Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 7, 40124

OsBT1 Encodes an ADP-glucose Transporter Involved in Starch Synthesis and Compound Granule Formation in Rice Endosperm

Affiliations

OsBT1 Encodes an ADP-glucose Transporter Involved in Starch Synthesis and Compound Granule Formation in Rice Endosperm

Sanfeng Li et al. Sci Rep.

Abstract

Starch is the main storage carbohydrate in higher plants. Although several enzymes and regulators for starch biosynthesis have been characterized, a complete regulatory network for starch synthesis in cereal seeds remains elusive. Here, we report the identification and characterization of the rice Brittle1 (OsBT1) gene, which is expressed specifically in the developing endosperm. The osbt1 mutant showed a white-core endosperm and a significantly lower grain weight than the wild-type. The formation and development of compound starch granules in osbt1 was obviously defective: the amyloplast was disintegrated at early developmental stages and the starch granules were disperse and not compound in the endosperm cells in the centre region of osbt1 seeds. The total starch content and amylose content was decreased and the physicochemical properties of starch were altered. Moreover, the degree of polymerization (DP) of amylopectin in osbt1 was remarkably different from that of wild-type. Map-based cloning of OsBT1 indicated that it encodes a putatively ADP-glucose transporter. OsBT1 coded protein localizes in the amyloplast envelope membrane. Furthermore, the expression of starch synthesis related genes was also altered in the osbt1 mutant. These findings indicate that OsBT1 plays an important role in starch synthesis and the formation of compound starch granules.

Figures

Figure 1
Figure 1. Characterization of the osbt1 mutant.
(a,e) Appearance of mature seeds of wild-type (WT) (a) and osbt1 (e); (b,f) Cross-sections of mature seeds of WT (b) and osbt1 (f); (c,g) SEM analysis of the periphery area of mature endosperm of WT (c) and osbt1 (g). The periphery areas shown are indicated as blue squares in (b,f); (d,h) SEM analysis of the central area of mature endosperm of WT (d) and osbt1 (h). The central areas shown are indicated as red squares in (b,f); Scale bars: 1 mm in (a,b,e,f); 20 μm in (c,d,g,h); (il) Quantification of WT and osbt1 seed size and grain weight including grain length (i), grain width (j), grain thickness (k) and 1000-grain weight (l). Data are given as means ± SD from three replicates. Statistical comparisons were performed using Student’s t-test; all data were compared with WT (*P < 0.05, **P < 0.01).
Figure 2
Figure 2. Abnormal compound granules formation in osbt1 seeds.
Semi-thin sections of WT (a–c) and osbt1 mutant (d–f) endosperm at 9 DAF. (a,b,d,e) The central region of endosperm cells; (c,f) The peripheral region of endosperm cells. Stars indicate aleurone cells in (c,f). Arrowheads in (e) indicate smaller, abnormal starch granules in cytosol. Scale bars: 10 μm.
Figure 3
Figure 3
Electron micrographs depicting amyloplast development in WT (a–d) and the osbt1 mutant (e,h) endosperms. (a,e) A representative amyloplast in peripheral endosperm cells of WT and osbt1 at 6 DAF; (b–d,f–h) An amyloplast in central endosperm cells of WT and osbt1 at 6 (b,f), 9 (c,g), and 12 DAF (d,h), respectively. White arrows indicate the stroma inside the amyloplast. Bars: 2 μm.
Figure 4
Figure 4. Seed properties and starch physicochemical characteristics in the osbt1 mutant.
(a–d) Quality trait parameters of the WT and osbt1. Values are means ± SD (n = 3). The asterisks indicate statistical significance between WT and the mutant, as determined by a Student’s t–test (*P < 0.05; **P < 0.01). (e) Differences in the amylopectin chain length distributions between the WT and osbt1. (f) Pasting properties of endosperm starch of WT (blue line) and osbt1 (red line). The viscosity value at each temperature is the average of three replicates. The gray line indicates the temperature changes during the measurements. (g) Gelatinization temperature of endosperm starch. To, Tp, and Tc represent the onset, peak, and conclusion gelatinization temperatures, respectively. All data are presented as means ± SD from three replicates. Two-tailed unpaired t-tests indicate the significant differences: *P < 0.05, **P < 0.01. (h) Gelatinization characteristics of starch from osbt1 mutant seeds. Starch powder was mixed with different concentrations (1–9 M) of urea solution. The most significant difference was observed for 3–4 M urea.
Figure 5
Figure 5. Map-based cloning, complementation of the osbt1 mutant and phenotype of transgenic lines.
(a) Fine mapping of the OsBT1 locus. The OsBT1 locus was mapped to a 39.5 kb region by markers Indel1-3 and Indel1-10 on chromosome 2 (Chr.2), which contained seven predicted genes. The molecular markers and the number of recombinants are shown. cM, centimorgan; ORF, open reading frame. (b) Gene structure and mutation site in osbt1. The cDNA sequence comparison shows the 18 bp insertion in osbt1 leading to six amino acid addition. (c) Identification of the 18 bp insertion between the WT and osbt1 using an Indel marker. M, Marker. (d) Complementation of the osbt1 mutation in transgenic lines (S1–S2) and overexpressing the OsBT1 in osbt1 (OX-1, OX-2) showing the completely restored WT seed appearance (d) and the starch granules morphology (e). RNAi of OsBT1 in WT genotype produce abnormal seed appearance. RNAi seeds (Ri-1, Ri-2) became chalkiness (d), and the starch granules became abnormal (e). Insets in (d) represent the transverse sections of representative grains. Bars: 1 mm in (d); 20 μm in (e).
Figure 6
Figure 6. Expression pattern of OsBT1.
(a) qRT-PCR analysis of OsBT1 expression level in various tissues and in developing endosperms of the WT and osbt1. Values are means ± SD (n = 3). The asterisks indicate statistical significance between the WT and the mutant, as determined by a Student’s t-test (*P < 0.05; **P < 0.01). (b–h) GUS expression in root (b), steam (c), leaf (d), leaf sheath (e), panicle (f), spikelet (g) and brown rice (h) driven by the OsBT1 promoter. Bars: 2 mm.
Figure 7
Figure 7. Subcellular localization of OsBT1.
(a) Free GFP used as a control. (b,c) Full-length coding region fusion protein OsBT1-GFP (b) and osbt1-GFP (c) in front of the GFP. 16 h after transformation, tobacco protoplasts were observed using a confocal laser scanning microscope. Green fluorescence signals (GFP), red chlorophyll autofluorescence (red), bright-field images, and an overlay of green and red signals are shown in each panel. (d) OsBT1-GFP fusion protein was expressed in rice under the control of the maize UBIQUITIN1 promoter and then the localization of OsBT1-GFP in developing endosperm was observed by confocal laser scanning microscope. The outer limit envelope of the amyloplasts is indicated by white arrowhead. Scale bars: 10 μm.
Figure 8
Figure 8. Expression profiles of rice starch synthesis genes during seed development in WT and osbt1.
Total RNA extracted from developing seeds at 6, 9, 12 and 15 DAF was used for qRT-PCR analysis. The expression of each gene in the 6 DAF WT seeds was set as reference value of 1. All data are shown as means ± SD from three replicates. The asterisks indicate statistical significance between the WT and the mutant, as determined by a Student’s t-est (*P < 0.05; **P < 0.01).

Similar articles

See all similar articles

Cited by 11 PubMed Central articles

See all "Cited by" articles

References

    1. Lopez-Juez E. & Pyke K. A. Plastids unleashed: their development and their integration in plant development. Int. J. Dev. Biol. 49, 557–577 (2005). - PubMed
    1. Sakamoto W., Miyagishima S. Y. & Jarvis P. Chloroplast biogenesis: control of plastid development, protein import, division and inheritance. Arabidopsis Book. 6, e0110 (2008). - PMC - PubMed
    1. Martin C. & Smith A. M. Starch biosynthesis. Plant Cell. 7, 971–985 (1995). - PMC - PubMed
    1. Hanashiro I. et al. . Granule-bound starch synthase I is responsible for biosynthesis of extra-long unit chains of amylopectin in rice. Plant Cell Physiol. 49, 925–933 (2008). - PubMed
    1. James M. G., Robertson D. S. & Myers A. M. Characterization of the maize gene sugary1, a determinant of starch composition in kernels. Plant Cell. 7, 417–429 (1995). - PMC - PubMed

Publication types

Feedback