OPAQUE3, encoding a transmembrane bZIP transcription factor, regulates endosperm storage protein and starch biosynthesis in rice

Abstract

Starch and storage proteins are the main components of rice (Oryza sativa L.) grains. Despite their importance, the molecular regulatory mechanisms of storage protein and starch biosynthesis remain largely elusive. Here, we identified a rice opaque endosperm mutant, opaque3 (o3), that overaccumulates 57-kDa proglutelins and has significantly lower protein and starch contents than the wild type. The o3 mutant also has abnormal protein body structures and compound starch grains in its endosperm cells. OPAQUE3 (O3) encodes a transmembrane basic leucine zipper (bZIP) transcription factor (OsbZIP60) and is localized in the endoplasmic reticulum (ER) and the nucleus, but it is localized mostly in the nucleus under ER stress. We demonstrated that O3 could activate the expression of several starch synthesis-related genes (GBSSI, AGPL2, SBEI, and ISA2) and storage protein synthesis-related genes (OsGluA2, Prol14, and Glb1). O3 also plays an important role in protein processing and export in the ER by directly binding to the promoters and activating the expression of OsBIP1 and PDIL1-1, two major chaperones that assist with folding of immature secretory proteins in the ER of rice endosperm cells. High-temperature conditions aggravate ER stress and result in more abnormal grain development in o3 mutants. We also revealed that OsbZIP50 can assist O3 in response to ER stress, especially under high-temperature conditions. We thus demonstrate that O3 plays a central role in rice grain development by participating simultaneously in the regulation of storage protein and starch biosynthesis and the maintenance of ER homeostasis in endosperm cells.

Keywords: ER stress; OPAQUE3; grain development; high temperature; rice.

Figure 1

Characterization of the opaque3 (o3) mutant. (A) Appearance of wild-type (WT) and o3 mutant brown rice. (B) Transverse sections of WT and o3 endosperm. (C) Fresh WT and o3 grains at various stages of development. DAF, days after fertilization. Scale bars, 2 mm in (A–C). (D) Dry weight of WT and o3 grains at various stages of grain filling. (E–G) WT and o3 seed setting rate (E), 1000-grain weight (F), and grain yield per plant (G). (H–K) The percent content of total starch (H), amylose (I), total protein (J), and lipid (K) in WT and o3 endosperm. (L) Storage protein content in WT and o3 milled rice. (M) SDS-PAGE profiles of total storage proteins in WT and o3 dry grain. pGlu, 57-kDa proglutelin; αGlu, 40-kDa glutelin acidic subunit; αGlb, 26-kDa α-globulin; βGlu, 20-kDa glutelin basic subunit; Pro, prolamin. (N) Immunoblot analysis of GluA, GluB, globulin, and the molecular chaperones BiP1 and PDIL1-1. Red arrows denote 57-kDa proglutelins. Blue arrows denote the glutelin acidic subunits. EF1α was used as a loading control. Data in (D–L) are means ± SD from at least three biological replicates. Statistically significant differences were determined using Student’s t-test (∗P < 0.05, ∗∗P < 0.01). P values are shown in (E–L) when statistically significant.

Figure 2

Protein body and starch grain development in WT and o3 mutant endosperm cells. (A) Endoplasmic reticulum (ER) and protein body I (PBI) were observed in WT endosperm cells at 12 DAF. (B and C) The Golgi apparatus and dense vesicles (DVs) in WT endosperm cells. (D–F) Small protein bodies (blue arrows) remaining in the terminal of ER or ER-derived vesicles and PBI or PBII were observed in o3 endosperm cells. (G) The Golgi apparatus and DVs in o3 endosperm cells. (H) The structure of fragmentized protein bodies (red dotted box) was observed in o3 endosperm cells. (I) The structure of the fusion of PBI and PBII was observed in o3 endosperm cells. (J–O) Semi-thin sections of WT (J–L) and o3(M–O) endosperm at 9 DAF. (J and M) The peripheral region of the endosperm. (K, L, N, and O) The central region of the endosperm. Stars indicate aleurone cells in (J) and (M). Red dotted lines indicate an endosperm cell of o3 filled with abundant single and dispersed starch granules (SGs). Scale bars, 1 μm (A–E), (H), and (I), 0.2 μm (F) and (G), and 20 μm (J–O).

Figure 3

Identification of Opaque3 (O3) using Map-based cloning and the MutMap method. (A) The O3 locus was first mapped to the long arm of chromosome 7 between the markers RM22097 and RM22166. (B) Identification of genomic regions possibly harboring causal mutations for o3 mutants using MutMap. The red curves represent SNP index plots on chromosome 7; the red arrowhead indicates a single peak detected in the primary mapping region. (C) Gene structure and mutation site in O3 (LOC_Os07g44950). A SNP (T to C) caused a change from Leu-139 to Pro-139 in the encoded protein. (D) Grain appearance and SG morphology of the WT, o3 mutant, complementation lines (o3 mutant expressing O3; Com1 and Com2), and O3 knockout lines (cr1 and cr2). Insets show transverse sections of representative grains. Scale bars, 2 mm (top) and 5 μm (bottom). (E) Relative expression level of O3 in developing endosperm of WT, o3, Com1, Com2, cr1, and cr2. (F–I) 1000-grain weight (F), total protein content (G), total starch content (H), and amylose content (I) of WT, o3, Com1, Com2, cr1, and cr2 grains. Data in (E–I) are means ± SD from at least three biological replicates. Significant differences are indicated by different letters according to Student’s t-test. (J) SDS-PAGE profiles of total storage proteins of WT, o3, Com1, Com2, cr1, and cr2 dry grains.

Figure 4

Expression pattern and subcellular localization of O3. (A)O3 expression levels in various tissues and in developing endosperm at 6, 12, 18, and 24 DAF. Values are means ± SD from three biological replicates. (B) GUS staining in roots, stems, leaves, leaf sheaths, spikelets, and developing grains (3, 6, 12, 18, and 24 DAF) driven by the O3 promoter. Scale bars, 2 mm. (C) Subcellular localization of O3. Free green fluorescent protein (GFP) and the full-length O3 fusion protein (O3-GFP or GFP-O3) were transiently expressed in rice protoplasts. O3-GFP or GFP-O3 co-localized with DAPI and HDEL-mCherry signals of the nucleus and ER, respectively. O3-GFP was mostly expressed in the nucleus after 2 h of dithiothreitol (DTT) treatment. GFP signals, various organelle marker signals, bright-field images, and merged images of GFP and marker signals are shown in each panel. Scale bars, 5 μm.

Figure 5

O3 directly binds to the promoters of OsBIP1 and PDIL1-1 to activate their transcription. (A) ChIP-seq results showing the distribution of O3 binding sites for OsBIP1 and PDIL1-1 loci, as shown in Integrative Genomics Viewer. Red arrowheads indicate significant peaks calculated by MACS2; positions of peaks are shown in Attachment data II. The input sample was used as a negative control. (B and C) ChIP-qPCR assay showing the enrichment of O3 at promoter regions of OsBIP1(B) and PDIL1-1(C). DNA samples acquired before immunoprecipitation were used as the input. (D) EMSA showing that O3 can bind to probes of OsBIP1 and PDIL1-1. The 5-bp consensus pUPRE-II sequence (TGACG) in the promoters of OsBIP1 and PDIL1-1 is indicated by red text. The mutated pUPRE-II motif in mut probes is showed in brackets. (E–G) LUC transient transactivation assay in rice protoplasts. Constructs used in the transient expression assays are shown in (E). O3 significantly activated transcription of OsBIP1(F) and PDIL1-1(G). O3-m represents the mutant form with a Leu-139 to Pro-139 substitution in the coding region. (H) qRT–PCR analysis of OsBIP1 transcript level in OE (OE-OsBIP1) lines in the o3 mutant background. (I) The grain appearance of osbip1 and pdil1-1 mutants in the ZhongJian100 (ZJ100) background. Scale bars, 5 mm. (J) The grain appearance of OE-OsBIP1 lines in the o3 background. Scale bars, 5 mm. Data in (B), (C), and (F–H) are means ± SD from at least three biological replicates. Statistically significant differences were determined using Student’s t-test (indicated by different lowercase letters (P < 0.05); ∗P < 0.05, ∗∗P < 0.01).

Figure 6

The o3 mutant is more prone to ER stress under high-temperature conditions. (A) Appearance of WT and o3 grains under high-temperature conditions (35°C, 12 h light/28°C, 12 h dark) and normal-temperature conditions (28°C, 12 h light/22°C, 12 h dark). Scale bars, 5 mm. (B–E) The grain weight (B), total starch content (C), amylose content (D), and total protein content (E) of WT and o3 grains under high- and normal-temperature conditions. (F) SDS-PAGE profiles of total storage proteins of WT and o3 dry grains. (G) Transcript levels of genes related to ER stress in WT and o3 grains at 9 DAF under high- and normal-temperature conditions. (H) Semi-quantitative PCR analysis of OsbZIP50 transcripts in WT and o3 endosperm cells under high- and normal-temperature conditions. U and S, the unspliced and spliced forms of the OsbZIP50 mRNA, respectively. Data in (B–E) and (G) are means ± SD from at least three biological replicates. Significant differences are indicated by different letters according to Student’s t-test.

Figure 7

Proposed model of the role of O3 in maintaining ER homeostasis and regulating endosperm storage protein and starch biosynthesis in rice. Under normal conditions in the WT, O3 is located in the ER and the nucleus. It simultaneously regulates ER protein processes and secretion as well as storage protein and starch biosynthesis in endosperm cells by binding to specific motifs, such as pUPRE-II, O2, and the GCN4 box, to activate transcription of UPR genes and storage protein and starch biosynthesis genes, ultimately ensuring normal development of rice grains. However, mutation of O3 leads to downregulated expression of ER stress-related genes, such as OsBIP1 and PDIL1-1, as well as genes related to storage protein and starch biosynthesis, resulting in ER stress and an impaired protein folding process in the ER. This leads to excessive accumulation of the 57-kDa glutelin precursor and reduces the contents of starch and storage proteins. High temperature can aggravate ER stress and lead to more abnormal grain development in the o3 mutant. As physiological feedback, more O3 (mutated) is transferred to the nucleus from the ER, together with OsbZIP50 and other unknown TFs, to activate expression of key UPR genes to maintain ER homeostasis, especially under high-temperature conditions.