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Underlying Mechanisms of Zymographic Diversity in Starch Synthase I and Pullulanase in Rice Developing Endosperm Yaling Chen, and Jinsong Bao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b06030 • Publication Date (Web): 09 Feb 2016 Downloaded from http://pubs.acs.org on February 15, 2016
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Journal of Agricultural and Food Chemistry
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Underlying Mechanisms of Zymographic Diversity in Starch Synthase I and
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Pullulanase in Rice Developing Endosperm
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Yaling Chen,† Jinsong Bao*†
4 5 6
1
7
Zhejiang University, Huajiachi Campus, Hangzhou, 310029, China
Institute of Nuclear Agricultural Science, College of Agriculture and Biotechnology,
8 9 10 11
*
Corresponding
author:
Jinsong
Bao,
Tel:
+86-571-86971932;
+86-571-86971421; E-mail:
[email protected].
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ABSTRACT: Amylopectin is synthesized by the coordinated actions of many
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(iso)enzymes, including AGPase, SSs, BEs and DBEs. Here, two polymorphic forms
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of SSI and pullulanase (PUL) in rice developing seeds, designated as SSI-1/SSI-2
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and PUL-1/PUL-2, were discovered for the first time by zymographic analysis. The
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SSI and PUL polymorphisms were strongly associated with the SSI microsatellite
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marker (P=3.6×10-37) and PUL InDel markers (P16 by branching lesser branched
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polyglucans.17 Loss of PUL in rice confers an increase in short chains (DP≦13) of
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amylopectin.18. BEI and PUL are members of the CAZy glycoside hydrolase family
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13 (GH13), which have three major domains, the catalytic domain, the N-terminal
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domain and the C-terminal domain.19 The crystal structures of barley PUL have been
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revealed by the X-ray crystallographic analysis.20 Although the crystal structures of
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PUL among different plants are highly similar, the amino acid variations in the three
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major domains may affect PUL activity and binding affinity with carbohydrates and
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then affect the starch properties.
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In this study, we reported for the first time that SSI and PUL display zymographic
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polymorphisms
among
different
rice
genotypes.
Western
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spectrometric analysis and co-immunoprecipitation analysis were carried out to
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discover the molecular mechanism underlying the polymorphisms existed in the SSI
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and PUL enzymes in rice endosperm. The results from this study will enhance our
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understanding of starch biosynthesis-related enzymes properties and provide helpful
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information for starch biosynthesis in rice endosperm.
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MATERIALS AND METHODS 5
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mass
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Materials. A total of 390 rice varieties from an association mapping panel and a
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reference rice Nipponbare were used in this study.21 The population structure was
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estimated using 100 microsatellite markers, and seven subpopulations (groups) were
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revealed.21 POP1, POP2, POP3, POP4, POP6 and POP7 consist of indica accessions,
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whereas POP5 consists of japonica accessions.21 Rice plants were grown at the
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Zhejiang University Farm in 2014. Developing grains from 10-15 day after flower
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(DAF) were collected and immediately frozen on ice, and then stored at -80℃ until
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use. Antiserums against rice SSI, PUL, BEI and BEIIb were kind gifts from Dr.
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Naoko
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Adenosine-5′-diphosphoglucose disodium salt, oyster glycogen and potato
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amylopectin were purchased from sigma (Australia).
Fujita
at
Akita
Prefectural
University,
Akita,
Japan.
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Extraction of Soluble Proteins. Soluble protein were extracted in 3 volume of
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extraction buffer containing 50 mM imidazole, pH 7.4, 8 mM MgCl2, 10% Glycerol,
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500 mM β-Mercaptoethanol followed by centrifugation at 12,000×g with
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microcentrifuge for 10 min at 4℃. The step was repeated twice using 2 volume of
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extraction buffer. The supernatant was collected and the protein concentration was
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estimated using NANODROP 2000 spectrophotometer (Thermo, Canada).
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Analysis of Zymogram. Native-polyacrylamide gel (PAGE) activity staining of SS
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was performed on 7.5% and 6.0% (w/v) acrylamide slab gel containing 0.8% (w/v)
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oyster glycogen according to Nishi et al.22 and Fujita et al.23,24 protocol. 6
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Journal of Agricultural and Food Chemistry
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Native-PAGE activity staining of DBEs was performed on 7.5% and 6.0%
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acrylamide slab gel containing 0.17% (w/v) rice amylopectin (sigma, Australia) as
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described in Fujita et al.25
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Mass Spectrometric Analysis. Protein bands representing different loci were
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incised. The protein samples were sent to Shanghai Applied Protein Technology
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Company (Shanghai, China). Through Tandem Mass Tag™ (TMT) method,
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enzymolysis of peptide fragment was recorded using Thermo Scientific Q Exactive
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(Thermo Finnigan, San Jose, Canada).
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Markers for Genotyping. Total DNA was extracted from fresh leaves using
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modified cetyl trimethyl ammonium bromide method.26 To detect a single-base
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substitution by dCAPS analysis, a mismatch was made in the sense primer (5’-
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CAAGTGTAAAGCTGAATTGAAG -3’) using dCAPS Finder 2.0 software.27 It
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generated an additional MboII site in the Nipponbare SSI allele (GAAGA) (Genbank
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accession: Os06g0160700) but not in the 93-11 allele (GAAGG) (Genbank accession:
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AY299404.1). The antisense primer was 5’- TTGAATATTGTCCCGCATGAGA -3’.
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Two units of MboII (Takara, Dalian, China) were added to 20 µl of PCR product and
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mixture was incubated for 4 h at 37 ℃. All amplifications were performed on a
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MG96G thermal cycler (Hangzhou LongGene Scientific Instruments Co. Ltd.,
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Hangzhou, China). The PCR products were separated by electrophoresis in 8%
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PAGE with 3.4% cross-linker (ratio of bisacrylamide to acrylamide). 7
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Co-immunoprecipitation. Immunoprecipitation experiments were conducted using
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the methods described by Crofts et al.28 with some modifications. The supernatant of
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450 µl of each sample (10 mg/ml ) was mixed with 20 µl of antibody of SSI or PUL
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for 2 h at 4 ℃. A 470 µl aliquot of reconstituted 50% protein A-Sepharose resin
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(TransGen)
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A-Sepharose-antibody-protein complex was centrifuged at 6000 g for 3 min at 4 ℃
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and the supernatant was discarded. The resin was then washed eight times with PBS
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(137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 1.8 mM KH2PO4, pH 7.4). Bound
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proteins were released by boiling for 10 min in 1×SDS buffer. After centrifugation at
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12 000 rpm for 3 min, 6µl was used for western blotting.
was
added
and
incubated
for
3
h
at
4℃.
The
protein
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Western Blotting. The protein bands were blotted onto polyvinylidene fluoride
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fluoride (PVDF) membranes by transblotter after Native-PAGE and SDS-PAGE.
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SDS-PAGE western blotting procedure was performed according to Crofts et al.29,
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and Native-PAGE western blotting procedure was modified appropriately.
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Statistical Analysis. Association analysis between marker alleles including 41
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markers for starch synthesis-related genes9 and the polymorphic forms of SSI or
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PUL was performed with TASSEL Version 2.1 software, taking the gross-level
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population structure (Q) and kinship (K) into account.30,31 The P value determining
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whether markers were associated with protein was set at P
