Serine protease NAL1 exerts pleiotropic functions through degradation of TOPLESS-related corepressor in rice

doi:10.21203/rs.3.rs-1892822/v1

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Serine protease NAL1 exerts pleiotropic functions through degradation of TOPLESS-related corepressor in rice

https://doi.org/10.21203/rs.3.rs-1892822/v1

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published 22 Jun, 2023

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NAL1 (NARROW LEAF 1) is a breeding-valuable pleiotropic gene that affects multiple agronomic traits in rice, but the molecular mechanism is largely unclear. Here, we report that NAL1 is a serine protease and displays a novel hexameric structure whose formation is mediated by ATP-containing positively charged pocket at the N-terminal region. Moreover, we identified TOPLESS-related corepressor OsTPR2 involved in multiple growth and development processes as the substrate of NAL1. We found that NAL1 degraded OsTPR2, thus modulating the expression of downstream genes related to hormone signaling pathways, eventually achieving its pleiotropic physiological function. An elite allele NAL1^A originated from wild rice could increase grain yield. Furthermore, the NAL1 homologs in different crops have a similar pleiotropic function to NAL1. Our study uncovers a NAL1-OsTPR2 regulatory module and provides gene resources for the design of high-yield crops.

Rice is a staple food for more than half of the world’s population, but global food security still faces great challenges in the future due to the continuous increase in population and decrease in arable land¹. Crop yield is a genetically complex trait consisting of numerous traits, and pleiotropy is defined as one gene influencing multiple traits. For example, IPA1 (Ideal Plant Architecture 1), encoding a SPL (SQUAMOSA promoter-binding protein-like) transcription factor, regulates different downstream genes, thereby enhancing effective tiller, panicle branching, and disease resistance, ultimately improving rice yield². Ghd7 (Grain number, plant height and heading date 7), encoding a CCT-domain transcription factor, increases grain number, plant height and nitrogen use efficiency by repressing the expression of OsTB1, ARE1, and other genes involved in the floral transition and lateral branch development^3–5. These pleiotropic genes have been widely used in modern rice breeding⁶. Therefore, the employment of pleiotropic genes simultaneously enhancing multiple agronomic traits is an effective approach in crop breeding.

NAL1 is a pleiotropic gene identified by multiple genetic analyses in rice. Significant loci containing NAL1 have been identified by analyzing different traits (grain yield, plant architecture, leaf morphology, root architecture, photosynthesis rate, chlorophyll content) via genome-wide association study (GWAS) in different natural populations^7–11. A major quantitative trait loci (QTL) allelic to NAL1 has been detected in different recombinant inbred lines or near-isogenic lines related to flag leaf traits, grain yield, chlorophyll content, and source leaf/sink capacity traits^12–17. A major natural variation associated with the pleiotropic function has been identified to be an A-to-G substitution in the coding region of NAL1, resulting in the conversion of amino acid His233 into Arg233 (H233R), and the biological function of NAL1 in controlling the pleiotropic traits has been confirmed using transgenic or introgression lines^7,15,18. NAL1 is expressed in all organs such as roots, leaf sheaths, leaves, culms, and young panicles. NAL1 protein contains a nuclear localization signal in its C-terminus, and it has been found to be concentrated in nuclear protein bodies¹⁹. The studies of different nal1 mutants have revealed that NAL1 affects cell division and expansion, thus influencing leaf width and adventitious root development. Furthermore, in nal1 mutants, the expression levels of genes involved in auxin transport and signaling are changed greatly^19–22. Recently, FZP (FRIZZY PANICLE) has been reported as a target of NAL1 to regulate secondary branching of inflorescences and grain yield in rice²³. In barley, a putative cyclophilin-type HvPPIase has been identified to interact with HvNAL1 to regulate tiller number and leaf morphology²⁴. Nevertheless, the molecular mechanism by which NAL1 regulates the multiple traits remains largely unknown.

The TPL/TPR (TOPLESS/TOPLESS-related) family proteins, belonging to Groucho/Tup1-type corepressors, are involved in hormone signaling pathways and other transcriptional regulation processes, thus affecting multiple traits such as plant height, leaf architecture, tiller number, and spike development²⁵. Different transcriptional repressors/adaptor proteins and histone deacetylase (HDACs) bind TPL/TPR family proteins respectively through LisH domain in the N terminus and the WD40-repeat domain in the C terminus to form complexes, and these resulting complexes decrease histone acetylation levels of downstream genes and repress their expression^26–29. TPL/TPR family proteins are pivotal for plant development, and thus the fine-tuned regulation of protein activity and stability is important for the exertion of their suppressive function³⁰. A recent report on Arabidopsis has shown that SUMOylation of TPR1 represses its activity and inhibits the constitutive activation of immune responses under non-pathogenic conditions³¹. However, how TPL/TPR protein levels are regulated to relieve the repression of downstream gene expression remains unclear.

In this study, we biochemically reconstituted NAL1 in vitro and solved its crystal structure. We found that NAL1 displayed a hexameric structure, and that ATP molecule played a key role in its oligomeric assembly. Structure comparison and biochemical assays suggested that NAL1 was a new plant-specific serine protease. We also found that NAL1 interacted with OsTPR2 (a TPL/TPR member in rice) via its C-terminal EAR-like motif to promote OsTPR2 degradation. OsTPR2 degradation by NAL1 increased the histone acetylation levels of the genes in auxin and strigolactone (SL) signaling pathways as well as development-related genes, thus up-regulating the expression of these genes. In addition, we found that an elite allele NAL1^A with a variation at the catalytic triad of the protease, originated from common wild rice (Oryza. rufipogon), was selected during rice breeding.

Crystal structure determination of NAL1 hexamer

In the past decade, several studies have demonstrated the pleiotropic roles of NAL1 in agronomic traits, and it has been identified as a valuable gene in rice breeding^9,12,15. In order to unveil NAL1 pleiotropic mechanism, we performed protein structure analysis since sequence alignment of NAL1 against known proteins could not predict a reliable biochemical function. The crystal structure of NAL1 (residues 59-463) was determined at a resolution of 2.6 Å (Fig. 1a, Extended Data Fig. 1a and Extended Data Table 1). Electron density of residues 78-84, 175-183, and 407-412 was undetectable, which might be due to their flexibility in solution. NAL1 was trimeric in one unit cell (Extended Data Fig. 1b), and NAL1 hexamer was assembled from two symmetric neighboring unit cells (Fig. 1a). The protomers of NAL1 shared almost identical architectures without apparent structural variation (Extended Data Fig. 1c). To further validate the oligomeric state of NAL1, analytical ultracentrifugation (AUC) experiments were performed. The results showed that the full-length NAL1 was mainly in a hexameric state, which was consistent with the crystallographic analysis (Extended Data Fig. 1d).

Dali (a structure comparison server) search revealed that NAL1 was a serine protease according to the structural similarity (Extended Data Fig. 1f, and Extended Data Table 2). Thus, each NAL1 protomer was divided into two segments: N-terminal domain (NTD, residues 59-197) and C-terminal serine protease domain, consisting of 10 α-helices and 22 β-sheets (Extended Data Fig. 1a, e). Within the NTD, an ATP molecule was found in a pocket surrounded by a3, a4, b4, and a loop (Fig. 1b, Extended Data Fig. 2a), and this pocket was stabilized by a positively charged network consisting of residues R120, R122, K123, K139, and K142 (Fig. 1c). This ATP molecule expelled a3 from one protomer to another, thus leading to a cation-p interaction between W144 and H248 from the neighboring protomers, eventually mediating the formation of a trimer. Our results showed that the oligomerization was abolished by W144A point mutation, further validating role of the cation-p interaction in oligomerization (Extended Data Fig. 2b). Further, we investigated the effect of the residues surrounding ATP on NAL1 oligomerization. The mutation of either R120 or R139 into alanine disrupted the oligomerization (Extended Data Fig. 2b), suggesting that ATP could mediate the oligomerization of NAL1.

The C-terminal serine protease domain of NAL1 consisted of two perpendicular six-stranded β-barrel subdomains, and the putative catalytic triad was composed of H233, D291, and S385, which were located in the crevice between the two lobes (Fig. 1d, Extended Data Fig. 2c). We noticed that the natural single nucleotide variation (A-to-G substitution) of NAL1 occurred exactly in the catalytic triad, resulting in the conversion of His233 to Arg233 (H233R), which was found to be associated with the pleiotropic traits^11,17. In this study, we designated the two alleles of NAL1 as NAL1^A (H233) and NAL1^G (R233), respectively. To genetically examine the functional differences between the alleles, we constructed two complementary lines NAL1^A-COM and NAL1^G-COM in the NAL1-knockout mutant (nal1-cri) background generated by CRISPR-Cas9 technique. Both complementary lines partially recovered phenotypes of nal1-cri, but the NAL1^A-COM line showed significantly stronger phenotypes than the NAL1^G-COM line, including wider flag leaf, higher plant height, larger root, larger panicle, and higher grain yield (Fig. 1e-j). These results suggested that the NAL1^A was a genetically stronger allele, and that the H233 in the catalytic triad might be a critical residue for NAL1 functions.

NAL1 physically interacts with OsTPR2

To unveil the underlying mechanism of NAL1’s pleiotropic functions, immunoprecipitation in combination with mass spectrometry (IP-MS) was used to identify NAL1-interacting proteins. Of the detected 31 proteins (Extended Data Table 3), 3 TPR family proteins (OsTPR1/2/3) were selected for further research since they regulate multiple growth and development processes^28,32.

To verify the interaction between NAL1 and OsTPR family proteins, we performed luciferase complementation imaging (LCI) assays in Nicotiana benthamiana leaves. The previously reported NAL1 mutant (nal1-1) with 10-AA deletion in the serine protease domain¹⁹ was used as a negative control. We found that NAL1, rather than nal1-1, interacted with all the three rice TPR proteins (Extended Data Fig. 3a). OsTPR2 was selected for further investigation since this gene has been reported to be involved in regulating multiple developmental and agronomic traits^33-36. Bimolecular fluorescence complementation (BiFC) assays showed that YFP fluorescence signal was detected in the cells co-expressing OsTPR2-nYFP and NAL1-cYFP, but not in the cells co-expressing OsTPR2-nYFP and nal1-1-cYFP (Fig. 2a). In addition, NAL1-GFP was co-immunoprecipitated with OsTPR2-Flag when they co-expressed in tobacco leaves (Fig. 2b). All above results suggested the interaction between NAL1 and OsTPR2.

OsTPR2 was divided into N-terminal TPD domain and C-terminal WD40 domain according to previous reports^32,37. LCI assay was performed to further determine which domain of OsTPR2 interacted with NAL1. The results showed that TPD, rather than the WD40 domain, interacted with NAL1 (Fig. 2c), implying that N-terminal TPD domain mediated the interaction between NAL1 and OsTPR2. Previous reports show that TPD interacts with proteins via an EAR-motif (LXLXL)^38-41. We detected two EAR-like motifs (EAR-1: 558-MSLHLGD-564; EAR-2: 576-SSLDLEK-582) in the C-terminus of NAL1 (Fig. 2d, Extended Data Fig. 3b). To further determine whether these two EAR-like motifs mediated the interaction between NAL1 and OsTPR2, a truncated NAL1 with the two EAR-like motifs deleted (residues 1-559) was constructed. Gel filtration assays showed that the deletion of two EAR-like motifs completely abolished the interactions (Extended Data Fig. 3c). Point mutations at the EAR-1 motif disrupted their interaction (Fig. 2d), whereas those at the EAR-2 motif did not (Extended Data Fig. 3c), suggesting the key role of EAR-1 in the interaction between NAL1 and OsTPR2. To further decipher the interaction mechanism between NAL1^EAR-1 and OsTPR2, we modeled a OsTPR2-NAL1^EAR-1complex structure, in which L560 and L562 of NAL1^EAR-1exhibited a hydrophobic interaction with F74, F104, L118, L130 of OsTPR2 (Extended Data Fig. 3d, e). Taken together, NAL1 C-terminal EAR-like motif and OsTPR2 N-terminal TPD domain jointly mediated the interaction between NAL1 and OsTPR2.

NAL1 promotes degradation of OsTPR2

To test whether NAL1, a structurally distinct serine protease, is capable of degrading OsTPR2, in vivo protein degradation assays were performed using rice protoplasts. The results showed that OsTPR2 was hardly degraded in nal1-cri protoplasts, whereas ~50% OsTPR2 was degraded within 5 h in the wild type (ZH11). As a negative control, GFP protein exhibited no change in nal1-cri and ZH11 protoplasts (Fig. 2e, Extended Data Fig. 4a). Moreover, the same assays were performed in the nal1-1 mutant and its corresponding wild type (rice cultivar ZF802)¹⁹, and similar results were observed (Extended Data Fig. 4b). Overall, our results showed that NAL1 could facilitate the degradation of OsTPR2 in vivo.

Subsequently, recombinant 6×His-tagged OsTPR2 was used as substrate to investigate its degradation by NAL1 in cell-free extracts from nal1-cri and wild type ZH11 plants. Compared to ZH11, the degradation of OsTPR2 was delayed in the nal1-cri extract (Fig.2f, Extended Data Fig. 4c). Similar results were observed in the assays of cell-free extracts of nal1-1 mutant and wild type ZF802 plants (Extended Data Fig. 4d). These results further suggested that NAL1 could promote OsTPR2 degradation. To further confirm whether NAL1 functions biochemically as a serine protease, different protease inhibitors were added into ZH11 cell-free extracts to examine the degradation of OsTPR2. The results showed that all the 3 serine protease inhibitors phenylmethanesulfonyl fluoride (PMSF), aprotinin, and leupeptin could almost abolish OsTPR2 degradation. In contrast, the metalloprotease inhibitor bestatin, the aspartic protease inhibitor pepstain, or the cysteine protease inhibitor E-64 could not inhibit OsTPR2 degradation (Fig. 2g, h). These results strongly suggested that NAL1 was a bona fide serine protease.

To examine the importance of the catalytic triad to the proteolytic activity of NAL1, wild type NAL1 and the triad-mutated NAL1 (mNAL1) were incubated with OsTPR2 in vitro, respectively. The results showed that wild type NAL1 degraded ~50% OsTPR2 within 1 h, whereas mNAL1 exhibited highly compromised degradation activity (Fig. 2i, Extended Data Fig. 4e). To further reveal the function of the catalytic triad of NAL1 in plants, we examined the proteolytic activity in the previously reported near isogenic lines NIL-IR and NIL-ZS containing the alleles NAL1^A and NAL1^G, respectively¹⁷. In vivo and cell-free protein degradation assays both showed that NAL1 protein from NAL1^A allele had stronger proteolytic activity than that from NAL1^G (Fig. 2j, k). In addition, the OsTPR2 abundance quantified by the anti-OsTPR2 antibody was significantly higher in nal1-cri than in ZH11 (Extended Data Fig. 4f), while the OsTPR2 mRNA level showed no difference between nal1-cri and ZH11 (Extended Data Fig. 4g). These results suggested the importance of the catalytic triad to the proteolytic activity of NAL1.

To clarify the genetic interaction between NAL1 and OsTPR2, we constructed OsTPR2 RNA interference (RNAi) lines in the nal1-cri background. The expression level of OsTPR2 in three positive OsTPR2-RNAi/nal1-cri lines was decreased to approximately half of that in nal1-cri (Extended Data Fig. 5a). Compared to those in nal1-cri, plant height, flag leaf width, root dry weight, and grain yield were significantly increased in the three OsTPR2-RNAi/nal1-cri lines (Fig. 3a-f). These results suggested that the pleiotropic function of NAL1 genetically depended on OsTPR2, which was consistent with the biochemical relationship between NAL1 and OsTPR2.

NAL1 exerts pleiotropic function by alleviating OsTPR2-mediated hormone signaling pathway suppression

TPL/TPR family proteins have been reported to regulate hormone signaling pathways by forming complexes with repressors and transcription factors, thus affecting histone acetylation levels of downstream target genes in Arabidopsis^{26-28,32,34,38,39}. To determine whether the rice homologs share the same mechanism, we investigated the potential complex formation from OsIAA3/17 (identified by a yeast-two-hybrid screening of OsTPR2), OsARF25 (OsIAA3/17-interacting transcription factor in auxin signaling pathway)^42,43, and OsTPR2 by in vitro pull-down assays. Meanwhile, the repressor D53³⁴ and its corresponding transcription factor IPA1⁴⁴ in SL signaling pathway were selected for examining their interactions with OsTPR2. The results showed that GST-tagged OsARF25 (or IPA1) was co-eluted with 6×His-tagged OsIAA3/17 (or D53) and StrepII-tagged OsTPR2, but not with StrepII-tagged OsTPR2 alone (Fig. 4a-c), suggesting that OsTPR2 might participate in the regulation of hormone signaling by forming a TF-adaptor-TPR complex like their homologs in Arabidopsis. Since NAL1 promoted OsTPR2 degradation, we speculated that NAL1 might affect histone acetylation, thus alleviating the OsTPR2-mediated suppression of genes in the hormone signaling pathways. To test the speculation, the histone acetylation levels in ZH11 and nal1-cri were examined. H3Ac, H3K9Ac, and H3K18Ac levels were lower in nal1-cri than in ZH11, with H3K18Ac exhibiting the most significant difference (Fig. 4d). Furthermore, a total of 13277 genes were found to have a lower H3K18Ac level in nal1-cri than in ZH11 by ChIP-seq analysis. Based on the published RNA-seq data of nal1 mutant and its wild type²⁰, 902 genes whose expressions were down-regulated in the nal1 mutant²⁰ also showed decreased H3K18Ac levels (Extended Data Fig. 5b, and Extended Data Table 4), further supporting that NAL1 fulfilled its pleiotropic functions by alleviating the OsTPR2-mediated gene expression suppression.

To further clarify how the NAL1-OsTPR2 module regulated gene expression, we investigated OsARF25-targeted genes (PIN1b, ERF3 and CKX4) in auxin signaling pathway and IPA1-targeted genes (SLR1 and DEP1) and CKX9 (affected by D53, but not the target gene of IPA1) in SL signaling pathway. The genome browser data showed that H3K18Ac level of these genes was significantly lower in nal1-cri than in ZH11, which was confirmed by H3K18Ac-ChIP qPCR assays (Fig. 4e, f, Extended Data Fig. 4c, d). We examined the effects of NAL1 on the expression of these genes. As expected, these genes showed lower expression in nal1-cri than in ZH11 (Fig. 4g). In the double mutant OsTPR2-RNAi/nal1-cri lines, down-regulating OsTPR2 partially alleviated the expression suppression of above-mentioned genes, compared to nal1-cri (Fig. 3g). These results jointly implied that NAL1 positively modulated histone acetylation and gene expression by degrading the co-repressor OsTPR2.

NAL1 and its homolog genes in other crops are highly breeding-valuable

In previous studies, the natural variation of NAL1, mainly two haplotypes corresponding to the alleles NAL1^A and NAL1^G, is genetically detected in cultivated Asia rice varieties^10,11,15. The difference of their protease activity (Fig. 1, 2) was in accordance with their biological function in vivo. It has been reported that the stronger allele NAL1^A is derived from the weaker allele NAL1^G in the ancestral population and has undergone artificial selection^8,18,45, but its detailed origin route is still unknown. Therefore, we analyzed whole-genome sequencing data of a large panel of accessions including 3243 O. sativa varieties and 460 O. rufipogon accessions^46,47. NAL1^A exists in 71.39% of japonica rice varieties, but only in 1.67% of indica rice varieties, indicating that the NAL1^A might be population-specific. Indica and japonica have been reported to be descended from common wild rice Or-I and Or-III, respectively⁴⁶. We noticed that NAL1^Aexisted only in 2 Or-III accessions, but NAL1^Gexisted in all the Or-I accessions (Fig. 5a, Extended Data Fig. 6a), implying that NAL1^A in japonica rice was originated from Or-III wild rice. Single-nucleotide diversity analysis showed that the diversity of japanica was almost 20 folds higher that of Or-III, but indica did not significantly differ from Or-I (Extended Data Fig. 6b), indicating that NAL1^A might undergo artificial selection during japanica rice domestication, which is consistent with previous report⁴⁵.

Since almost all indica rice varieties contain the weak allele NAL1^G, we wondered whether the functionally strong allele NAL1^A has potential in indica rice breeding. To this end, we introduced the NAL1^Aallele into Huang-hua-zhan (HHZ), an elite indica variety widely cultivated in China, to replace its NAL1^Gallele. Morphological traits including plant height, flag leaf width, root and panicle size were significantly improved in the near-isogenic line HHZ-NAL1^A, compared to those in HHZ. As a result, grain yield per plant was significantly increased (Fig. 5b-g), indicating promising application value of the NAL1^Aallele in indica rice breeding.

Since NAL1 is a breeding-valuable pleiotropic gene in rice, we further examined the conservativeness of the function of its homologs in other crops. A phylogenic tree of NAL1 and its homolog proteins from wheat, maize, soybean, and rapeseed showed that all the four crops had homologs of NAL1 (Extended Data Fig. 7a). One homolog from each crop was selected to complement nal1-cri. Compared to nal1-cri, the complementary lines exhibited significant recovery in plant height, flag leaf morphology, root system, and grain yield (Extended Data Fig. 7b-g), indicating that the NAL1 homologs from main crops may have conserved functions.

Although numerous genetic studies of NAL1 in rice have been reported, the molecular mechanism by which NAL1 regulates plant growth and development remains unknown. Here, we revealed the crystal structure and biochemical function of NAL1, providing direct evidence that NAL1 was a bona fide serine protease (Fig. 1, 2). The protein domain organization of NAL1 is reminiscent of the well-known high temperature requirement A (HtrA) serine proteases which generally consist of one protease domain and one or two PDZ domains, and the protease domain of HtrA is critical for its cage-like oligomerization (Extended Data Fig. 1g)⁴⁸. Different from HtrA family proteases, NAL1 forms a stacked trimeric ring under mediation of both protease domain and the NTD in which ATP molecule is found to be located in a positively charged pocket (Fig. 1, Extended Data Fig. 2). This special oligomeric architecture suggested that NAL1 was a serine protease with a new hexameric structure. ATP is required for NAL1 oligomeric assembly, suggesting a possible correlation between oligomerization and the plant energetic states, but it still needs further investigation.

Since NAL1 is a serine protease, the identification of its potential substrate is important for understanding its pleiotropic mechanism. However, identification of protease substrates is challenging mainly because of the momentary nature of the interaction and instability of the substrates⁴⁹. Using IP-MS, we identified several NAL1-interacting proteins including OsTPR family members from NAL1-Flag overexpressing lines. In addition, we found that the interaction between NAL1 and OsTPR2 was mediated by both NAL1 C-terminal EAR-like motif and OsTPR2 N-terminal TPD domain (Fig. 2, Extended Data Fig. 3), and this binding mode is consistent with previous reports^40,41,50. TPL/TPR family proteins play important roles in numerous developmental processes, and they can form transcriptional repression complexes to modulate multiple hormone signaling pathways²⁵. Therefore, controlling TPL/TPR protein homeostasis is important for plant growth. The quality control of repressors involved in plant hormone signaling is mostly dependent on ubiquitination degradation system⁵¹. Our in vivo and cell-free protein degradation assays showed that OsTPR2 was proteolytically degraded by NAL1 (Fig. 2, Extended Data Fig. 4), which was distinct from the ubiquitination degradation. This finding extends our knowledge of protein homeostasis regulation of important repressors.

In Arabidopsis, the canonical TF-adaptor-TPL/TPR regulatory modules function in auxin, jasmonic acid (JA), SL signaling pathways²⁵. The canonical ARF-IAA-TPL/TPR complex in auxin signaling and MYC2-JAZ/NINJA-TPL/TPR complex in JA signaling have been intensively investigated for their roles in regulating plant growth, development, and stress responses^28,39,52,53. In SL signaling, TPL/TPR has been reported to interact the adaptor protein SMXL and unknown TFs to regulate SL-responsive genes^34,54. In this study, the canonical regulatory modules OsARF25-IAA3/17-OsTPR2 and IPA1-D53-OsTPR2 in rice were identified (Fig. 4), implying that TF-adaptor-TPL/TPR might be a conserved regulation module in both monocot and dicot plants.

The NAL1-OsTPR2 module regulates the expression of auxin and SL signaling genes such as PIN1b, ERF3, CKX4, SLR1, DEP1, and CKX9 (Fig. 4). PIN1b is involved in the development of adventitious root, and it is a downstream gene of OsARF25 and IPA1^44,55. In this study, we found that PIN1b histone acetylation was regulated by OsARF25-IAA3/17-OsTPR2 and/or IPA1-D53-OsTPR2 modules (Fig. 4), providing insight into how PIN1b is involved in the crosstalk between auxin and SL signaling. CKX4 and CKX9 play a pivotal role in crown root formation and shoot architecture^43,56,57. ERF3 promotes crown root elongation^42,58. SLR1 and DEP1 have been reported to be involved in rice shoot and panicle development⁴⁴. In addition to the genes involved in hormone signaling, we also examined the expression of ACL1, a downstream gene of URL1-OsTPR2, involved in leaf development³⁵. Our results showed that the expression of ACL1 was decreased in nal1-cri, but this decrease was alleviated in OsTPR2-RNAi/nal1-cri lines (Extended Data Fig. 8), which further supported the phenotypes of OsTPR2-RNAi/nal1-cri lines that were partially recovered compared to nal1-cri (Fig. 3). Knockout of OsTPR2 resulted in severe developmental defects and even infertility, which deprived us of exploring their genetic interaction by OsTPR2 knockout. In addition, other NAL1-interacting proteins or substrates including the paralogs of OsTPR2 (OsTRP1/3) (Extended Data Table 3) might also partially contribute to the pleiotropic phenotypes. For example, FZP regulating the secondary panicle branches could be degraded by NAL1²³, but this protein was not identified in our IP-MS experiment. Therefore, we cannot rule out the possibility that there might exist other NAL1 substrates, and further investigation of these potential substrates is likely to provide insight into NAL1’s pleiotropic function. Taken together, we propose a simplified working model for the NAL1-OsTPR2 module. NAL1, as a plant-specific serine protease, can directly degrade the co-repressor OsTPR2, thereby resulting in the increase of histone acetylation level and expression levels of downstream genes related to hormone signaling pathways (such as auxin and SL examined in this study), ultimately modulating plant growth and development and improving multiple agronomically important traits such as plant, leaf, and root architecture, and grain yield (Fig. 6).

The natural variation (NAL1^A to NAL1^G) was found to change one of the catalytic triad (H233 to R233), and our genetic complementation assays indicated the importance of H233 for NAL1 proteolytic activity (Fig. 1). This might be the main reason why NAL1^A/NAL1^G alleles were identified by GWAS of different traits^7–11. In addition, both in vivo and in vitro protein degradation assays showed that NAL1 protein encoded by NAL1^A allele had higher proteolytic activity than that encoded by NAL1^G (Fig. 2, Extended Data Fig. 4). NAL1^A allele originated from wild rice might undergo artificial selection in japonica rice cultivars, but not in indica. Interestingly, NAL1^A exhibited a potential in promoting agriculture traits as evidenced by introgression lines (Fig. 5)^12,15. In addition, NAL1^A can also increase nitrogen use efficiency in indica rice background⁴⁵. However, a recent study has revealed that the weak allele NAL1^G is conducive to the maintenance of the balance between leaf photosynthesis and plant architecture for increasing biomass and grain yield under low planting density in indica rice variety 9311⁵⁹. Compared to japonica, indica rice cultivar has been conventionally planted with lower plant density. Therefore, we speculate that NAL1^A may have been selected during japonica rice domestication because of its stronger pleiotropic function leading to a relatively more compact architecture, thus allowing high-density planting. Nevertheless, the detailed origin and selection mechanism of NAL1^A deserves further studies.

NAL1 is a plant-specific serine protease and its homologs are highly conserved. Our genetic complementation experiments indicated that NAL1 genes had conservative function in main crops (Extended Data Fig. 7). Recently, NAL1 homologs in barley and sugarcane have also been reported to affect leaf width^24,60. However, there are no reports that NAL1 homologs affect agronomic traits in main crops except rice. Interestingly, protein sequence analysis showed that the amino acid residues corresponding to H233 (or R233) of NAL1 were all arginine in the other main crops (Extended Data Fig. 9). In addition, we found no natural variation altering this amino acid residue based on sequencing databases from other species. It will be interesting to explore why the H233R variation occurs only in rice and whether there are potential important variations at NAL1 other sites in the plants. Our data showed that the grain yield of NAL1^A complementary line was significantly higher than that of NAL1^G and NAL1-homologs complementary lines (Fig. 1, Extended Data Fig. 7), indicating that the function of NAL1^A was not only stronger than that of NAL1^G but also than that of the NAL1 homologs from other main crops. Thus, we propose that the functionally stronger allele of NAL1 may be used to improve agronomic traits of other crops by gene editing technology. In summary, this study reveals the molecular mechanisms by which NAL1-OsTPR2 module regulates rice growth and development and identifies the key residues affecting NAL1 proteolytic activity, and these findings are valuable for crop breeding design.

Materials

To construct NAL1-knockout line by CRISPR/Cas9 technology, the 20 bp specific sgRNA target sequence of NAL1 was designed and cloned into the TKC vector driven by OsU6 promoter ⁶¹, and then the resultant vector was transformed into rice calli of wild type Zhonghua11 (ZH11, O. sativa ssp japonica) through Agrobacteria-mediated transformation. The homozygous mutants, named nal1-cri, were identified by sequencing and PCR. Primers used were shown in Extended Data Table 5.

To construct the complement lines in nal1-cri background, NAL1 homologs from maize, wheat, soybean and rapeseed, NAL1^A, and NAL1^G were cloned into pCAMBIA2300 vector and these genes were driven by NAL1 native promoter from ZH11. The resultant vectors were transformed into rice calli of homozygous nal1-cri through Agrobacteria-mediated transformation. The positive transgenic lines were identified by PCR. Primers used in the PCR were shown in Extended Data Table 5.

To generate NAL1^Aintrogression line, we crossed indica cultivar Huang-Hua-Zhan (HHZ) and japonica rice W118. Progenies containing the NAL1^A allele (from W118) were backcrossed to HHZ for four generations and then self-crossed for three generations. Plants homozygous for NAL1^A were selected from BC₄F₃ population and identified the phenotype in pot and field experiments.

Protein expression and purification

NAL1 was cloned into a modified pET15D vector and expressed in E. coli as 6×His-fusion. The construct was transformed into E. coli BL21 (DE3) and the cell cultures were grown at 37°C to OD600 of 1.0 - 1.2. Isopropyl-β-D-thiogalactoside of 0.2 mM was added to induce protein expression at 16°C for 12-16 h. The E. coli cells were harvested, homogenized in buffer A (25 mM Tris–HCl, pH 8.0, 150 mM NaCl), and lysed using a high-pressure cell disrupter (JNBIO, China). The cell lysates were centrifuged at 14,000 rpm for 1 h at 4°C. The supernatant containing soluble proteins was loaded onto a column equipped with Ni²⁺ affinity resin (Ni-NTA), washed with buffer B (25 mM Tris–HCl, pH 8.0, 150 mM NaCl, 15 mM imidazole), and eluted with buffer C (25 mM Tris–HCl, pH 8.0, 150 mM NaCl, 250 mM imidazole). The 6×His tag was removed by DrICE digestion. Then the digested protein was separated by cation exchange chromatography (Source 15Q, GE Healthcare) using buffer A with a linear NaCl gradient. The purified protein was concentrated and subjected to gel filtration chromatography (Superose 6, GE Healthcare) in a buffer containing 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM dithiothreitol. Proteins during all purification processes were subjected to SDS-PAGE and visualized by Coomassie blue staining. The peak fractions were collected and stored at -80°C. NAL1 boundaries and mutants, as well as the seleno-methionine labeled proteins were purified similarly as the wild-type.

Crystallization, data collection, and structure determination

Protein was concentrated to 10 mg ml^-1before crystallization trials. Crystallizations were performed using the sitting-drop vapor diffusion method at 18°C by mixing an equal volume (1 μL) of protein with reservoir solution. Since our initial attempts to crystallize the full length NAL1 (residues 1–582) were unsuccessful, a series of truncations and cysteine mutations were performed. After several rounds of optimization, the crystal of a NAL1 construct (Q59-A463, C61S/C193S/C198S/C326S/C337S/C364S) appeared overnight and grew to full size within 3 days in the well buffer containing 0.1 M MES, pH 6.2, 0.1 M calcium acetateand 4% PEG6000. The crystals were flash-frozen in liquid nitrogen and cryoprotected by adding glycerol to a final concentration of 10%-20%.

The diffraction data were all collected at Shanghai Synchrotron Research Facility (SSRF) on beamline BL17U or BL19U. The data were integrated and processed with the HKL2000 program suite and XDS packages ⁶². Further data processing was carried out using CCP4 suit ⁶³. The crystal structure of NAL1 was solved via single-wavelength anomalous diffraction (SAD) method at a resolution of 2.6 Å. The structure was iteratively built with COOT ⁶⁴ and refined with PHENIX program ⁶⁵. Data collection and structure refinement statistic were summarized in Extended Data Table 1. All figures were generated using the program PyMOL (http://www.pymol.org/).

Analytical ultracentrifugation (AUC) analysis

The stoichiometry of NAL1 oligomer was investigated by AUC experiments, which were performed in a Beckman Coulter XL-I analytical ultracentrifuge using two-channel centrepieces. NAL1 was in solutions containing 25 mM Tris-HCl, pH 8.0, 150 mM NaCl. Data were collected by absorbance detection at 18°C for proteins at a concentration of approximately 0.6 mg mL^-1 at a rotor speed of 35,000 rpm. The SV-AUC data were globally analyzed using the SEDFIT program and fitted to a continuous c(s) distribution model to determine the molecular mass.

IP-MS

Immunoprecipitation in combination with mass spectrometry (IP-MS) was performed to identify NAL1-interacting proteins. Total protein was extracted from the seedlings of NAL1-overexpression transgenic plants ⁷ and wild type ZH11. The extracted total protein was precipitated by anti-Flag magnetic agarose (A36797, Thermo Scientific) and washed 5 times with a PBS buffer (3 mM Na₂HPO₄, 155 mM NaCl, 1 mM KH₂PO₄, pH 7.4, 1×complete protease inhibitor cocktail). The beads were added to a loading buffer and boiled for 10 min, then were subjected to SDS-PAGE. After in-gel digestion, the samples were used for mass spectrometry (Thermo Scientific, nano LC-Q EXACTIVE). Information about interacting proteins of NAL1 by IP-MS was shown in Extended Data Table 3.

BiFC and LCI assays

NAL1 and 10-AA deleted mutant nal1-1 were respectively cloned into the C-terminal YFP fusion vector pSPYCE(R) and OsTPR2 was cloned into N-terminal YFP fusion vector pSPYNE(R)173. The constructs were transformed into Agrobacteria GV3101. Different combinations as indicated were transiently expressed in tobacco leaves. After 48 h, the fluorescence was captured using a confocal microscope (LSM980, ZEISS).

NAL1 and 10-AA deleted mutant nal1-1 were respectively cloned into the pCAMBIA-1300-cLuc and the full-length and truncations of OsTPR2 were cloned into the pCAMBIA-1300-nLuc vectors. The constructs were transformed into Agrobacteria GV3101. Different combinations as indicated were transiently expressed in tobacco leaves. After 48 h, 1 mM luciferin was applied to the injected regions of the tobacco leaves. The Luc fluorescence signal was observed with CCD-imaging instrument (Tanon Science and Technology). Relative primer sequences are shown in Extended Data Table 5.

Co-immunoprecipitation

NAL1 and the 10-AA deleted mutant nal1-1 were cloned into the GFP fusion vector HGF, and OsTPR2 was cloned into the Flag fusion vector pCAMBIA-1300-Flag. Different combinations as indicated were transiently expressed in tobacco leaves. After 48 h, total protein from the infiltrated tobacco leaves was independently extracted in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, 1 mM PMSF and 1×complete protease inhibitor cocktail). Each protein extract was incubated with anti-Flag magnetic agarose (A36797, Thermo Scientific) for 4 h at 4°C, and washed 5 times with lysis buffer. The beads were added to loading buffer and boiled for 10 min, then samples were detected by western blot with anti-Flag and anti-GFP antibody.

Size exclusion chromatography

To further map the crucial residues that mediate the interactions between NAL1 and OsTPR2, a series of truncations or mutants were constructed and purified to homogeneity. Proteins as indicated were mixed, incubated for 1 h at room temperature, and further subjected to gel filtration chromatography (Superose 6, GE Healthcare) in a buffer containing 25 mM Tris, pH 8.0, 150 mM NaCl, 5 mM dithiothreitol. The relevant fractions of each independent injection were collected for SDS-PAGE analysis.

Cell-free protein degradation assays

The cell-free protein degradation assay was performed as described previously ²³. Total protein was extracted from the seedlings of ZH11 and nal1-cri, ZF802 and nal1-1, or NIL-IR and NIL-ZS with degradation buffer (25 mM Tris-HCl, pH 7.5, 10 mM NaCl, 10 mM MgCl₂, and 10 mM ATP). After two 10 min centrifugations at 12,000 rpm at 4°C, the supernatant was collected and the protein concentration was determined by A280 using NanoDrop 2000 Spectrophotometer (Thermo Scientific). Each cell-free degradation assay was performed in 250 μL degradation buffer including 500 μg total proteins, and 100 ng purified OsTPR2-N-His. For the different protease inhibitors treatment, total protein extracts from ZH11 were used and serine protease inhibitor (5 mM PMSF, 1 mM aprotinin or 1 mM leupeptin), metalloprotease inhibitor (0.5 mM bestain), aspartic protease inhibitor (1 μM pepstain) and cysteine protease inhibitor (10 μM E-64) was separately added into each mixture. The mixtures were incubated at 37°C. Equal volume of protein mixture was collected at indicated time (0, 10, 20, and 30 min) and subjected to western blot with anti-His antibody. Plant actin was used as the loading control.

In vivo protein degradation assays

30 μg OsTPR2-Flag or GFP plasmids were transfected into ZH11 and nal1-cri, ZF802 and nal1-1, or NIL-IR and NIL-ZS protoplasts. After 16 h incubation, protoplasts were treated with 200 μM protein synthesis inhibitor cycloheximide (CHX; Sigma-Aldrich) and gently mixed. Equivalent protoplasts were collected at indicated time (0, 1, 3, and 5 h). Total proteins were extracted with extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% IGEPAL CA-630, 1×complete protease inhibitor cocktail) and subjected to western blot with anti-Flag antibody (F3165, Sigma-Aldrich) and anti-GFP antibody (ab290, Abcam). GFP protein was used as a negative control.

In vitro protein degradation assays

The proteolytic activity of NAL1 to OsTPR2 was assayed in a reaction buffer (25 mM Tris-HCl, pH 7.5, 10 mM NaCl, and 10 mM MgCl₂, 10 mM ATP) including 0.2 mg purified His-tagged NAL1 or the triad-mutated NAL1 (mNAL1) and 0.2 mg purified StrepII-tagged OsTPR2 in a total volume of 200 μL. The mixtures were incubated at 37°C. Equal volume of protein mixture was collected at indicated time (0, 15, 30, and 60 min) and subjected to SDS/PAGE. Subsequently, the gels were stained with Coomassie Brilliant Blue R-250.

In vitro pull-down assays

OsARF25 and IPA1 were separately cloned into GST-tagged pGEX-6p-1 vector. OsIAA3/17 and D53 were separately cloned into His-tagged pET32a vector. OsTPR2 was cloned into StrepII-tagged pMlink vector. In the StrepII pulldown assays, purified recombinant proteins were added in StrepII-binding buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.5% Triton X-100, 0.5% glycerol, 1 mM PMSF and 1×complete protease inhibitor cocktail) as indicated. Each protein mixture was incubated with StrepII beads (Sigma-Aldrich, 41246) for 4 h at 4°C and washed 5 times with binding buffer. After incubation, the beads were added to a 1×loading buffer and boiled for 10 min, and then samples were detected separately by western blot with anti-His and anti-GST antibody. The OsTPR2-StrepII was detected by Coomassie Brilliant Blue R-250.

ChIP-seq and ChIP-qPCR

3 g of ZH11 and nal1-cri seedlings were harvested and fixed for 30 min in 1% (v/v) formaldehyde under vacuum. Fixed tissues were homogenized, and chromatin was isolated and sonicated to produce DNA fragments around 300 base pairs. Then, anti-H3K18Ac (A7257, ABclonal) antibody with Dynabeads Protein A (10002D, Invitrogen) were added to the sonicated chromatin followed by incubation overnight at 4°C to precipitate antibody-bound DNA fragments. DNA was eluted and sequenced by Novogene Inc. (Beijing, China) with Illumina HiseqXten-PE150 and a paired-end sequencing strategy. The raw sequencing reads were filtered by fastp v0.23.1 (with the parameter “-q 30”) ⁶⁶ and mapped to rice genome assembly MSU v7.0 by bowtie2 v2.4.5 ⁶⁷ with default parameters. Peak calling was performed on nonredundant, uniquely mapped reads by MACS2 v2.2.7.1 with parameters “-f BAMPE -q 0.05 --call-summits” ⁶⁸. Peaks called in at least two replicates were used for differential-modification analysis by R package DiffBind v3.6.1 ⁶⁹ with its built-in DESeq2 analyzer. Peaks with |log2(fold change)| > 1 and FDR < 0.05 were defined as differentially modified regions and their associated genomic features were annotated by R package ChIPseeker ⁷⁰. For genomic track visualization, relative reads coverage of IP vs input was processed by bamCompare in deepTools v3.5.1 ⁷¹ with parameters “--scaleFactorsMethod SES -bs 1” and visualized in IGV ⁷².

For ChIP-qPCR, DNA fragments were sonicated to 500-1000 base pairs. After DNA was eluted, relative enrichment of each fragment was determined by qPCR. The ChIP DNA sample was quantified in triplicates. Primers used for the ChIP assays were listed in Extended Data Table 5.

RT-qPCR

Total RNA was extracted from seedlings of ZH11, nal1-cri and double mutant lines using TransZol (TransGen, Beijing, China). Reverse transcription was carried out with a cDNA synthesis kit (TransGen, Beijing, China). qPCR was performed using SYBR Green mix (Kuke, Wuhan, China) on an Applied Biosystems 7500 Fast Real-Time PCR System. Quantitative variations were calculated by the relative quantification method (2^-ΔΔCT) and four independent repeated experiments were performed for each sample. Primers were shown in Extended Data Table 5.

ACKNOWLEDGMENTS

We thank the staffs of the BL17U1/BL19U1 beamline of the National Center for Protein Sciences Shanghai (NCPSS) at the Shanghai Synchrotron Radiation Facility for assistance during data collection, and research associate Dr. Delin Zhang at the Center for Protein Research, Huazhong Agricultural University, for technical support.

This work was supported by Innovative Research Group Project of the National Natural Science Foundation of China (grant 31821005), the Key Program of National Natural Science Foundation of China (grant 31930080), the Foundation of Hubei Hongshan Laboratory (grant 2021hszd011, 2021hskf003), and the China Postdoctoral Science Foundation (grant 2019M652669). We thank the BaiChuan fellowship of College of Life Science and Technology, Huazhong Agricultural University, for funding support.

AUTHOR CONTRIBUTIONS

W.L., J.Y., P.Y., and L.X. designed the study; W.L., J.Y., Y.Z., F.Z., Y.Y., X.L., and H.W. performed all experiments. W.L., J.Y., Z.G., Y.C., and H.T. analyzed data. W.L., J.Y., H.X., X.L., and L.X. wrote and revised the article.

DECLARATION OF INTERESTS

The authors declare no competing interests.

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Serine protease NAL1 exerts pleiotropic functions through degradation of TOPLESS-related corepressor in rice

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Journal Publication

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Abstract

Figures

Introduction

Results

Crystal structure determination of NAL1 hexamer

NAL1 physically interacts with OsTPR2

NAL1 promotes degradation of OsTPR2

NAL1 exerts pleiotropic function by alleviating OsTPR2-mediated hormone signaling pathway suppression

NAL1 and its homolog genes in other crops are highly breeding-valuable

Discussion

Methods

Materials

Protein expression and purification

Crystallization, data collection, and structure determination

Analytical ultracentrifugation (AUC) analysis

IP-MS

BiFC and LCI assays

Co-immunoprecipitation

Size exclusion chromatography

Cell-free protein degradation assays

In vivo protein degradation assays

In vitro protein degradation assays

In vitro pull-down assays

ChIP-seq and ChIP-qPCR

RT-qPCR

Declarations

References

Additional Declarations

Supplementary Files

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Journal Publication

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