OsbZIP58, a basic leucine zipper transcription factor, regulates starch biosynthesis in rice endosperm

Abstract

Starch composition and the amount in endosperm, both of which contribute dramatically to seed yield, cooking quality, and taste in cereals, are determined by a series of complex biochemical reactions. However, the mechanism regulating starch biosynthesis in cereal seeds is not well understood. This study showed that OsbZIP58, a bZIP transcription factor, is a key transcriptional regulator controlling starch synthesis in rice endosperm. OsbZIP58 was expressed mainly in endosperm during active starch synthesis. osbzip58 null mutants displayed abnormal seed morphology with altered starch accumulation in the white belly region and decreased amounts of total starch and amylose. Moreover, osbzip58 had a higher proportion of short chains and a lower proportion of intermediate chains of amylopectin. Furthermore, OsbZIP58 was shown to bind directly to the promoters of six starch-synthesizing genes, OsAGPL3, Wx, OsSSIIa, SBE1, OsBEIIb, and ISA2, and to regulate their expression. These findings indicate that OsbZIP58 functions as a key regulator of starch synthesis in rice seeds and provide new insights into seed quality control.

Keywords: Endosperm; OsbZIP58; coordination; rice; starch biosynthesis..

Fig. 1.

Assay of binding of OsbZIP transcription factors to the Ha-2 fragment of the Wx promoter and the C53 fragment of the SBE1 promoter in yeast. (A) Diagram of the p178-C53/p178-Ha2 reporter constructs and pPC86-bZIP bait construct. P _CYC1, the minimal promoter of the yeast cytochrome C1 gene; GAL4 AD, GAL4 activation domain; P _ADH1, a constitutively active ADH1 promoter; T _ADH1, ADH1 transcription termination signal. (B) Detection of interaction between OsbZIP transcription factors and the chimeric promoters by yeast one-hybrid analysis. The blue yeast colonies indicate positive interactions. (C) Quantitative assays of β-galactosidase (β-gal) activity in different yeast transformants. Data are presented as means ±standard deviation (SD) from six replicates in two assays. Light grey columns indicate pPC86-bZIP transformed into EGY48 (p178-Ha2); dark grey columns indicate pPC86-bZIP transformed into EGY48 (p178-C53). (This figure is available in colour at JXB online.)

Fig. 2.

Gene structure of OsbZIP58, and grain size and weight of the osbzip58 mutants and CLs. The CLs are transformants containing a wild-type OsbZIP58 gene in the osbzip58-1 mutant background. (A) Diagram of the OsbZIP58 gene structure and T-DNA insertion positions in osbzip58-1 (PFG_1B-15317.R) and osbzip58-2 (PFG_3A-09093.R). Exons are shown as black boxes. Both mutants had a T-DNA insert in the first intron. Primers used in the genotype analysis are indicated by black arrows. GE0279 and GE0301 are gene-specific primers, and GE0295 is the T-DNA specific-primer. (B–E) Grain weight (B), grain length (C), grain width (D), and grain thickness (E). Fifty seeds were analysed for seed size, and data are presented as means ±SD. The 1000-grain weight was determined by counting ten replicates of 100-grain samples independently on an electronic balance. Data are shown as mean ±sd. Two-tailed unpaired tests indicate significant differences in 1000-grain weight and seed size. **P <0.01.

Fig. 3.

Altered seeds phenotype of the osbzip58 mutants and CLs. (A–D) Dongjin; (E–H) osbzip58-1; (I–L) osbzip58-2; (M–P) CL1; (Q–T) CL2. The appearance of mature seeds is shown in (A), (E), (I), (M) and (Q). Cross-sections of mature seeds are shown in (B), (F), (J), (N) and (R). SEM of the central area of mature endosperm is shown in (C), (G), (K), (O) and (S), from the cross-sections in (B), (F), (J), (N), and (R), respectively, indicated by a red square. SEM of the ventral area of mature endosperm is shown in (D), (H), (L), (P) and (T), from the cross-sections in (B), (F), (J), (N), and (R), respectively, indicated by a blue square. Bars: 1mm (A, B, E, F, I, J, M, N, Q, R; 10 μm (C, D, G, H, K, L, O, P, S, T).

Fig. 4.

Altered starch granules morphology in the wild-type Dongjin and the osbzip58-1 mutant examined using semi-thin sections. Immature seeds were fixed in FAA and stained with ammonium methylbenzene blue. (A, C) Dongjin; (B, D) osbzip58-1. (A, B) 10 DAF; (C, D) 15 DAF. a, Amyloplast; c, endosperm cell; p, protein body; s, starch granule. Bars, 50 μm.

Fig. 5.

Altered starch content and fine structure of amylopectin in mutants of OsbZIP58. (A) Total starch content in endosperm (n=5). (B) Apparent amylose content in endosperm (n=5). (C) Soluble sugar content in endosperm (n=5). (D) Differences in the chain length distributions between Dongjin and osbzip58-1 / osbzip58-2. (E) Differences in the chain length distributions between Dongjin and CL1/CL2.

Fig. 6.

Expression pattern of OsbZIP58. (A) Expression patterns of OsbZIP58 in roots, stems, leaves, flowers, seedlings, and seeds analysed by qRT-PCR. The developmental stage of the seed is indicated by DAF. Rice OsAct1 was used as a control. (B, C) Detection of OsbZIP58 mRNA in cross-sections of a maturing rice seed by in situ hybridization at 5 DAF (B) and 7 DAF (C). The region expressing OsbZIP58 is shown in purple. Antisense strand was used as a probe. (D) In situ hybridization with a sense-strand probe in maturing rice seed at 7 DAF. P, Pericarp; DV, dorsal vascular; E, endosperm. Bars, 100 μm (B); 200 μm (C, D).

Fig. 7.

Expression profiles of rice starch synthesis genes during seed development in wild-type Dongjin and osbzip58-1 mutant. Total RNA was extracted from seeds at 3, 5, 7, 10, 15, and 20 DAF. The expression of each gene in the 3 DAF seeds of Dongjin was used as a control. All data are shown as means ±SD from five biological replicates. Two-tailed unpaired t-tests were used to determine significant differences. *P <0.05; **P <0.01.

Fig. 8.

OsbZIP58 broadly bind to the promoters of rice starch metabolism genes in vivo. (A) Diagram of the promoter region from –2000bp upstream of the putative transcription initiation site to the translation start site ATG in the ten rice starch metabolism genes. Vertical black lines indicate the ACGT elements. Arrowheads indicate the putative transcription initiation site. Vertical arrows indicate the translation start site ATG. PCR fragments are indicated by thick lines. (B) Quantitative real-time PCR assay of chromatin immunoprecipitated DNA. Normal rabbit IgG was used for the negative control. ‘% input’ represents the qPCR signals that were derived from the ChIP samples versus qPCR signals that were derived from the input sample taken early during the ChIP procedure. All data are shown as means ±SD from six biological replicates. Two-tailed unpaired t-tests were used to determine significant differences. *P <0.05; **P <0.01. (C) Detection of interactions between OsbZIP58 and the chimaeric promoters by yeast one-hybrid analysis. The plasmids pPC86-OsbZIP58 and p178 were transformed into EGY48, and colonies were selected on selection medium (SD/–Ura–Trp+X-gal). The blue yeast colonies indicate positive interactions. (D) Quantitative assays of β-galactosidase (β-gal) activity in different yeast transformants. Data are presented as means ±SD from six replicates in two assays. (This figure is available in colour at JXB online.)