NF-YB1-YC12-bHLH144 complex directly activates Wx to regulate grain quality in rice (Oryza sativa L.)

Abstract

Identification of seed development regulatory genes is the key for the genetic improvement in rice grain quality. NF-Ys are the important transcription factors, but their roles in rice grain quality control and the underlying molecular mechanism remain largely unknown. Here, we report the functional characterization a rice NF-Y heterotrimer complex NF-YB1-YC12-bHLH144, which is formed by the binding of NF-YB1 to NF-YC12 and then bHLH144 in a sequential order. Knock-out of each of the complex genes resulted in alteration of grain qualities in all the mutants as well as reduced grain size in crnf-yb1 and crnf-yc12. RNA-seq analysis identified 1496 genes that were commonly regulated by NF-YB1 and NF-YC12, including the key granule-bound starch synthase gene Wx. NF-YC12 and bHLH144 maintain NF-YB1 stability from the degradation mediated by ubiquitin/26S proteasome, while NF-YB1 directly binds to the 'G-box' domain of Wx promoter and activates Wx transcription, hence to regulate rice grain quality. Finally, we revealed a novel grain quality regulatory pathway controlled by NF-YB1-YC12-bHLH144 complex, which has great potential for rice genetic improvement.

Keywords: G-box; Wx gene; grain quality; heterotrimer complex; nuclear factor Y; rice (Oryza sativa L.).

Figure 1

Spatial‐expression pattern of NF‐YB1 and phenotypical characterization of crnf‐yb1s and WT. (a) Schematic presentation of the NF‐YB1 gene structures and conserved protein domains. Boxes: exons; blue box: coding sequences; red box: conserved HLD; line: intron; α1, α2, α13 and αC are four conserved α‐helix structures. (b) qRT‐PCR analysis of NF‐YB1 transcription abundances in various tissues and stages. The expression level of callus was set as 1. (c) mRNA in situ hybridization analysis of NF‐YB1 on 7 DAP seeds. E: embryo; En: endosperm; Al: aluerone layer. Scale bar = 1 mm. (d) Negative CK of (c) using sense probe for hybridization. (e) Plant morphology at grain‐filling stage. (f‐k) comparison of the seed length (f), width (g), chalkiness (h), 1000‐grain‐weight (i), percentage of Grain With Chalkiness (j) and Degree of Endosperm Chalkiness (k) of crnf‐yb1s and WT. Data are shown as Means ± SD of at least three biological replicates. *: P ≤ 0.05 by the Student's t test. (l) Pasting properties of seeds analysed by Rapid Visco Analyzer (RVA).

Figure 2

Protein–protein interaction analysis of NF‐YB1, NF‐YC12 and bHLH144. (a) Bacterial‐two‐hybrid analysis of NF‐YB1 with NF‐YCs. LGF2‐pBT and Gal11‐pTRG were used as CK+, pBT and pTRG empty vector was used as CK‐. (b) Quantification of the interaction intensities between NF‐YB1‐pTRG and NF‐YCs‐pBT. The values on the Y axis represent the ratio (%) between colony numbers grow out on medium M9/‐HIS/+Cm/+Ter/+Strep and M9/+Cm/+Ter. (c) Interaction test of truncated NF‐YB1 and NF‐YC12 by bacterial‐two‐hybrid. The numbers underneath indicate the positions of the truncated regions on the protein. The position of HLD on NF‐YB1 is 31‐93, and on NF‐YC12 is 53‐115. ⊿HLD indicates HLD was truncated out. (d) in vitro GST pull‐down assay of HIS‐NF‐YB1 and GST‐NF‐YC12. (e) Y3H analysis of NF‐YB1, NF‐YC12 and bHLH144. (f‐g) qRT‐PCR analysis of NF‐YC12 and bHLH144. (h) in vitro GST pull‐down assay of HIS‐NF‐YB1, GST‐NF‐YC12 and HIS‐FLAG‐bHLH144. (i) BiFC assay of NF‐YB1, NF‐YC12 and bHLH144 interactions. Two tested proteins were constructed in vector pDOE‐BiFC, which generates YFP fluorescence when the two bind with each other. The third protein or empty control was fused with mcherry to generate red fluorescence. BF: bright field. Scale bar = 20 μm.

Figure 3

Phenotypical characterization of crnf‐yc12s, crbhlh144s and WT. (a) Plant morphology of crnf‐yc12s and WT at grain‐filling stage. (b‐c) comparison of the seed length (b) and width (c) of crnf‐yc12s with WT. (d‐f) comparison of the seed width (d), length (f) and plant morphology of crnf‐yc12s and WT at grain‐filling stage (e). (g) Comparison of chalkiness of crnf‐yc12s and crbhlh144s with WT. Percentage of Grain With Chalkiness (h) and Degree of Endosperm Chalkiness (i) of crnf‐yc12s, crbhlh144s and WT. Data are shown as Means ± SD of at least three biological replicates. *: P ≤ 0.05 by the Student's t test.

Figure 4

qRT‐PCR analysis of the transcriptional abundances of starch biosynthesis‐related DEGs and master regulators in 7 DAP seeds of crnf‐yb1s, crnf‐yc12s, crbhlh144s and WT. The asterisk represents significant difference with the WT at P ≤ 0.05 as determined by the Student's t test.

Figure 5

NF‐YB1 directly binds to the G‐box of Wx promoter and activates its transcription. (a) Y1H assay of NF‐YB1, NF‐YC12 and bHLH144 to the promoter of Wx. NF‐YB1‐pB42AD and SUT4‐pLACZ1 were used as CK+. (b) LUC transient transcriptional activity assay in rice protoplast. Reporter: proWx:LUC ; Effectors: pro35S:NF‐YB1:tNOS , pro35S:NF‐YC12:tNOS and pro35S:bHLH144:tNOS . Values are mean ± SD with biological triplicates. **: P < 0.01 by the Student's t test. (c) Probe positions on Wx promoter. Transcription Starting Site (TSS) was set as 0; Numbers indicate the distances (bps) of probes to the TSS. Red letters indicate the core element sequences in the position. (d) EMSA assay to show NF‐YB1 binds with the probe 1 on the promoter of Wx. (e) EMSA assay showing the binding of NF‐YB1 with mutated G‐box probe 1. (f) ChIP qRT‐PCR assay showing the NF‐YB1 bind to Wx promoter regions. Values are mean ± SD with biological triplicates. The enrichment values were normalized to Input. IgG immunoprecipitated DNA was used as a CK. **: P < 0.01 in comparison with the IgG mock samples. (g) EMSA assay showing the binding intensities of NF‐YB1 with probe 1 in the presence of NF‐YC12 and bHLH144. (h) Degradation assay of HIS‐NF‐YB1 under the absence or presence of GST‐NF‐YC12 and/or HIS‐bHLH144‐FLAG. Equal starting amount of the total proteins were used for the degradation as indicated by the Commassie blue staining. (i) Degradation curve of HIS‐NF‐YB1deduced from the result of (h).

Figure 6

A working model for NF‐YB1‐YC12‐bHLH144 complex regulating starch synthesis in rice seeds. NF‐YB1 binds to NF‐YC12 to form a dimer, and then bind with bHLH144 to form a heterotrimer. The trimer complex protects NF‐YB1 against the ubiquitin/26S proteasome‐mediated degradation. Stable NF‐YB1 activates the transcription of the key granule‐bound starch synthase gene Wx by directly binding to the “G‐box” domain of its promoter, hence to regulate the starch synthesis.