ^**P < 0.01, or ^***P < 0.001). Statistical comparisons among samples were performed using two-way one way analysis of variance, followed by Bonferroni’s post-hoc test at the P < 0.05 level, and different letters with the same superscript mark indicate significant differences.

Accession numbers

Sequence data from this article can be found in the rice genome annotation project databases under the following accession numbers: OsNAC016 (LOC_Os01g01430); GKS2 (LOC_Os05g11730); SAPK4 (LOC_Os01g64970); SAPK8 (LOC_Os03g55600); SAPK9 (LOC_Os12g39630); SAPK10 (LOC_Os03g41460); OsPUB3 (LOC_Os01g60860); OsPUB16 (LOC_Os01g66130); OsPUB24 (LOC_Os03g45420); OsPUB28 (LOC_Os01g67500); OsPUB33 (LOC_Os02g33590); OsPUB43 (LOC_Os02g34410); and OsPUB75 (LOC_Os03g13010).

Supplemental data

The following materials are available in the online version of this article.

Supplemental Figure S1. Comparison of the growth and development between wild-type and osnac016 T-DNA insertion mutant.

Supplemental Figure S2. Spatio-temporal expression profile of OsNAC016 and OsPUB43.

Supplemental Figure S3. Morphological phenotypes and molecular identification of OsNAC016 knockout mutants generated by CRISPR/Cas9 system.

Supplemental Figure S4. Full scanning of OsNAC016 with different functional domains and their autoactivation assays in yeast.

Supplemental Figure S5. Sketches of phosphorylation motifs recognized by GSK3s and SnRK2s in OsNAC016.

Supplemental Figure S6. Performance of wild-type and osnac016 mutant under drought stress.

Supplemental Figure S7. Seed germination and root hair elongation in response to ABA in wild-type (DJ) and osnac016 mutant.

Supplemental Figure S8. Phosphorylated OsNAC016-GFP (OsNAC016-GFP-P) in OsNAC016-GFP plants.

Supplemental Figure S9. The interaction of OsNAC016 with multiple rice U-box/ARM proteins by an Y2H assay.

Supplemental Figure S10. The expression of OsNAC016 in the loss-of-function mutant osbzr1.

Supplemental Figure S11. The interaction analysis in a Y2H assay.

Supplemental Table S1. DEGs in osnac016 and OE2 from RNA-seq analysis.

Supplemental Table S2. DEGs for BR signaling in OE2 from RNA-seq analysis.

Supplemental Table S3. DEGs for ROS scavenging in osnac016 and OE2 from RNA-seq analysis.

Supplemental Table S4. Primers used in this study.

Supplemental Method S1. Propidium iodide (PI) staining.

Supplemental Method S2. Water loss rate measurement.

Supplemental Method S3. Phylogenetic Analysis.

Funding

This work was supported by National Natural Science Foundation of China (32071985 and 31771747), Natural Science Foundation of Chongqing, China (cstc2020jcyj-msxmX0656), and Fundamental Research Funds for the Central Universities, China (2020CDJ-LHZZ-034).

Conflict of interest statement. The authors declare no conflict of interest.

 

H.J. conceived and designed the research. W.Q. performed plant growth and responses to abiotic stress, BiFC, Co-IP, phosphorylation, ubiquitination, and ChIP assays. L.Y and X.Z. performed protein purification and pull-down analysis. Y. B. performed stomatal opening experiment. S.Y. helped to construct vectors. W.Q. wrote the manuscript, and H.J. revised it.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/general-instructions) is Junli Huang (huangjunli@cqu.edu.cn).