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

Nature Plants volume 9, pages 1130–1142 (2023)Cite this article

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Abstract

NARROW LEAF 1 (NAL1) is a breeding-valuable pleiotropic gene that affects multiple agronomic traits in rice, although the molecular mechanism is largely unclear. Here, we report that NAL1 is a serine protease and displays a novel hexameric structure consisting of two ATP-mediated doughnut-shaped trimeric complexes. 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 signalling pathways, eventually achieving its pleiotropic physiological function. An elite allele, NAL1A, which may have originated from wild rice, could increase grain yield. Furthermore, the NAL1 homologues 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.

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Fig. 1: Crystal structure of NAL1 hexamer.
Fig. 2: NAL1 targets OsTPR2 for degradation.
Fig. 3: OsTPR2 functions genetically downstream of NAL1.
Fig. 4: NAL1 affects expression of auxin and SL signalling pathway genes.
Fig. 5: Functionally strong allele NAL1A originating from common wild rice improves grain yield.
Fig. 6: A proposed working model for the NAL1 pleiotropic function through OsTPR2 degradation.

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Data availability

ChIP-seq data generated in this study have been deposited in the GEO database under accession no. GSE207132. Coordinates and structure factors of NAL1 in this study have been deposited in the Protein Data Bank under accession code 7Y77. The structure data are obtained from public data in the Protein Data Bank under accession code 1P01, 1SGP, 2R3Y, 3K6Y and 5IL9. The orthologues of wheat, maize, soybean, oil crop were downloaded from Ensembl plants (https://plants.ensembl.org/Oryza_sativa/Gene/Compara_Ortholog?db=core;g=Os04g0615000;r=4:31205267-31214632;t=Os04t0615000-01). Materials used in this study are available upon request. Source data are provided with this paper.

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Acknowledgements

We thank the staff 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 D. Zhang at the Center for Protein Research, Huazhong Agricultural University, for technical support. This work was supported by the National Natural Science Foundation of China (grants 31930080 to L.X., 31821005 to L.X., 32270255 to J.Y.), the Foundation of Hubei Hongshan Laboratory (grants 2021hszd011, 2021hskf003 to J.Y.), and the China Postdoctoral Science Foundation (grant 2019M652669 to W.L.). We thank the BaiChuan fellowship of College of Life Science and Technology, Huazhong Agricultural University, for funding support. The computations in this paper were run on the bioinformatics computing platform of the National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University.

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  1. These authors contributed equally: Wenjing Li, Junjie Yan.

Authors and Affiliations

  1. National Key Laboratory of Crop Genetic Improvement, Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan, China

    Wenjing Li, Junjie Yan, Yu Zhang, Fei Zhang, Zeyuan Guan, Yilong Yao, Yu Chang, Haifu Tu, Xiaokai Li, Huaijun Wang, Haiyan Xiong, Xuelei Lai, Ping Yin & Lizhong Xiong

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  1. Wenjing Li
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Contributions

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

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Correspondence to Lizhong Xiong.

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Extended data

Extended Data Fig. 1 NAL1 forms a hexamer.

a, The domain organization of NAL1. b, NAL1 trimer in asymmetric unit. c, Analytical ultracentrifugation characterization of NAL1 oligomerization. d, Structural alignment of determined NAL159–463 protomers with the AlphaFold2 predicted NAL11–582. e, Structure superposition of NAL1 with other serine proteases. f, Secondary structural elements of NAL1 protomer. g, Density map of ATP molecules. h, Size-exclusion chromatography analysis of NAL1 mutants. Fractions with the same elution volume from each injection were subjected to SDS-PAGE. The experiment in h was repeated independently three times with similar results. i, Structural alignment of catalytic triad in each protomer. The arrow indicates the orientations of the imidazole ring of H233 are relatively rotated. j, Oligomeric architecture comparison with other Deg proteases.

Source data

Extended Data Fig. 2 Plant architecture and yield-related traits of wild type (ZH11), complementary lines (NAL1A-COM and NAL1G-COM), and NAL1-knockout line (nal1-cri).

a, Flag leaf morphology, bar = 5 cm. b, Root system, bar = 5 cm. c, Panicle structure, bar = 5 cm.

Extended Data Fig. 3 C-terminal EAR-1 motif of NAL1 mediates the interaction with OsTPR2.

a, NAL1 interacts with OsTPR family proteins. NAL1 interacts with OsTPR family proteins (OsTPR1, OsTPR2 and OsTPR3) in LCI assays in tobacco leaves. The 10-AA deletion mutant nal1-1 was used as a negative control. b, A schematic domain organization of NAL1 and TPD. c, Size-exclusion chromatography analysis of the interactions between TPD and NAL1 truncations and mutants. Deletions of the C-terminal EAR motifs abolished their interactions. The last EAR motif had no effect on their interaction. The experiment in c was repeated independently three times with similar results. d, Structural model of OsTPD and NAL1EAR-1 complex. The model was obtained based on the structure of OsTPD and NINJA (PDB ID: 6C6V). e, Interaction details between OsTPD and NAL1EAR-1. Key residues were labeled.

Source data

Extended Data Fig. 4 OsTPR2 was degraded by NAL1 and partially rescue nal1-cri’s phenotype.

a, Degradation of OsTPR2 in ZF802 and nal1-1 protoplasts after CHX treatment. Transfected rice protoplasts were incubated for 16 h, and then treated with (50 g ml−1) CHX to block protein synthesis. Equal volume protoplasts were collected at different time points for immunoblotting detection of OsTPR2. b, Degradation of OsTPR2 in total proteins extracted from ZF802 and nal1-1 seedlings using cell-free protein degradation assays. His-OsTPR2 was respectively added to extracts from ZH11 and nal1-cri plants. Equal volume protein mix was collected at different time points for immuno-blotting detection of OsTPR2. c, Degradation of OsTPR2 in NIL-IR (NAL1A) and NIL-ZS (NAL1G) protoplasts after CHX treatment. Transfected rice protoplasts were incubated for 16 h, and then treated with (50 g ml-1) CHX to block protein synthesis. Equal volume of protoplasts were collected at different time points for immunoblotting detection of OsTPR2. d, Degradation of OsTPR2 in total protein extract from NIL-IR and NIL-ZS seedlings by cell-free protein degradation assays. His-OsTPR2 was respectively added to total protein extracts from NIL-IR and NIL-ZS plants. Equal volume of protein mix was collected at different time points for immunoblotting detection of OsTPR2. e, Degradation of OsTPR2 in vitro. NAL1 (encoded by NAL1A) or the NAL1H233R (encoded by NAL1G) were separately incubated with OsTPR2 for 0, 30, 60 or 120 min at 37 °C. After terminating the reaction, the reaction mixtures were subjected to SDS/PAGE. The location of OsTPR2 was indicated in the gel. f, OsTPR2 protein levels in ZH11 and nal1-cri in vivo. Anti-OsTPR2 antibody was used to detect OsTPR2 protein level in equal total protein extracts from ZH11 and nal1-cri seedlings. Actin was used as a control. g, OsTPR2 expression levels in ZH11, nal1-cri and the OsTPR2-knockdown lines in the nal1-cri background (named TN lines) seedlings (n = 4). RT-qPCR was repeated at least three times. Data are means ± SD. Different lowercase letters above bars indicate significant difference at P < 0.05 level by one-way ANOVA. The exact P values are listed in Supplementary Table 6. h-j, Plant architecture and yield-related traits of wild type (ZH11), nal1-cri, and OsTPR2-knockdown lines in the nal1-cri background (named TN lines). (h) Flag leaf morphology, bar = 5 cm. (i) Root system, bar = 5 cm. (j) Panicle structure, bar = 5 cm. k, The oligomerization of NAL1 decreased its proteolytic activity. Wild type or the mutated NAL1 (NAL1R120A, NAL1K139A and NAL1W144A) proteins were separately incubated with OsTPR2 for 0, 30, 60 or 120 min at 37 °C. After terminating the reaction, the reaction mixtures were subjected to SDS/PAGE. The location of OsTPR2 was indicated in the gel. The experiments in a-f, k were repeated independently three times with similar results.

Source data

Extended Data Fig. 5 NAL1 affects H3K18Ace levels of auxin and SL signaling pathway genes.

a, Relative histone acetylation and histone levels of nal1-cri mutant in Fig. 4d were normalized based on that of ZH11. The Integrated density of each interested protein band was carried out with FIJI/ImageJ. Data are means ± SD of three biological replicates. * and ** indicate significant differences between ZH11 and nal1-cri at P < 0.05 and 0.01 levels, and ns indicates no significant difference by two-sides Student’s t-test. b, Venn diagram of genes with significantly decreased expression and H3K18Ace levels. RNA-seq and ChIP-seq were used to analyse the expression and H3K18Ace levels, respectively, in ZH11 and nal1-cri seedlings. c, Genome browser traces of H3K18Ace ChIP-seq data in ZH11 and nal1-cri from representative genes. The fragments examined by ChIP-qPCR were indicated. d, H3K18Ace levels of Actin in ZH11 and nal1-cri seedlings. Data are fold-changes relative to levels in ZH11 seedlings. ChIP-qPCR was repeated at least three times. Data are means ± SD of three biological replicates. ns indicates no difference by two-sides Student’s t-test. e, Relative expression levels of auxin and SL signaling pathway genes in the seedlings of nal1-cri and TN lines. RT-qPCR was repeated for four times (n = 4). * and ** indicate significant differences between each complementary line and nal1-cri at P < 0.05 and 0.01 levels, and ns indicates no significant difference by two-sides Student’s t-test. The exact P values are listed in Supplementary Table 6.

Source data

Extended Data Fig. 6 Genetic analysis of NAL1 in the indica, japonica and O. rufipogon populations.

a, Representative genotypes of NAL1. b, Nucleotide diversity of NAL1. c, Single-nucleotide diversity.

Source data

Extended Data Fig. 7 Conservativeness of NAL1 homolog functions in different crops.

a, Phylogenic tree of NAL1 homologues from different crops. A neighbor-joining tree was built by MEGA-X using a Poisson correction model with gaps to complete deletion. Topological robustness was assessed by bootstrap analysis with 1000 replicates. The bar is an indicator of genetic distance based on branch length. Genes, which were indicated by red box, were selected to construct complementary lines. b-k, Phenotypes of yield-related traits of wild type ZH11, nal1-cri and the complementary (COM) lines constructed by NAL1-homologous genes from wheat (Ta), maize (Zm), rapeseed (Bn), and soybean (Gm). (b) Plant morphology, bar = 20 cm. (c) Flag leaf morphology, bar = 5 cm. (d) Root system, bar = 5 cm. (e) Panicle structure, bar = 5 cm. (f) Plant height (n = 10), (g) Flag leaf length (n = 8), (h) Flag leaf width (n = 8), (i) Root dry weight (n = 8), (j) Spikelet number per panicle (n = 10), (k) Grain yield per plant (n = 10). Data are means ± SD. Different lowercase letters above bars indicate significant difference at P < 0.05 level by one-way ANOVA. The exact P values are listed in Supplementary Table 6.

Source data

Extended Data Fig. 8 The expression of ACL in ZH11, nal1-cri and TN lines seedlings (n = 4).

RT-qPCR was repeated at least three times. Data are means ± SD. ** indicates significant differences between ZH11 and nal1-cri at P < 0.01, and * indicates significant differences between each TN lines and nal1-cri at P < 0.05 by two-sides Student’s t-test. The exact P values are listed in Supplementary Table 6.

Source data

Extended Data Fig. 9 Sequence alignment of plant NAL1 homologs.

Putative catalytic triad, ear motif, and interface residues are labeled with red star, blue circle, and green rectangle, respectively.

Supplementary information

Reporting Summary

Supplementary Tables 1–6

Table 1: Data collection, refinement and structural determination. Table 2: Homologous structure search for NAL1 by Dali. Table 3: NAL1 interaction proteins identified by IP–MS. Table 4: List of genes with significantly reduced expression and H3K18Ace levels in NAL1 mutants compared with wild-types. Table 5: Primers used in this study. Table 6: Individual P values of each figure.

Source data

Source Data Figs. 2 and 4 and Extended Data Figs. 1, 3 and 4

Unprocessed western blots and gels.

Source Data Table

Statistical source data for Figs. 1, 3, 4 and 5 and Extended Data Figs. 4, 5, 6, 7 and 8.

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Li, W., Yan, J., Zhang, Y. et al. Serine protease NAL1 exerts pleiotropic functions through degradation of TOPLESS-related corepressor in rice. Nat. Plants 9, 1130–1142 (2023). https://doi.org/10.1038/s41477-023-01449-2

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