NPR1 promotes blue light-induced plant photomorphogenesis by ubiquitinating and degrading PIF4

Abstract

Light is a major determinant of plant growth and survival. NONEXPRESSER OF PATHOGENESIS-RELATED GENES 1 (NPR1) acts as a receptor for salicylic acid (SA) and serves as the key regulator of SA-mediated immune responses. However, the mechanisms by which plants integrate light and SA signals in response to environmental changes, as well as the role of NPR1 in regulating plant photomorphogenesis, remain poorly understood. This study shows that SA promotes plant photomorphogenesis by regulating PHYTOCHROME INTERACTING FACTOR 4 (PIF4). Specifically, NPR1 promotes photomorphogenesis under blue light by facilitating the degradation of PIF4 through light-induced polyubiquitination. NPR1 acts as a substrate adaptor for the CULLIN3-based E3 ligase, which ubiquitinates PIF4 at Lys129, Lys252, and Lys428, and leading to PIF4 degradation via the 26S proteasome pathway. Genetically, PIF4 is epistatic to NPR1 in the regulation of blue light-induced photomorphogenesis, suggesting it acts downstream of NPR1. Furthermore, cryptochromes mediate the polyubiquitination of PIF4 by NPR1 in response to blue light by promoting the interaction and ubiquitination between NPR1 and PIF4. Transcriptome analysis revealed that under blue light, NPR1 and PIF4 coordinately regulate numerous downstream genes related to light and auxin signaling pathways. Overall, these findings unveil a role for NPR1 in photomorphogenesis, highlighting a mechanism for posttranslational regulation of PIF4 in response to blue light. This mechanism plays a pivotal role in the fine-tuning of plant development, enabling plants to adapt to complex environmental changes.

Keywords: NPR1; PIF4; photomorphogenesis; salicylic acid; ubiquitination.

Fig. 1.

NPR1 and PIF4 play important roles in the SA-mediated inhibition of hypocotyl elongation in seedlings. (A and B) The phenotype and hypocotyl lengths of Col-0 seedlings grown for 4 d under white light (10 μmol m⁻² s⁻¹) (A) and darkness (B) with different concentrations of SA treatment. (C) The phenotype and hypocotyl lengths of pifq, pif4-2, and 35S::PIF4-MYC seedlings grown for 4 d under white light (10 μmol m⁻² s⁻¹) with different concentrations of SA treatment. (D) The phenotype and hypocotyl lengths of Col-0, npr1-1, and 35S::NPR1-GFP npr1-1 seedlings grown for 4 d under white light (10 μmol m⁻² s⁻¹) with or without SA treatment. Error bars represent SD (n ≥ 25). Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test; *P < 0.05, ****P < 0.0001, ns, no significance.

Fig. 2.

NPR1 negatively regulates hypocotyl elongation under blue light in a PIF4-dependent manner. (A) Phenotype and hypocotyl lengths of Col-0 and npr1-1 seedlings after 4 d of growth in darkness, white, blue, red, and far-red light conditions. (Scale bar, 0.5 mm.) Error bars represent SD (n ≥ 30). (B) Cotyledon area of 4-d-old Col-0, npr1-1, and 35S::NPR1-GFP npr1-1 seedlings under the white and blue light. Error bars represent SD (n ≥ 60). (C and D) The phenotype and hypocotyl lengths of Col-0, npr1-1, npr1-crispr-5, pif4-2, pifq, npr1-crispr pif4-2-9 and npr1-crispr pifq-12, and pif4-2 npr1-1 seedlings after 4 d of growth under white light (10 μmol m⁻² s⁻¹). (E) Phenotype and hypocotyl lengths of Col-0, npr1-1, npr1-crispr-5, pif4-2, npr1-crispr pif4-2-9, and pif4-2 npr1-1 seedlings after 4 d of growth under blue light (5 μmol m⁻² s⁻¹). (Scale bar, 0.5 mm.) Error bars represent SD (n ≥ 30). Statistical significance was analyzed using one-way ANOVA with Tukey’s multiple comparison test; *P < 0.05, **P < 0.01, ****P < 0.0001, ns, no significance.

Fig. 3.

NPR1 physically interacts with PIF4 in vitro and in vivo. (A) BiFC assays demonstrate the interaction between NPR1 and PIF4 in N. benthamiana leaf cells. (Scale bar, 200 μm.) nYFP, N-terminal fragment of Yellow Fluorescent Protein (YFP); cYFP, C-terminal fragment of YFP. (B) The Split-Luc assay reveals the interaction between NPR1 and PIF4 in N. benthamiana leaf cells. nLuc, N-terminal fragment of luciferase; cLuc, C-terminal fragment of luciferase. (C) The Split-Luc assay reveals the interaction between NPR1 and PIF4 in Arabidopsis protoplasts. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparison test; ****P < 0.0001. (D) Co-IP assays demonstrate the association of NPR1 with PIF4 in vivo under blue light. (E) PIF4-MYC interacts with NPR1-mCherry in Col-0 protoplasts. (F) Co-IP assays demonstrate that the in vivo association between NPR1 and PIF4 is significantly stronger under blue light compared to darkness. Asterisk, nonspecific band. Numbers represent quantifications from three biological replicates. (G) Pull-down assays show that GST-PIF4, but not GST, can pull down His-NPR1 in vitro. (H) Pull-down assays show that His-NPR1-MYC can pull down GST-PIF4 in vitro. Asterisk, nonspecific band.

Fig. 4.

The CUL3-NPR1 complex mediates the polyubiquitination of PIF4 at Lys129, Lys252, and Lys428. (A) In vitro ubiquitination by a CUL3 E3 ligase complex with recombinant MBP-NPR1-MYC and GST-PIF4 showed that NPR1 mediates the ubiquitination of PIF4. The numbers represent quantifications from three biological replicates. (B) Semi-in vitro ubiquitination assays using purified GST-NPR1 proteins and protein extracts from 35S::PIF4-MYC seedlings demonstrated that NPR1 mediates the ubiquitination of PIF4. (C) In vivo ubiquitination assays in Arabidopsis (Col-0) protoplasts confirmed the ubiquitination of PIF4-MYC by NPR1-mCherry. (D) In vivo ubiquitination assays were performed in 14-d-old 35S::PIF4-MYC, 35S::PIF4-MYC npr1-1, and 35S::PIF4-MYC 35S::NPR1-GFP seedlings under white light. The numbers represent the quantification of three biological replicates. (E) In vitro ubiquitination assays with a CUL3 E3 ligase complex showed that NPR1 ubiquitinates PIF4 but not PIF4(3KR), with quantifications from three biological replicates. (F) Semi-in vitro ubiquitination assays showed that GST-PIF4 is ubiquitinated in the presence of total protein extracts from Col-0 but not from npr1-1, while GST-PIF4(3KR) showed no detectable ubiquitination in either background. (G) In vivo ubiquitination assays in Arabidopsis (Col-0) protoplasts confirmed that NPR1-mCherry mediates the ubiquitination of PIF4-MYC, but not PIF4(3KR)-MYC.

Fig. 5.

NPR1 is responsible for the ubiquitination and subsequent degradation of PIF4 under blue light. (A) Semi-in vitro degradation assays revealed that GST-PIF4(3KR) degrades significantly slower than GST-PIF4 in Col-0 extracts, with no significant difference observed in the npr1-1 mutant; this degradation was notably inhibited by the 26S proteasome inhibitor MG132 (150 μM). The data on the right represent quantifications from three independent biological replicates. (B) In vivo degradation assays showed that PIF4-MYC was rapidly degraded in the presence of NPR1-Flag, which was fully blocked by the 26S proteasome inhibitor MG132, whereas PIF4(3KR) remained resistant to NPR1-Flag-mediated degradation. The data on the right represent quantifications from three independent biological replicates in N. benthamiana. (C) Hypocotyl lengths of Col-0, npr1-1, pif4-2, cry1 cry2, and PIF4pro:PIF4 (3KR)-MYC pif4-2 seedlings were measured after 4 d of growth under blue light (5 μmol m⁻² s⁻¹). Error bars represent the SD (n ≥ 30). (D) Hypocotyl elongation in PIF4pro:PIF4(3KR)-MYC seedlings was insensitive to SA compared to PIF4pro:PIF4-MYC. (E) PIF4(3KR)-MYC is more stable than PIF4-MYC in 4-d seedlings transitioning from dark to blue light. The data below represent quantifications from three independent biological replicates. (F) Western blot analysis was conducted on 4-d-old dark-grown Arabidopsis (Col-0) and npr1-1 seedlings under various durations of blue (B), red (R), and far-red (FR) light treatment and constant blue (cBlue), red (cRed), and far-red (cFR) light for 4 d. The data on the right represent quantifications from three independent biological replicates. (G) Detection of PIF4 protein during the transition from darkness to blue light exposure. Dark-grown Col-0 and npr1-1 seedlings were pretreated with CHX (100 μM), MG132 (100 μM), or CHX+MG132 for 1 h in liquid 1/2 MS medium after vacuuming for 5 min. The 4-d-old seedlings were then subjected to blue light treatment for 30 min. (H) In vivo ubiquitination assays were conducted on 4-d-old 35S::PIF4-MYC, 35S::PIF4-MYC npr1-1, and 35S::PIF4-MYC 35S::NPR1-GFP seedlings under dark and blue light conditions.

Fig. 6.

Cryptochromes enhance the NPR1-PIF4 interaction, facilitating PIF4 degradation and enabling NPR1 to regulate the expression of light-signaling genes in a PIF4-dependent manner. (A and B) The Split-Luc assay revealed that HF-CRY1 enhances the interaction between NPR1 and PIF4 in N. benthamiana leaf cells (A) and in Arabidopsis protoplasts (B). nLuc, N-terminal luciferase fragment; cLuc, C-terminal luciferase fragment. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test; ****P < 0.0001. (C) In vivo degradation assays were conducted using N. benthamiana leaf cells transiently expressing PIF4-MYC or PIF4(3KR)-MYC and different concentrations of HF-CRY1 and NPR1-Flag, with or without MG132. (D) Semi-in vitro ubiquitination assays confirmed that CRYs enhanced NPR1-mediated ubiquitination of PIF4. (E) Venn diagram illustrating the overlap in the number of differentially expressed genes (DEGs, P < 0.05, fold change > 1.5) identified when comparing npr1-1 versus Arabidopsis (Col-0), pif4-2 versus Col-0, and pif4-2 npr1-1 versus Col-0 under blue light. (F) Heatmap showing 1,359 genes regulated by NPR1 under blue light when comparing npr1-1 versus Col-0, pif4-2 versus Col-0, and pif4-2 npr1-1 versus Col-0. (G) Gene Ontology (GO) enrichment analysis of DEGs in npr1-1 and pif4-2 plants grown under blue light. (H) Venn diagram illustrating the overlap in the number of DEGs (P < 0.05, fold change > 1.5) identified when comparing npr1-1 versus Col-0, pif4-2 versus Col-0, and pif4-2 npr1-1 versus Col-0 under red light. (I) Heatmap showing 976 genes regulated by NPR1 under red light when comparing npr1-1 versus Col-0, pif4-2 versus Col-0, and pif4-2 npr1-1 versus Col-0. (J) GO enrichment analysis of DEGs in npr1-1 plants grown under blue and red light conditions. (K) A working model showing that NPR1 regulates plant photomorphogenesis by mediating the ubiquitination and degradation of PIF4. E2, ubiquitin conjugating enzyme; Ub, ubiquitin; CUL3, CULLIN3; RBX1, RING-box 1; CRYs, cryptochromes.