Molecular Structure and Physicochemical Properties of Starches from

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Molecular structure and physicochemical properties of starches from rice with different amylose contents resulting from modification of OsGBSSI activity Changquan Zhang, Shengjie Chen, Xinyu Ren, Yan Lu, Derui Liu, Xiuling Cai, Qianfeng Li, Jiping Gao, and Qiaoquan Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05448 • Publication Date (Web): 27 Feb 2017 Downloaded from http://pubs.acs.org on February 28, 2017

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Journal of Agricultural and Food Chemistry

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Molecular structure and physicochemical properties of starches

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from rice with different amylose contents resulting from

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modification of OsGBSSI activity

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Changquan Zhang , Shengjie Chen , Xinyu Ren , Yan Lu , Derui Liu , Xiuling

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Cai‡,§, Qianfeng Li†,§, Jiping Gao‡,§, Qiaoquan Liu*,†,§

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†,§

†

†

†

†

‡

Key Laboratory of Crop Genetics and Physiology of Jiangsu Province, Key Laboratory of Plant

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Functional Genomics of the Ministry of Education, Yangzhou University, Yangzhou 225009,

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China ‡ National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology,

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Chinese Academy of Sciences, Shanghai 200032, China

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§

Co-Innovation Center for Modern Production Technology of Grain Crops of Jiangsu Province /

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Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of

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Education, Yangzhou University, Yangzhou 225009, China

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*

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Yangzhou 225009, China. Tel.: +86 514 8797 9242. e-mail: [email protected]

Corresponding Author. Address: College of Agriculture, Yangzhou University,

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E-mail addresses:

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Chagnquan Zhang: [email protected]

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Shengjie Chen: [email protected]

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Xinyu Ren: [email protected]

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Yan Lu: [email protected]

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Qianfeng Li: [email protected]

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Derui Liu: [email protected]

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Xiuling Cai: [email protected]

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Jiping Gao: [email protected]

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ACS Paragon Plus Environment


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ABBREVIATIONS USED.

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AAC, apparent amylose content; AC, amylose content; ATR-FTIR, attenuated total

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reflectance-fourier transform infrared; BDV, breakdown viscosity; CPV, cool paste

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viscosity; DP, degree of polymerization; DSC, diﬀerential scanning calorimeter; ECQ,

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eating and cooking quality; GBSSI, granule-bound starch synthase I; GC, gel

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consistency; GPC, gel permeation chromatography; HPAEC, high-performance

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anion-exchange chromatography; Ptemp, pasting temperature; PC, protein content;

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RVA, Rapid Visco Analyzer; To, onset temperature of gelatinization; ∆H, enthalpy of

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gelatinization; XRD, X-ray powder diffraction; SXAS, small-angle X-ray scattering.

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ABSTRACT

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OsGBSSI, encoded by the Waxy (Wx) gene, is the key enzyme in synthesis of amylose

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chains. Transgenic rice lines with varying GBSSI activity were previously developed

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via site-directed mutagenesis of the Wx gene in the glutinous cultivar

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Guanglingxiangnuo (GLXN). In this study, grain morphology, molecular structure,

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and physicochemical properties were investigated in four transgenic lines with

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modified OsGBSSI activity and differences in amylose content. A milky opaque

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appearance was observed in low and non-amylose rice grains due to air spaces in the

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starch granules. Gel permeation chromatography (GPC) and high-performance

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anion-exchange chromatography (HPAEC) analyses showed that while OsGBSSI can

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synthesize intermediate and extra-long amylopectin chains, it is mainly responsible

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for the longer amylose chains. Amylose content was positively correlated with trough 2


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viscosity, final viscosity, setback viscosity, pasting time, pasting temperature and

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gelatinization temperature, and negatively with gel consistency, breakdown viscosity,

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gelatinization enthalpy and crystallinity. Overall, the findings suggest that OsGBSSI

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may be also involved in amylopectin biosynthesis, in turn affecting grain appearance,

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thermal and pasting properties, and the crystalline structure of starches in the rice

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endosperm.

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KEYWORDS: Oryza sativa L., amylose, OsGBSSI, physicochemical properties,

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starch fine structure, crystallinity

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INTRODUCTION

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Starch is the major energy source in cereal grains. Normal cereal starch is made up of

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long-chain linear amylose with a few branches and large numbers of highly branched

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short-chain amylopectin.1 The amylopectin branches can further be divided into three

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groups, A-, B- and C-types, depending on their organization in the amylopectin

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clusters.2 Some branches are arranged in a double helical conformation, packing into

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crystallites that form alternating crystalline and amorphous layers, while amylose is

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typically amorphous and often forms a single helical complex combined with lipid

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molecules. These semi-crystalline starch granules form a layered organization with

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alternating semi-crystalline and amorphous growth rings.3

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In rice, amylose content (AC), gel consistency (GC) and gelatinization

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temperature (GT) are the key components affecting eating and cooking quality 3



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(ECQ).4 Amylose in the rice endosperm is mainly synthesized by granule-bound

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starch synthase I (OsGBSSI), which is encoded by the Waxy (Wx) gene located on

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chromosome 6.5 Thus, AC is controlled mainly by expression levels of Wx as well as

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activity of OsGBSSI.6,7 In addition to AC regulation, the Wx gene is also responsible

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for GC, GT and other physicochemical properties of rice starch.8,9 In general, a higher

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AC is usually associated with a harder texture of cooked rice10. Expression levels of

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Wx and OsGBSSI activity in the endosperm are therefore used as key determinants of

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ECQ variation.4,11 Moreover, there is accumulating evidence to suggest that GBSSI is

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also involved in the biosynthesis of extra-long amylopectin chains in wheat,12 rice13

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and maize.14

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Based on the AC, rice varieties are classified into waxy (0-2%), very low (3-9%),

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low (10-19%), intermediate (20-25%), and high (>25%) amylose types.15 Rice

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cultivars with specific amylose types are required to match the needs of the market in

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different countries. To the best of our knowledge, most of our observed AC diversity

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is due to the various aspects of the natural allelic variation at the Wx locus. In line

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with this, at least seven Wx alleles have so far been identified: Wxa, Wxb, Wxin, Wxmp,

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Wxmq, Wxop and wx.16−21 Several studies have investigated the physicochemical

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properties and starch structure of different rice cultivars with altered AC.22−24

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However, there is a lack of clear information on the relationship between GBSSI

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activity and AC and starch physicochemical properties because of the great genetic

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difference between different rice cultivars.

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We recently developed a method aimed at altering GBSSI enzymatic activity 4


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using site-directed mutagenesis, and subsequently generated eight amino acids

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substitution of OsGBSSI transgenic rice lines with different GBSSI activities and

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different apparent amylose contents (AAC) in the glutinous rice cultivar

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Guanglingxiangnuo (GLXN) background.11 We found all the amino acid substitutions

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caused a reduction in GBSSI activity and AAC compared to wild type.11 Although the

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enzymatic basis of different OsGBSSI mutants has been illustrated in vivo,13 the effect

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of OsGBSSI activity variation on the molecular structure and physicochemical

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properties of rice grain starch remains unclear. In this study, starches from five typical

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transgenetic rice lines with clearly different AAC were used for the fine structure and

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physicochemical properties analysis. The data will help determine the structural and

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physiological properties of rice with different OsGBSSI activity, while providing new

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information for precise engineering of rice grain quality improvement.

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MATERIALS AND METHODS

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Rice samples and growth conditions. Five transgenic rice lines11 and their wild-type,

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the glutinous japonica rice cultivar GLXN, were used in this study (Table 1). As a

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negative control, the transgenic rice line P1300 was used. It carried an empty

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pCAMBIA1300 vector and showed the same glutinous phenotype as the wild-type

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GLXN. The remaining transgenic lines, Y268F, R408G, E410D, and CDS, were

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modified to give a different AAC via mutation of the Wx gene, as well as different

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OsGBSSI activity.11 As reported by Liu et al., CDS represents the wild-type OsGBSSI,

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while Y268F, R408G and E410D represent amino acid substitutions at residue No.

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268, 408 and 410 in the wild-type OsGBSSI protein, respectively.11 The rice lines 5



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were planted in the same field at the experimental farm of Yangzhou University in

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Yangzhou, Jiangsu, China. Plots were arranged in a randomized block pattern, with

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two replications of 10 rows per plot and 10 plants per row. Growing conditions,

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including field management practices, were similar throughout the growing season.

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Seeds were harvested at maturity from 10 plants in the middle of each plot and

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air-dried. Data of each sample represent the mean of the two plots.

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Flour and starch preparation. Mature seeds were air-dried, dehusked with a rice

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huller (Model SY88-TH, Korea), and subsequently polished using a grain polisher

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(Kett, Tokyo, Japan). Milled rice samples were then stored in sealed bags under

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refrigeration at 4°C until analysis. Polished rice samples were then ground in a mill

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(FOSS 1093 Cyclotec Sample Mill, Sweden) with a 0.5 mm screen, and starch

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samples prepared from the milled rice endosperm using the neutral protease method

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as described previously.10

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Composition and physicochemical analyses. The starch content of the milled rice

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flour was analyzed using a total starch assay kit (K-TSTA, Megazyme; Wicklow,

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Ireland). AAC was determined using the iodine colorimetric method,8 and the true AC

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was calculated based on gel permeation chromatography (GPC) analysis (see below

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for details). GC was measured according to the method of Tan et al.8 The crude

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protein content was calculated from the nitrogen content of the rice flour using a

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nitrogen determinator (FOSS TECATOR Kjeltec2300) according to AOAC standard

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method 990.03.25 Moisture content was measured using a halogen moisture analyzer

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(METTLER TOLEDO MJ33, Switzerland), and pasting properties were determined

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using a Rapid Visco-Analyzer (RVA) (Techmaster, Newport Scientific; Warriewood,

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Australia) using the methods of Zhu et al.26 The thermal properties were measured

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with a differential scanning calorimeter (DSC200F3, Netzsch Instruments NA LLC; 6


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Burlington, MA) according to our previous report.10 All tests were performed in

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triplicate.

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Scanning electron microscopy. Grains were randomly selected for phenotypic

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analysis. To obtain cross-sections, grains were frozen in liquid nitrogen, mounted on

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aluminum specimen stubs with adhesive tabs, coated with gold and examined using

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an environmental scanning electron microscope (SEM, Philips XL-30). For SEM

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observations of the starch granules, samples were suspended in ethanol and mounted

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on an aluminum stub using double-sided adhesive tape. The starch samples were then

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observed and photographed after coated with gold using a sputter coater. To

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determine the size distribution, more than 500 complete granules were analyzed per

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sample using Image J software (http://rsbweb.nih.gov/ij/) based on the SEM images.

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GPC and High-Performance Anion-Exchange Chromatography (HPAEC).

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Purified rice starch was debranched with isoamylase (EC3.2.1.68, E-ISAMY,

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Megazyme) and the relative molecular weight distribution of the debranched starch

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determined by GPC with a PL-GPC 220 system (Polymer Laboratories Varian, Inc.

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Amherst, MA). The PL-GPC 220 system included three columns (PL110-6100, -6300,

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-6525) with a differential refractive index detector (DRI）according to our recent

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report.27 The GPC data used for drawing the molecular weight distribution curves was

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transformed through integral equations based on dextrans of known molecular

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weights （2800, 18 500, 111 900, 192 410 000, 1 050 000, 2 900 000, and 6 300 000）.

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With use of dextran standards, the GPC data with DRI detection, which are

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distributions of molecular size, are reported as dextran-equivalent molecular weight

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denoted MW. For comparison between AP1, AP2 and AM, two replicate

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measurements were performed, and the normalized to have the same area under the

7



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curve. The debranched starch was also quantitatively analyzed using HPAEC

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(Thermo ICS-5000, Thermo Corp, Sunnyvate, CA) equipped with a pulsed

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amperometric detector, a guard column, a CarboPacTM PA200 analytical column,

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and an AS-DV auto sampler according to our recent report.26

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X-ray Powder Diffraction. X-ray powder diffraction (XRD) analysis of the starches

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was investigated on a D8 ADVANCE type X-ray diffractometer (D8, Bruker,

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Germany). All samples were treated in a desiccator with a saturated solution of NaCl

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to maintain a constant humidity (relative humidity = 75%) for 7 days prior to XRD

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analysis. The relative crystallinity of the starches was determined as described by Wei

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et al.28

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Small-angle X-ray scattering. Small-angle X-ray scattering (SAXS) analysis of

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isolated starch samples was performed according to Cai et al.24 SAXS measurements

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were obtained using a Bruker Nano Star SAXS instrument equipped with a Vantec

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2000 detector and pinhole collimation for point focus geometry. The SAXS data were

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analyzed using Diffract Plus Nano Fit software. SAXS was implemented using the

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method of Yuryev et al.29 Bragg spacing (D), which represents the lamellar distance,

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was calculated from the position of the peak (qo) using D = 2π/qo. Experiments were

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carried out in duplicate.

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Attenuated Total Reflectance-Fourier Transform Infrared. Attenuated total

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reflectance-Fourier transform infrared (ATR–FTIR) analysis of the starch was carried

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out using a Varian 7000 FTIR spectrometer with a DTGS detector equipped with an

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ATR single reflectance cell containing a germanium crystal (45° incidence angle;

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PIKE Technologies) using an attenuated total reflectance accessory according to our

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previous method.27 Absorbance values at 1047 and 1022 cm−1 were extracted from the 8


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spectra after correction.

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Statistical Analysis. For sample characterization, at least two replicate measurements

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were performed unless otherwise specified. All data represent the means ± standard

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deviation (means ± SD) of the two plots. Data were subjected to one way ANOVA

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and Tukey’s multiple comparison analysis using SPSS 16.0 statistical software

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program, and Pearson’s bivariate correlations using the same software. Results with a

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corresponding probability value of p < 0.05 were considered statistically significant.

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RESULTS AND DISCUSSION

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Amylose and other components in the milled rice. The AAC of wild-type GLXN

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and its negative control transgenic line P1300 was very low (Table 1), and there was

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no effect on the general grain quality after transformation of the empty vector p1300

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into the GLXN background. In the transgenic lines E410D, Y268F and R408G, the

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AAC of the milled rice flour was 8.27, 10.03 and 16.34%, respectively (Table 1),

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which was relatively lower than that of the CDS transgenic rice with the wild-type

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OsGBSSI (17.57%). These data are consistent with our previous report.11 The true AC

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(AM) of the isolated starches was also estimated using GPC analysis (Table 2). No

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amylose was detected in the glutinous GLXN or P1300 rice starches, while the

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remaining four samples exhibited a relatively low level compared with

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the corresponding AAC results. This was possibly due to absorption of similar

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wavelengths by the amylopectin-iodine and amylose-iodine complexes during

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colorimetry analysis.

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To determine the effect of amylose variation on rice ECQ, the major components

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and physicochemical characteristics of milled rice flour were investigated and 9



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compared. All flour and starch samples had similar moisture and crude protein

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contents (Table 1). A total starch content assay showed that rice grains with a higher

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AC tended to contain a relatively higher total starch content, although the differences

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were not significant. In terms of GC (Table 1), a strong negative correlation with the

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AAC was observed in all six rice flours (r = -0.95, p < 0.01), confirming that the Wx

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gene is the major gene controlling AC and GC.30

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Appearance of grains and starch granules. Figure 1A shows the phenotype of

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milled rice from the test rice samples. P1300 rice, which was transformed with the

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empty vector, showed a typical opaque phenotype identical to that of wild-type

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GLXN. Grains from E410D showed a non-transparent phenotype, but had a

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distinguishable white-belly. Grains from Y268F showed a milky phenotype similar to

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that of low amylose mutants such as Milky Queen.17 Grains of both R408G and CDS

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grains were translucent. In fact, except for GLXN and P1300, all grains showed a

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clear white-belly, possibly due to the loosely-packed, round starch granules and large

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air spaces.31 These phenotypes were also likely affected by the complicated genetic

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background of the GLXN cultivar, not just modification of OsGBSSI activity. Overall,

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these results suggest that as AC decreases, so too does grain transparency.

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SEM was used to further investigate the opaque appearance and determine whether

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there was a significant difference in starch granule morphology among grains with a

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different appearance. Figure 1B1–G4 shows the SEM micrographs of transverse

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sections of mature grains. The typical characteristics of chalky regions are observed,

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with the compound starch granules loosely packed, with air spaces within or between

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them in all the white-belly parts of tested grains (Figure 1B–G2). All starch granules

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in the non-chalky region were packed tightly together (Figure 1B3–G3); however,

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some holes in the core of starch granules were observed in glutinous and low AC 10


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grains (Figure 1B3, 4–E3, 4). Both GLXN and P1300 grains showed the same number

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of holes, while grains from Y268F and E410D had few holes. Like most regular

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japonica and indica rice, grains from R408G and CDS, which contained an

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intermediate AC, showed no holes in their starch granules (Figure 1F1–G4). These

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observations suggest that the air space in the granule center causes the milky and

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opaque appearance due to refraction. We also concluded that as the AC decreased in

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the glutinous and low AC lines, the number of air spaces increased, thereby resulting

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in the opaque appearance. Because P1300 was a negative control of the transgenic

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rice and showed the same appearance as GLXN, it was subsequently used as a

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glutinous control line during further analyses.

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We also used SEM to compare the morphology of isolated starch granules from

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grains of the five transgenic rice lines. All five lines showed a similar morphology;

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that is, a polyhedral and irregular shape with sharp angles and edges (Figure 2A–E).

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However, small holes were observed in some of the granules, possibly in poorly

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developed starch from rice grown in chalky regions. Size distribution was

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subsequently investigated revealing a unimodal peak with all five samples (Figure 2F).

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Moreover, there was no significant difference in the average particle size, which

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ranged from 4.77 to 5.00 µm.

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Pasting properties of the rice flours and starch granules. RVA pasting properties

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reflect changes in the apparent viscosity of a sample during heating and cooling in

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sufficient water, thereby predicting the texture of cooked rice. Flour and starch pasting

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properties are shown in Figure 3, with a summary provided in Supplementary Table

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S1. Though flours and starches from different transgenic rice had significant amylose

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levels, the RVA profiles of peak viscosity (PKV) showed no correlation with the AC

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of the non-glutinous sample. This is in agreement with other studies suggesting a lack 11



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of correlation between AC and PKV.10,16 In line with this, many factors are known to

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affect PKV such as α-amylase activity, protein structure and lipid content.32,33 Flour

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and starch from P1300 showed the lowest PKV, in agreement with the easily

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disintegrated and lower rigidity of glutinous starch granules, which in turn might

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reduce the resistance to shear force, consequently decreasing the peak viscosity.34

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Other characteristics including trough viscosity, final viscosity, setback viscosity,

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pasting time and pasting temperature showed a good correlation with AC, increasing

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with increasing AC, except in P1300. This finding confirms the suggestion that rice

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starch with a low AC is more prone to gelatinization, in agreement with previous

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reports on rice and maize.14,35 These results also suggest that a high level of amylose

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leached from higher amylose starch and reorganized during cooling results in a higher

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setback value. In contrast, as for the breakdown viscosity, a decreasing trend was

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observed with increasing AC, possibly because amylose intertwines with amylopectin,

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helping maintain the integrity of the starch granules and thereby reducing the

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breakdown of swollen starch-granules.36,37

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Fine structure of the different starches. The relative molecular weight distributions

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of rice starch from the five samples were determined by GPC. Except for P1300, a

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trimodal distribution with low, mid, and high molecular weight peaks was observed;

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namely, AP1, AP2 and AM fractions, respectively (Figure 4A). The AM fraction

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represents amylose and consists of two peaks, AM1 and AM2, which represent the

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relatively short and long amylose chains, respectively.38 In general, the area ratio of

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AM to (AM+AP1+AP2) represents the true AC. As shown in Table 2, excluding

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P1300, starch from E410D had the lowest AC followed by Y268F, R408G and CDS.

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Interestingly, the AM1-to-AM2 ratio showed a significant decreasing trend (r = -0.95,

287

p < 0.05) with increasing GBSSI activity (Figure 4B and Table 2). This was 12


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consistent with a recent study in which rice starch from samples with a high AC was

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found to have a relatively higher number of long amylose chains (AM2) in

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comparisons between Oryza. barthii, Oryza. glaberrima and Oryza. sativa.39 It has

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also been reported that GBSSI is responsible for the biosynthesis of extra-long

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amylopectin branch chains in normal starches from various crops including rice,

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wheat and maize.12−14 Therefore, the AM1 fraction perhaps contains extra-long branch

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chains since both extra-long branch chains and short amylose chains share a similar

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molecular weight.39,40 Though other key enzymes such as starch branching enzyme

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(SBE) almost certainly play a role in amylose structure, affecting the length of

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amylose chains through branching,40 the above results suggest that longer amylose

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chains are mainly synthesized by GBSSI in normal cultivars. However, in some wild

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rice, there can be a large number of short amylose chains, most likely due to a new

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Wx allele yet to be identified.39 Because short amylose chains have significant effects

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on functional properties such as mouth-feel and digestibility,9,41 this finding has

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potential application in rice grain quality improvement and analysis of the amylose

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biosynthesis mechanism, which remains largely unknown.

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Most studies suggest that the AP1 and AP2 fractions indicate amylopectin, the

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AP1 fraction containing short starch chains such as A and short B chains (A + B1

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chains) and the AP2 fraction consisting of long B chains with higher molecular

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weight molecules.42 GPC analysis suggests that the glutinous starch contained a

308

significantly lower number of amylopectin chains in the AP1 fraction compared with

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other non-glutinous starch (Figure 4A). Therefore, the presence of GBSSI might have

310

caused elongation of the short chains into intermediate chains in the AP1 fraction,

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consistent with previous findings in rice and maize.12,14 The AP1-to-AP2 area ratio

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can be used as an index of the extent of amylopectin branching; the higher the ratio, 13



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the higher the branching degree.43 As shown in Table 2, the AP1-to-AP2 area ratio

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was significantly different among rice starches with different GBSSI activity. The

315

short chains of amylopectin (AP1) ranged from 63.08% to 75.19%, the long chains of

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amylopectin (AP2) from 21.27% to 24.81%, and the branching degree of amylopectin

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from 2.97 to 3.30. Moreover, a significantly negative correlation was observed

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between short (r = −0.90, p < 0.05) and long branched amylopectin chains (r = −0.98, p

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< 0.01) and AC. In terms of the amylopectin branching degree, a similar negative

320

correlation was also observed (r = −0.95, p < 0.01). Similar results were reported in a

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previous study using different cultivars with a varying AC: that the AC showed a

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significant negative correlation with amylopectin short chains.24 The above results

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provide evidence that GBSSI is not only involved in the biosynthesis of both short

324

and long amylose chains but also in the biosynthesis of amylopectin.

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Figure 4C shows the chain length distributions of the debranched amylopectin as

326

determined by HPAEC. Alterations in the degree of polymerization (DP) of the peak

327

chain length distribution of waxy and non-waxy rice starch exhibited different

328

tendencies. The proportion of amylopectin short chains ranging from DP 6 to 14 was

329

lower in all non-waxy debranched starches, in agreement with the GPC data.

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Compared with P1300, starch from CDS and R408G showed a similar curve, with

331

low amounts of short amylopectin chains and a high amount of intermediate chains

332

(DP 15−35). On the other hand, starch from E410D and Y268F showed a similar

333

curve but the amount of change was relatively low. These results suggest that GBSSI

334

is also responsible for the synthesis of intermediate (DP 15−35) amylopectin chains

335

via elongation of short chains (DP 6−14). A similar result was previously reported in

336

maize using the dosage effects of the Wx gene.14 Overall, these findings confirm that

337

as GBSSI activity increases, more intermediate amylopectin chains are synthesized. 14


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Crystalline structure of the different starches. The supra-molecular structure of the

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rice starches was characterized using XRD. XRD patterns were similar among the

340

five starch samples (Figure 5A), with a typical A-type diffraction pattern displaying

341

strong diffraction peaks at 15, 17, 18, 20 and 23° 2θ. The differences in intensity

342

reflect variation in the degree of crystallinities among the tested starches. The relative

343

crystallinity was therefore calculated based on the diffraction intensity, revealing a

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range in crystallinity from 28.37% to 33.88% (Table 3). The amylopectin content of

345

the starches is proportional to the degree of crystallinity and amylopectin is generally

346

thought to be responsible for starch crystallinity, while amylose disrupts the

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crystalline packing of amylopectin.36 Therefore, glutinous starch has the highest

348

crystallinity. Here, crystallinity of the non-glutinous starches showed an obvious

349

negative correlation with AC (r = −0.96, p < 0.01). These results are consistent with

350

previous studies on rice and maize starches.14,20

351

ATR-FTIR is used to explore the short-range molecular order structure near the

352

granular surface of rice.44 The FTIR intensity ratio of bands at 1022 and 1045 cm−1

353

indicates the amount of ordered to amorphous starch and has been linked to the

354

characteristics of amorphous and crystalline structures in different starches,

355

respectively. The intensity ratio of these bands (1045/1022 cm−1) can therefore be

356

used as a convenient index of FTIR compared to other measurements of starch

357

conformation.44 In the present study, the FTIR spectra of the five rice starches in

358

800-1200 cm-1 (Figure 5B) appeared similar. However, based on the calculation of

359

relative intensities at 1,045 and 1,022 cm-1 from the baseline to peak height (Table 3),

360

starches from P1300 rice had the highest ratios among the tested samples, in

361

agreement with previous studies.24,44 In the remaining samples, a clear decreasing

362

trend was also observed with increasing AC (r = −0.90, p < 0.05), in agreement with 15



Page 16 of 37

363

the XRD data. Overall, these results suggest that amylopectin predominantly denotes

364

the amount of double helices in waxy and low AC rice starches, although some

365

opposing results have been found in high AC starches (>20%).44

366

The semicrystalline layers of starch consist of a lamellar arrangement of

367

crystalline and amorphous regions with a repeat distance of 90–100 Å.45 The lamellar

368

structures of transgenic rice starches with diverse AC were therefore investigated

369

using SAXS (Figure 5C). The well-resolved main scattering peak around a scattering

370

vector (qo) of approximately 0.06 Å−1 is thought to arise from the periodic

371

arrangement of alternating crystalline and amorphous lamellae of amylopectin,

372

corresponding to the lamellar repeat distance or Bragg spacing.45 All starches are

373

scaled to equal intensity at a high qo (qo = 0.2 Å−1) to account for variations in sample

374

concentrations.46 The lamellar peak intensity and lamellar distance of the five rice

375

starches in the present study are shown in Table 3. The peak intensities showed

376

significant differences, ranging from 265.0 to 338.0, while the average lamellar

377

distance ranged from 9.0 to 9.2 nm. Moreover, a clear decrease in peak intensity was

378

observed with increasing AC (r = −0.94, p < 0.01), while an opposite trend was

379

observed with the average lamellar distance (r = 0.91, p < 0.05). These results are in

380

agreement with previous SAXS data on rice and wheat,24,29 which was found to be

381

due to an accumulation of amylose tie-chains with increasing AC, and thus, a decrease

382

in the electron density difference between crystalline and amorphous regions.46

383

Thermal properties of the different starches. The gelatinization properties of the

384

different starches were determined by DSC. The DSC parameters of endotherms

385

associated with gelatinization of the different rice samples are presented in Table 4.

386

The glutinous rice starch of P1300 had a distinct broad endothermic peak, whereas

387

narrow melting peaks were detected in the non-glutinous samples (Figure 6A). P1300 16


Page 17 of 37


388

starch also showed the lowest gelatinization properties including onset temperature

389

(To) and peak temperature (Tp); however, it had the highest enthalpy (∆Hgel) compared

390

with the non-glutinous starches (Table 4). It is suggested that endothermic transition

391

is the result of a loss of crystallites, which are mainly formed by amylopectin.47

392

Amylopectin short chains can reduce the efficiency of packing in starch crystallinity,

393

causing a decrease in the stability of the double helix, which can induce a lower

394

gelatinization temperature.47 The higher degree of crystallinity found in the P1300

395

starch therefore explains the high gelatinization enthalpy. In the non-glutinous

396

samples, an increase in gelatinization properties such as To, Tp and conclusion

397

temperature (Tc) was observed, with a significantly positive correlation with AC (To: r

398

= 0.99, p < 0.01, Tp: r = 0.99, p < 0.01 and Tc: r = 0.96, p < 0.05) increased. In

399

contrast, ∆Hgel showed a decreasing trend with increasing AC (r = −0.95, p< 0.01).

400

Glutinous rice starch consists mostly of crystalline regions, requiring less energy

401

to begin melting, whereas non-glutinous rice starch is a polymeric mixture of amylose

402

and amylopectin, and requires more energy since crystalline regions restrict hydration

403

of amorphous regions and delay initiation of gelatinization.48 Therefore, gelatinization

404

temperatures increased with increasing AC in the above samples. As for ∆Hgel,

405

contradictory results have been found with regard to the influence of amylose. For

406

example, Kong et al.49 reported a negative correlation between the gelatinization

407

enthalpy of rice starch and AC, while Cai et al.24 reported a slight increase in

408

gelatinization enthalpy with increasing AC. These seemingly contradictory results

409

might have resulted from the use of starch samples from different rice cultivars with

410

complicated genetic backgrounds. Nevertheless, the present study suggests that ∆Hgel

411

decreases with increasing AC in rice with the same genetic background but different

412

GBSSI activity. 17



Page 18 of 37

413

Retrogradation of the five starch samples after heating in water to 125 °C and

414

storage at 4 °C for 7 days was analyzed by DSC (Figure 6B and Table 4).

415

Retrogradation endotherms were observed in all samples, with a relatively higher To

416

but lower Tp and Tc in P1300 compared with the remaining samples. It has been

417

reported that a higher proportion of short amylopectin chains contributes to more

418

recrystallized domains in glutinous starch.26 Thus, the higher To in glutinous P1300

419

starch during retrogradation might be due to the higher AP1-to-AP2 ratio (Table 2). In

420

the non-glutinous starches, similar gelatinization curves were observed, with no

421

significant differences in To, Tp and Tc. However, an increase in retrogradation ∆Hret

422

was observed with increasing AC (r = 0.95, p < 0.01). It is well known that amylose

423

forms double helical associations of 40–70 glucose units during retrogradation,

424

whereas amylopectin crystallization occurs by association of the outermost short

425

branches.50 Though the lowest proportion of short-branch amylopectin was observed

426

in CDS starch, it showed the highest ∆Hret, suggesting that amylose contributes more

427

to double helical association during retrogradation of starch in rice cultivars with the

428

same genetic background.

429

In conclusion, this study provides clear information on the role of GBSSI activity

430

in grain morphology and amylose and amylopectin fine structure, as well as in the

431

relationships between amylose and amylopectin and on the gelatinization properties of

432

starch. Holes in the starch granule core were found to be the main factor causing an

433

opaque appearance but not poorly developed starch granules. Accumulating evidence

434

suggests that GBSSI is also involved in amylopectin synthesis, especially the

435

formation of extra-long chains. The present study supports the suggestion that GBSSI

436

can synthesize extra-long chains; however, it is mainly responsible for the synthesis

437

of longer amylose chains. The results also suggest that GBSSI can increase the 18


Page 19 of 37


438

proportion of intermediate amylopectin chains with a DP of 15-35, decreasing the

439

proportion of short chains with a DP < 14. In terms of starch physicochemical

440

characteristics, it was found that AC was positively correlated with trough viscosity,

441

final viscosity, setback viscosity, pasting time, pasting temperature and gelatinization

442

temperature, as well as with GC, breakdown viscosity and gelatinization enthalpy.

443

Furthermore, crystallinity, as determined by XRD, ATR-FTIR and SAXS, was found

444

to be significantly negatively correlated with AC, and thus, amylopectin short chains.

445

Overall, the results suggest that GBSSI is responsible for the biosynthesis of both

446

amylose and amylopectin, which in turn affects the grain appearance, thermal and

447

pasting properties and crystalline structure of the starch. These findings could

448

therefore prove beneficial in both food and non-food industries.

449

450

Funding

451

This study was financially supported by the National Natural Science Foundation

452

(31561143008 and 31401354), the Ministry of Agriculture (2014ZX08009-024B and

453

2016ZX08009003-004-009), the Jiangsu Natural Science Foundation (BK20140481,

454

BK20140484), the Ministry of Education (20133250120001), and the Jiangsu

455

Department of Education (PAPD, 201411117015Z, and KYLX15_1372).

456 457

References

458

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genotypes. Carbohydr. Polym. 2006, 64, 433–443.

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(47) Vandeputte, G. E.; Vermeylen, R.; Geeroms, J.; Delcour, J. A. Rice starches. I.

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gelatin-isation behaviour of granular starch. J. Cereal Sci. 2003, 38, 43–52.

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(48) Park, I.; Ibáñez, A. M.; Zhong, F.; Shoemaker, C. F. Gelatinization and pasting properties of waxy and non-waxy rice starches. Stärke 2007, 59, 388–396.

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(49) Kong, X. L.; Zhu, P.; Sui, Z. Q.; Bao, J. S. Physicochemical properties of starches

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from diverse rice cultivars varying in apparent amylose content and gelatinisation

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temperature combinations. Food Chem. 2015, 172, 433–440.

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(50) Singh, N.; Singh, J.; Kaur, L.; Sodhi, N. S.; Gill, B. S. Morphological, thermal

614

and rheological properties of starches from different botanical sources. Food

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Chem. 2003, 81, 219–231. 26


Page 27 of 37


616

FIGURE CAPTIONS

617

Figure 1. Appearance of polished grains (A) and micrographs of grain transverse

618

sections obtained by scanning electron microscopy of GLXN and five transgenic rice

619

lines (magnifications: 35 X for B1–G1; 2500 X for B2, 3–G2, 3 and 5000 X for

620

B4–G4.). Panels B–G indicate GLXN, P1300, E410D, Y268F, R408G and CDS,

621

respectively. CR and TR represent chalky and translucent regions, respectively.

622

Arrows indicate air spaces in the center of a whole starch granule. Scale bar = 0.5 mm

623

for B1–G1, 10 µm for B2, 3–G2, 3 and 5 µm for B4–G4.

624

Figure 2. Micrographs of purified starches obtained by scanning electron microscopy

625

(A–E) and the size distribution of the starch granules (F). Panels A–E represent

626

GLXN, P1300, E410D, Y268F, R408G and CDS, respectively. Scale bar = 10 µm.

627

Figure 3. Rapid viscosity profiles of the flours (A) and purified starches (B) from the

628

five transgenic rice lines.

629

Figure 4. Fine structure of debranched starches from the five transgenic rice lines.

630

Panels A and B show the fine structure as determined by GPC, with graphs in B

631

representing the amylose components in panel A. Panel C shows the changes in chain

632

length distribution of amylopectin from isoamylase-debranched starch as determined

633

by HPAEC. MW represents the apparent molecular weight relative to the standards.

634

Figure 5. XRD patterns (A), ATR–FTIR spectra (B), and SAXS spectra (C) of rice

635

starches from the five transgenic lines with different AC.

636

Figure 6. Gelatinization (A) and retrogradation (B) properties of purified starches

637

from the five transgenic lines at a 66.7% (w/v) water content as determined by

638

differential scanning calorimetry.

27



639

Tables

640

Table 1. The genetic background and physicochemical characteristics of rice samples used in this study a. Amino acid

Apparent

Crude protein Moisture content

Rice line

Transgenic construct

substitution

amylose content

at OsGBSSI

(%, w/w)

Total starch Gel consistency

content (%, w/w)

641

Page 28 of 37

content (mm)

(%, w/w)

(%, w/w)

GLXN

Wild-type

/

3.08±0.24e

14.23±0.13a

7.84±0.21a

83.26±2.45a

85.29±1.08a

P1300

Empty vector p1300

/

3.22±0.13e

14.11±0.16a

7.96±0.11a

82.50±0.79a

85.51±0.57a

E410D

pE410D

E410D

8.27±0.18d

14.02±0.09a

7.99±0.09a

80.33±1.15b

85.72±0.61a

Y268F

pY268F

Y268F

10.03±0.33c

13.87±0.09a

8.11±0.08a

74.33±4.21c

85.68±0.45a

R408G

pR408G

R408G

16.34±0.10b

13.76±0.15a

8.05±0.13a

66.34±4.12d

85.81±0.37a

CDS

pCDS

WT

17.57±0.15a

13.53±0.07a

8.17±0.07a

59.33±4.62e

86.23±0.69a

a

Data represent means ± standard deviations. For each column, values not displaying the same letter are significantly different (p < 0.05).

28


Page 29 of 37

642


Table 2. GPC parameters of the five rice starches with different amylose contentsa. GPC peak area (%)

Moisture content

Crude protein content

(%, w/w)

(%, w/w)

AP1/AP

AP2/AP

P1300

11.65±0.12a

0.71±0.03a

75.19±0.16a

E410D

11.45±0.09a

0.73±0.06a

Y268F

10.94±0.16b

R408G CDS

Area ratio

Sample AM/(AM+AP1+AP2)

AP1/AP2

AM1/AM2

24.81±0.16a

——

3.30±0.08a

——

74.21±0.19b

23.23±0.15b

2.56±0.03d

3.19±0.03b

1.03±0.02a

0.79±0.05a

73.30±0.36c

22.22±0.27c

4.49±0.11c

3.05±0.03c

0.81±0.03b

11.24±0.15a

0.77±0.02a

65.22±0.23d

21.36±0.08d

13.41±0.14b

3.03±0.02c

0.60±0.01c

10.92±0.10b

0.80±0.08a

63.08±0.08e

21.27±0.13d

15.57±0.12a

2.97±0.01d

0.55±0.02d

643

a

644

0.05). AP, AP1, AP2, AM, AM1, AM2 correspond to the amylopectin, short branch chains of amylopectin, long branch chains of amylopectin, the amylose, short

645

chains of amylose, and long chains of amylose, respectively.

Data represent means ± standard deviations. n = 2 for the GPC parameters. For each column, values not displaying the same letter are significantly different (p

Molecular Structure and Physicochemical Properties of Starches from

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