The E3 ligase OsPUB33 controls rice grain size and weight by regulating the OsNAC120-BG1 module

Abstract

Grain size and weight are important determinants of crop yield. Although the ubiquitin pathway has been implicated in the grain development in rice (Oryza sativa), the underlying genetic and molecular mechanisms remain largely unknown. Here, we report that the plant U-box E3 ubiquitin ligase OsPUB33 interferes with the OsNAC120-BG1 module to control rice grain development. Functional loss of OsPUB33 triggers elevated photosynthetic rates and greater sugar translocation, leading to enhanced cell proliferation and accelerated grain filling. These changes cause enlarged spikelet hulls, thereby increasing final grain size and weight. OsPUB33 interacts with transcription factor OsNAC120, resulting in its ubiquitination and degradation. Unlike OsPUB33, OsNAC120 promotes grain size and weight: OsNAC120-overexpression plants harbor large and heavy grains, whereas osnac120 loss-of-function mutants produce small grains. Genetic interaction analysis supports that OsPUB33 and OsNAC120 function at least partially in a common pathway to control grain development, but have opposite functions. Additionally, OsNAC120 transcriptionally activates BIG GRAIN1 (BG1), a prominent modulator of grain size, whereas OsPUB33 impairs the OsNAC120-mediated regulation of BG1. Collectively, our findings uncover an important molecular framework for the control of grain size and weight by the OsPUB33-OsNAC120-BG1 regulatory module and provide promising targets for improving crop yield.

Figure 1.

OsPUB33 is a negative modulator of grain size and weight in rice. A) Schematic diagram indicating targeted mutagenesis of OsPUB33 by the CRISPR/Cas9 strategy against ZH11 background. PAM, protospacer adjacent motif. The arrow refers to the gRNA sequence. The “T” or “A” means a single-base insertion. The “CGG” refers to PAM. B) Mature paddy grains (left) and brown rice grains (right) of ospub33 mutants and ZH11. Scale bars, 1 cm. C, D) Grain length and width (n ≥ 15). E) Quantification of 1000-grain weight (n = 3). F) Grains from the whole plant. Scale bar, 5 cm. G) Grain yield per plant (n = 10). H) Mature paddy grains (left) and brown rice grains (right) of ospub33-1 and the complementation lines (Com-L1 and Com-L2). Scale bars, 1 cm. I, J) Grain length and width (n ≥ 20). K) Quantification of 1000-grain weight (n = 3). Data are shown as means ± SD. In C, D, E, G, I to K, different lowercase letters indicate significant differences (P < 0.05, one-way ANOVA with Tukey's HSD test).

Figure 2.

Overexpression of OsPUB33 reduces grain size and weight. A) Mature paddy grains (left) and brown rice grains (right) of wild type (NIP) and OsPUB33-overexpression lines (OE1, OE2, OE3, and OE4). Scale bars, 1 cm. B, C) Grain length and width (n ≥ 15). D) Quantification of 1000-grain weight (n = 3). E) Grains from the whole plants. Scale bar, 5 cm. F) Grain yield per plant (n = 10). Data are shown as means ± SD. In B to D, F, different lowercase letters indicate significant differences (P < 0.05, one-way ANOVA with Tukey's HSD test).

Figure 3.

OsPUB33 controls the spikelet hull size by inhibiting cell division. A, B) Young spikelet hulls from various genetic backgrounds. Scale bars, 3 mm. The horizontal lines indicate the cross-sectional positions. C, D) Cross sections of spikelet hulls as indicated in A and B. Scale bar, 500 μm. E, F) Magnified view of the cross-sectional area boxed in C and D. Scale bar, 50 μm. G to L) Total lengths (G, H), cell numbers (I, J), and average length of each cell (K, L) in the outer parenchyma layer in spikelet hulls from various genetic backgrounds (n = 10). M to T) Expression of the genes involved in cell cycle regulation in panicles from various genetic backgrounds (n = 3). OsEF1a was used as the internal reference. Data are shown as means ± SD. In G to L, significant differences were determined by Student's t test (***P < 0.001; ns, not significant). In M to T, different lowercase letters indicate significant differences (P < 0.05, one-way ANOVA with Tukey's HSD test).

Figure 4.

OsPUB33 controls the leaf size, photosynthetic rate, and grain filling. A, B) Flag leaf from wild type (ZH11 or NIP), ospub33 mutants (ospub33-1 to ospub33-4), and OsPUB33-overexpression lines (OE1 to OE4). Top scale bars, 5 cm. Bottom scale bars, 1.5 cm. C to F) Flag leaf length and width of various genetic backgrounds (n ≥ 15). G, H) Net photosynthetic rate in flag leaves of various genetic backgrounds (n = 6). I, K) Grain weight of various genetic backgrounds measured during grain-filling stage (n = 10). DAP, day after pollination. J, L) Grain morphology of various genetic backgrounds during grain-filling stage. Scale bar, 1 cm. M to P) Sucrose content (M, O) and starch content (N, P) in flag leaves of various genetic backgrounds (n = 3). FW, fresh weight. Q, R) Iodine staining of temporary starch accumulation in the flag leaves of various genetic backgrounds. Scale bars, 1 cm. Data are shown as means ± SD. In (I, K), significant differences were determined by Student's t test (*P < 0.05, **P < 0.01, or ***P < 0.001; ns, not significant). In C to H, M to P, different lowercase letters indicate significant differences (P < 0.05, one-way ANOVA with Tukey's HSD test).

Figure 5.

Protein interaction between OsPUB33 and OsNAC120. A) Protein interaction between OsPUB33 and OsNAC120 in an Y2H assay. DDO: SD/-Leu-Trp. QDO: SD/-Ade-His-Leu-Trp. BD-p53, positive control; BD-Lam, negative control. B) Subcellular localization of OsPUB33 in the epidermal cells of N. benthamiana leaves. HY5-RFP was used as a nuclear marker. Scale bars, 100 µm. C) The OsPUB33–OsNAC120 interaction indicated by BiFC assays in N. benthamiana. Scale bars, 10 µm. D) SLC assays showing that OsPUB33 interaction with OsNAC120 in N. benthamiana. Scale bars, 2 cm. E) GST pull-down assays showing OsPUB33 interaction with OsNAC120. F) Semi-endogenous Co-IP analysis showing the interaction of His-OsPUB33 with endogenous OsNAC120–GFP. Protein lysates extracted from GFP or OsNAC120–GFP transgenic plants were incubated with His-OsPUB33, followed by immunoprecipitation with anti-GFP antibody. The immunoprecipitated proteins were detected with anti-His and anti-GFP antibodies, respectively. Immunoblotting analysis of Histone H3.1 (H3.1) was used as a control.

Figure 6.

OsPUB33 mediates OsNAC120 ubiquitination and degradation. A) Examination of E3 ubiquitin ligase activity of OsPUB33 in vitro. GST-OsPUB33 was incubated at 30 °C for 1 h with or without E1 (UBA2), E2 (UBC25), ATP, and ubiquitin (Ub). B) Semi-endogenous ubiquitination analysis showing OsNAC120 ubiquitination mediated by OsPUB33. Protein extracts from GFP or OsNAC120–GFP transgenic plants were incubated with His-OsPUB33, followed by immunoprecipitation with anti-GFP antibody. The immunoprecipitated proteins were detected with different antibodies. C) OsPUB33-mediated ubiquitination of OsNAC120 in N. benthamiana transiently co-expressing OsNAC120-3×FLAG with OsPUB33–GFP. D, E) OsPUB33-mediated His-OsNAC120 degradation in cell-free degradation assays in the protein extracts of wild type (ZH11; NIP), OsPUB33-overexpression line (OE1) and ospub33-1 mutant. F) OsNAC120 degradation through 26S proteasome. G) OsPUB33-mediated OsNAC120 degradation in N. benthamiana. In D to G, equal amount of protein stained by Coomassie brilliant blue (CBB) was used as a loading control. For immunoblotting analysis, 2 independent experiments were performed, and similar results were obtained. The relative intensity of protein bands was calculated from 2 independent experiments by using ImageJ.

Figure 7.

OsNAC120 positively regulates rice grain size and weight. A) Sequencing of the mutated sites indicating targeted mutagenesis of OsNAC120 by CRISPR/Cas9 strategy against NIP background. The “C” means a single-base insertion. The “AGG” refers to PAM. B to D) Grain length and width in OsNAC120 knockout mutants (osnac120-cr1 to osnac120-cr3) and NIP (n ≥ 20). In B, scale bars, 1 cm. E) Quantification of 1000-grain weight (n = 3). F) Grain phenotype of wild type (Hwayoung, HY; Dongjin, DJ) and T-DNA mutants of OsNAC120 (osnac120-1, osnac120-2). Scale bars, 1 cm. G to I) Grain length and width in ZH11 and OsNAC120-OE lines (OE1 to OE4) (n ≥ 20). In H, scale bars, 1 cm. J) Quantification of 1000-grain weight (n = 3). K, Q) Young spikelet hulls from various genetic backgrounds. Scale bars, 3 mm. The horizontal lines indicate the cross-sectional positions. L, R) Cross sections of spikelet hulls from various genetic backgrounds. Scale bar, 500 μm. M, S) Magnified view of the cross-sectional area boxed in M and S, respectively. Scale bar, 50 μm. N to P, T to V) Total lengths (N, T), average length of each cell (O, U), and cell numbers (P, V) in the outer parenchyma layer in spikelet hulls from various genetic backgrounds (n = 10). Data are shown as means ± SD. In C to E, H to J, different lowercase letters indicate significant differences (P < 0.05, one-way ANOVA with Tukey's HSD test). In N to P, T to V, the significant difference was determined by Student's t test (**P < 0.01, ns, not significant).

Figure 8.

OsPUB33 and OsNAC120 work in a common pathway to regulate rice grain. A) Schematic diagram indicating targeted mutagenesis of OsNAC120 by CRISPR/Cas9 strategy against ospub33-1 background. The dashes mean a 50-bp deletion. B) Mature paddy grains (left) and brown rice grains (right) of various genetic backgrounds (ZH11, ospub33-1, osnac120-cr1, and osnac120 ospub33-1). Scale bars, 1 cm. C, D) Grain length and width in various genetic backgrounds (n ≥ 20). E) Quantification of 1000-grain weight (n = 3). F) Grain phenotype of various genetic backgrounds (ZH11, OsNAC120-OE#1, ospub33-1, OsNAC120-OE#1/ospub33-1, and OsNAC120-OE#2/ospub33-1). OsNAC120–GFP was overexpressed in ospub33-1 mutant to generate OsNAC120-OE/ospub33-1 lines (OsNAC120-OE#1/ospub33-1 and OsNAC120-OE#2/ospub33-1). Scale bars, 1 cm. G, H) Grain length and width in various genetic backgrounds (n ≥ 10). I) Quantification of 1000-grain weight (n = 3). J)OsNAC120 expression in various genetic backgrounds (n = 3). OsActin1 was used as the internal reference gene. K) Function loss of OsPUB33 leads to increased OsNAC120 protein abundance in planta. H3.1 was a loading control. L) Function loss of OsPUB33 leads to reduced ubiquitination of OsNAC120 in planta. Immunoprecipitation was performed using anti-GFP antibody in OsNAC120-OE#1 and OsNAC120-OE#1/ospub33-1 plants, and the immunoprecipitated proteins were subjected to immunoblotting with anti-GFP and anti-Ub, respectively. Data are shown as means ± SD. In C to E, G, H, different lowercase letters indicate significant differences (P < 0.05, one-way ANOVA with Tukey's HSD test).

Figure 9.

OsNAC120 transcriptionally activates BG1 to regulate grain size in rice. A, B) Expression of grain size-related genes in young panicles from various genetic backgrounds (n = 3). OsActin1 was used as an internal control. C) ChIP-qPCR analysis of BG1 promoter fragments enriched by OsNAC120 in OsNAC120–GFP transgenic plants (n = 3 independent experiments). P0 to P7 represent the regions in the schematic diagram of BG1 promoter region (−2 kb upstream) showing the positions of CACG box recognized by NAC transcription factors. D) EMSA showing OsNAC120 binding to CACG-box motifs in BG1 promoter specifically. BG1_pro-probe: −1,705 to −1,745 bp in BG1 promoter. E, F) Transactivation of BG1 by OsNAC120 indicated by dual-luciferase reporter assays. G, H) OsPUB33 inhibits the transcriptional activity of OsNAC120. In E, G, n = 3 independent experiments. In F, H, scale bars, 2 cm. Data are shown as means ± SD. In A to C, E, significant differences were determined by Student's t test (*P < 0.05, **P < 0.01, or ***P < 0.001; ns, not significant). In G, different lowercase letters indicate significant differences (P < 0.05, one-way ANOVA with Tukey's HSD test). I) Proposed working model for OsPUB33 controlling rice grain size by interfering with the OsNAC120–BG1 module. Function loss of OsPUB33 leads to increased abundance of OsNAC120 protein, which enhances the transcriptional regulation of the downstream target gene BG1, thus promoting cell division and elongation and increasing grain size and weight.