Accepted Manuscript Effects of grain development on formation of resistant starch in rice Xiaoli Shu, Jian Sun, Dianxing Wu PII: DOI: Reference:

S0308-8146(14)00717-1 http://dx.doi.org/10.1016/j.foodchem.2014.05.014 FOCH 15795

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Food Chemistry

Received Date: Revised Date: Accepted Date:

30 December 2013 4 May 2014 6 May 2014

Please cite this article as: Shu, X., Sun, J., Wu, D., Effects of grain development on formation of resistant starch in rice, Food Chemistry (2014), doi: http://dx.doi.org/10.1016/j.foodchem.2014.05.014

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Effects of grain development on formation of

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resistant starch in rice

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Xiaoli SHU, Jian SUN, Dianxing WU*

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State Key Lab of Rice Biology and Key Lab of the Ministry of Agriculture for

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Nuclear Agricultural Sciences, Institute of Nuclear Agricultural Sciences, Zhejiang

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University, Hangzhou 310029, P. R. China

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* Author for correspondence:

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Telephone and Fax: +86-571-87971594

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E-mail: [email protected]

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Abstract

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Three rice mutants with different contents of resistant starch (RS) were selected to

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investigate the effects of grain filling process on the formation of resistant starch.

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During grain development, the content of RS was increased with grain maturation and

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showed negative correlations with the grain weight and the starch molecular weight

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(Mn, Mw) and a positive correlation with the distribution of molecular mass

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(polydispersity, Pd). The morphologies of starch granules in high-RS rice were almost

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uniform in single starch granules and exhibited different proliferation modes from

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common rice. The lower activities of ADP-glucose pyrophosphorylase and starch

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branching enzyme and the higher activity of starch synthase and starch de-branching

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enzyme observed in high-RS rice might be responsible for the formation of small

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irregular starch granules with large spaces between them. In addition, the lower

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molecular weight and the broad distribution of molecular weights lead to differences

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in the physiochemical properties of starch.

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Key Words: Rice development; Resistant starch; Starch synthesis; Starch granules

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1. Introduction

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Rice is the staple food for 60% of the world’s population, and 76-78% of the

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endosperm is composed of starch. Based on its digestibility, starch is classified into

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readily digestible starch (RDS), slowly digestible starch (SDS), and resistant starch

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(RS) (Englyst & Hudson, 1996). Resistant starch (RS) is the portion of starch that

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passes undigested through the small intestine of healthy individuals and is fermented

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in the large intestine. In addition, RS is subsequently classified into four groups: RS1,

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RS2, RS3, and RS4 (Perera, Meda, & Tyler, 2010). RS1 represents the starch in

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unprocessed food with a physically inaccessible form. RS2 is the starch with a natural

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status and a granular form, and RS3 is formed when foods containing starch are

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cooked and cooled, during which process the starch granules undergo gelatinisation

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and retrogradation; RS4 included those starch modified by various types of chemical

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treatments (Brown, 2004). RS has been proven to be favourable for improving

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glycaemic and insulinaemic responses, exhibits special functions in the management

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of metabolic disorders, such as diabetes and hyperlipidemia, and the prevention of

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cardiovascular and colonic diseases (Perera et al., 2010). Screening of a large set of

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barley varieties (Shu, Backes, & Rasmussen, 2012) show a wide range of RS content,

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many high-amylose cultivars in rice, maize, and barley that were developed via

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mutation breeding or biotechnology breeding (Bird, Jackson, King, Davies, Usher, &

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Topping, 2007; Hallstrom, Sestili, Lafiandra, Bjorck, & Ostman, 2011; H. Jiang, Lio,

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Blanco, Campbell, & Jane, 2010) have been proven to contain a high RS content and

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exhibit a low starch in vitro enzymatic hydrolysis, which leads to slower glucose

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release and can provide functional control of the glycemic index (GI).

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The starch granule structure, the ratio of amylose to amylopectin, and the fine

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structure of amylopectin markedly influence the amounts of RS (Perera, et al., 2010), 3

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which exert great impacts on the digestion of high-RS rice mutants (Yang, et al.,

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2006). Both high-amylose and high-RS rice mutants show apparently different

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morphologies and physiochemical properties compared with normal rice (C. Wei, F.

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Qin, L. Zhu, et al., 2010; Yang, et al., 2006). The starch granule of high-amylose rice

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TRS (C. Wei, F. Qin, L. Zhu, et al., 2010) is semi-compounded and contains many

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subgranules, whereas the high-RS mutant contains smaller, round, and irregular starch

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granules and shorter amylopectin (Shu, et al., 2007). Starch granules containing

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amylose and amylopectin are formed in the amyloplast during grain filling (J.S. Jeon,

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N. Ryoo, T.R. Hahn, H. Walia, & Y. Nakamura, 2010) under interactions among

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ADP-glucose pyrophosphorylase (AGP), starch synthase (SS), starch branching

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enzymes (BE) and starch de-branching enzymes (DBE), and the different

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morphologies and changes in the physiochemical properties of starch isolated from

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different cultivars during grain filling might reflect diversities in the grain quality (Cai,

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Liu, Wang, & Cai, 2011) and significantly affect the final properties of the rice and the

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digestibility of the starch. The loss or the partial reduction of the activities of one or

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several of these enzymes might result in a marked difference in the morphology and

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physiochemical properties of the starch granules (J.S. Jeon, et al., 2010). Both starch

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branching enzymes and starch synthases potentially influence the resistant starch

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content in rice (Butardo, et al., 2011; Zhang, et al., 2011). However, there were

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elusive studies on the interactions among RS, starch granules formation and key

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enzymes participating in starch synthesis during kernel formation. Jiang et al. (2010)

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found that the RS content increases with kernel maturation and an increase in the

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amylose/intermediate component content in high-amylose maize.

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In this study, we investigated the physiochemical properties, starch granule

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morphology, starch molecule mass weight and distribution, activities of the key

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enzymes involved in starch biosynthesis, and their interactions during the

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development of grain kernels in three rice mutants exhibiting different RS contents.

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This approach can provide new insights into the mechanism of RS formation and

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accumulation during grain development and will highlight possibilities for the

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breeding of rice with high RS through starch modifications.

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2. Materials and methods

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2.1 Rice materials

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Three indica rice mutants, namely MR4, MR1, and MR7), with RS content of

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9.04%, 4.5%, and 0.8% respectively were used in the present study. All of the

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materials were derived from RS111 by mutation breeding which may eliminate the

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influences by irradiation itself and grown in the field of Zhejiang University,

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Hangzhou, China, during the crop season of 2009. The panicles were harvested 5, 10,

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15, 20, 25, and 30 days after flowering (DAF) and divided into two portions. One of

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the portions was frozen with liquid nitrogen immediately after harvest and stored at

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-70°C until use for enzyme activity analysis. The other portion was dried at 35°C, and

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the plump grains were manually de-hulled and stored in a dryer until use. Due to the

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blighted endosperm in the early milky stages, which is hard to mill after drying, all of

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the samples were analysed as brown rice.

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2.2 RS, AAC, reducing sugars, and grain weight

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To determine the contents of RS, the apparent amylose content (AAC), and the

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amount of reducing sugars, the dried brown rice was ground into flour using an Udy

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Cyclone Mill (Fort Collins, CO, USA), passed through a 100-mesh sieve, and stored

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in a dryer until analysis. The RS was measured according to the methods described by

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Yang et al. (2006), briefly, samples were digested by 1% amylase for 16h at 37 oC after

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digestion by 600UI pepsin at 37 oC for 1h, the residues were collected by centrifuging 5

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and washed with 50% ethanol, then hydrolysed with 4M KOH for 20min on ice, after

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digesting by 100U amyloglucosidase at pH 4.5 for 1h at 60oC. The RS content were

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determined by measuring the released glucose with GOPOD kit. The AAC was

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measured by a simplified I2/KI assay described by Yang et al. (2006). Four standard

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samples, each with a different AAC (1.2%, 11.2%, 16.8%, and 26.8%), were provide

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by the China National Rice Research Institute (CNRRI). The reducing sugars were

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extracted twice with 85% ethanol at 50°C and analysed through the dinitrosalicylic

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acid (DNS) assay (Bailey, 1988) using maltose as the standard. The starch was

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isolated through the alkaline extraction method (Yang et al., 2006). The grain weight

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of 100 plump grains was measured. Each analysis was performed in triplicate.

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2.3 Scanning electron microscopy (SEM)

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The brown rice was broken manually. After coating with Pt ions in an argon

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atmosphere for 30 min (IB-5 ion coater, Eiko Co.), the cross-sections were stuck on

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double-adhesive tape fixed to a metallic stud. The starch granules were visualised

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with an electron microscope (TM-1000, Tabletop Microscope, Hitachi, Ltd, Japan) at

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15 kV.

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2.4 Preparation and assay of enzymes involved in starch synthesis

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The panicles stored at -70°C were de-hulled manually, and the fresh weight of 20

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grains was recorded. The enzymes were extracted and analysed according to the

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methods described by Chen et al. (2001). Four key enzymes in the starch synthesis

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process, namely ADP-glucose pyrophosphorylase (AGP), starch synthase (SS), starch

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branching enzyme (BE), and debranching enzyme (DBE), were analysed. All of the

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procedures were performed in triplicate and on ice.

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2.5 Starch molecular weight analysis with GPC

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The molecular weight of the polyglucans was determined using the method 6

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developed by Kubo et al. (Kubo, Fujita, Harada, Matsuda, Satoh, & Nakamura, 1999)

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with minor modifications. Briefly, 100 mg of rice flours was suspended in 2 mL of 1

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M NaOH. After agitation for 30 min at room temperature, 2 mL of distilled water was

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added to the sample suspension, and the mixture was centrifuged at 2000 g and 25°C

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for 5 min. Then, 50 µL of the supernatant was applied to a Waters GPC 515 separation

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system with a 2410 refractive index detector (Waters, Milford, USA). The column

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used was TSK-Gel4000SWXL (7.8×300 mm) and was equilibrated with 0.1 M

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NaNO3 prior to use. The samples were eluted with 0.1% NaCl solution at a flow rate

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of approximately 0.7 mL/min at room temperature. Five glucans (Sigma) with MP of

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2000 KDa, 188 KDa, 76.9 KDa, 43.2 KDa, and 10.5 KDa were used to construct the

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standard curve, and the molecular weights of the samples were calculated using the

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software associated with the analysis machine.

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2.6 Statistic analysis

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Principle Component Analysis was performed using the SPSS 16.0 software

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(SPSS Inc., Chicago IL, USA). Correlation matrix was evaluated to compare the

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correlations between each parameter.

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3. Results

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3.1 RS, AAC, reducing sugars, and grain weights

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The RS content in MR4, MR7, and MR1 increased from 3.3%, 0.17%, and 3.21%

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at 5 DAF to 9.04%, 0.8%, and 4.5% at 25 DAF, respectively, and this increase was

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accompanied by gradual increases in the AAC and the grain weight (Table 1). The

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grain grew fast and showed the highest filling rates during the first 5 days, and the

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grains of the high-RS mutants MR1 and MR4 developed slower than the low-RS

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mutant MR7 (Table 1). At the same developing stages, the RS content, AAC in MR4

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and MR1were significantly higher than in MR7. The content of reducing sugars in 7

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three materials were all markedly decreased from 5 DAF to 15 DAF and then

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remained stable at a low level (Table 1), which showed reverse changes as RS content

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and AAC. When analysed with PCA, the RS content clustered with AAC and grain

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weight, which indicated that they correlated positively to each other and the RS

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content showed negative correlation with the content of reducing sugars as they were

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in the opposite directions (Figure 1). That the increase in the RS content and the AAC

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with the initiation of grain filling indicates that starch is synthesised to fill the whole

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grain and that the grain development contributed to the formation of RS.

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3.2 Starch granules

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Rice grains developed through the pre-milk, milky, dough, yellow ripe and

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mature stages. The developing grains filled rapidly as the size of the starch granule

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sizes increased during the first 10 DAF. In this study, only MR4 and MR7 were

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compared because the morphologies of the starch granules of MR4 and MR1 were

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similar to each other and different from those of MR7.

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At 5 DAF, the starch granules were small, immature, and expected to further

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increase in size with different proliferation model. The starch granules of MR4 grew

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as either protrusions or fissions. In addition, some of these granules were

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dumbbell-shaped and others resembles beads-on-a-string, whereas none of these type

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of starch granules were observed in MR7 (Figure 2A, 2B). This finding might reflect

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the differential proliferate modes of starch granules in rice endosperm between the

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high-RS mutants and the low-RS mutant (common rice cultivar). During the grain

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filling process, the starch granules grew rapidly, assumed their shape, became stable,

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and had filled almost all the entire endosperm cells at 15 DAF (Figure 2E, 2F). The

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developing starch granules of MR7 at 10 DAF and 15 DAF were large and several

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single starch granules were compacted together (Figure 2D, 2F). In contrast, the starch 8

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granules of MR4 were small and extended into irregular directions, and most were

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single starch granules (Figure 2C, 2E).

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The mature compound granules of MR7 in the outer endosperm were polyhedral

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(Figure 2H, 2J). The individual granules were generally similar in size, which

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suggests that the synthesis of starch granules in amyloplast is synchronous.

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Furthermore, the starch granules of MR7 were tightly packed into compact,

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compound, and angular volumes and exhibited fewer spaces between each other

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(Figure 2D, 2F, 2H, 2J). However, the starch granules of MR4 were pleomorphic,

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small and round with large spaces between them, which indicated a loss in the

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compound granular organisation (Figure 2C, 2E, 2G, 2I). Obvious differences were

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also observed between the isolated pure starch granules: the starch granules of MR7

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were polyhedral, sharp-edged and detached (Figure 2L), and the starch granules of

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MR4 were malformed with an irregularly shape and small size (Figure 2K).

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3.3 Starch molecular mass determination

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The molecular weight distribution in a polymer describes the relationship

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between the number of moles of each polymer species and the molar mass of the

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species. The molecular weight (Mp) and percent increase in the area with graining

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filling were compared among the three rice mutants. The weight average molecular

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weight (M w), the number average molecular weight (Mn), and percent area of MR4

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were found to be the lowest, whereas the MR7 exhibited the highest values regardless

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of the developing stages. However, the polydispersity (Pd) of MR4 was the highest of

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the three materials (Table 2). This percent area is related to the uniformity of the

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molecular polymerisation. The lowest percent area that was found for the starch of

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MR4 was consistent with its highest AAC (Table 2). To investigate the differences of

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molecular weight among all of these materials during kernel development, a PCA was 9

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conducted. The differences of all materials with different development stages were

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shown in Figure 3A; the variations among MR7 and MR1, MR4 almost can be

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explained by PC1. According to the loading plots of the first two factors, besides RS,

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Pd could mainly explain the first variance and Mn, Mw could explain the second

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variance. RS showed positive correlation to Pd and negative correlation to Mn and Mw

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(Figure 3B).

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3.4 Activities of AGP, SSS, BE, and DBE

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The activity of AGP increased gradually from 0 DAF to 15 DAF and then began

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to decrease; however, this enzyme retained some activity until 25 DAF. The

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increasing rate found for MR4 was lower than that obtained for the other two mutants.

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The activity of AGP in MR7 was the highest, whereas that of the AGP in MR4 was

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the lowest throughout the grain development process. Especially at 10 DAF and 15

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DAF, the activities of AGP were significantly different among MR1, MR7 and MR4

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(Figure 4A), the activities of AGP exhibited a slightly negative correlation to the AAC

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and RS content and a positive correlation to the grain weights (Figure 1).

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Similarly, the activities of SSS reached a maximum at 10 DAF and then

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decreased gradually. MR4 exhibited the highest SSS activities at 5 DAF, and no

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significant differences were observed among the three materials after 15 DAF (Figure

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4B). MR1 exhibited a higher BE activity than MR4 before 15 DAF, and there were no

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clear differences in the BE activities after 15 DAF. While after 15DAF, the activities

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of BE in MR1 and MR4 were significant lower than those found in MR7 (Figure 4C).

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The SSS activity negatively affected the RS content slightly while no correlations

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between BE and RS were found (Figure 1).

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The activity of DBE increased rapidly from 5 DAF to 10 DAF and then

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decreased gradually. The DBE activity were significantly different among the three 10

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material after 10 DAF and the activity of DBE in MR4 was the highest and remained

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at a medium level until 25 DAF (Figure 4D). In addition, this activity showed positive

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correlations to the RS content and the AAC (Figure 1).

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4. Discussion

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4.1 RS and AAC, reducing sugar and grain weight

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RS increased with grain filling and the positive correlations between RS and

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AAC, RS and grain weight (Figure 1) were consistent with a previous study, which

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showed that the RS content increased with kernel maturation (Jiang et al., 2010) . The

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higher AAC in the high-RS mutants might result in changes in the grain filling

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process, i.e., partial filling and lower products compared with normal rice, because the

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AAC is correlated with the grain plumpness (Zhong & Li, 1994).

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As we supposed, RS increased with reducing sugars decreased (Table 1, Figure

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1), glucose and fructose, two major sugars in rice grain (Shu, Frank, Shu, & Engel,

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2008) which can be measured as reducing sugars by DNS, acted as the precursors of

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the ADP glucose that participates in starch synthesis. The relationship between free

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sugars and starch accumulation previously demonstrated, and the same trends were

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found in our samples. During grain filling, the sugars were transformed into starch

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and reached a stable level in the mature grains. Content of reducing sugars in MR4

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and MR1 was slightly higher than that of MR7 at the early filling stages, the lower

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grain weight of MR4 and MR1 might be due to some factors that restrict the increase

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of granules in the amyloplast and not a lack of precursors for starch synthesis. What’s

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more, the high content of reducing sugars might also negatively regulate ADP glucose

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and affect starch synthesis.

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4.2 RS and starch granule structure

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The marked differences between the starch granules of MR7 and MR4 (Figure 2)

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might result from the different amyloplast size or an altering of the amyloplast

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division process. The inhibition of the amyloplast division process can restrain the

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increase in the amyloplast diameter and result in pleomorphic amyloplasts with small

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starch granules (Yun & Kawagoe, 2009) or elongated starch granules (H. Jiang, et al.,

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2010). The protrusion of plastid was found as a unique form of plastid division in

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wheat and might be necessary for the initiation of B-type and C-type starch granules

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(Cunxu Wei, et al., 2010), B-type starches contain B-polymorphs having both trapped

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water with low mobility and “weakly bound” water with higher mobility while A-type

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starches contain A-polymorphs having only “trapped water”, C-type starches

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contained both A-polymorphs and B-polymorphs (Bogracheva, Wang, Wang, &

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Hedley, 2002). Both B-type and C-type starches showed more resistant to amylase

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than A-type starches (C. Wei, F. Qin, W. Zhou, et al., 2010). The protrusion growth of

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the partial starch granules of MR4 might be of benefit to the formation of B-type or

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C-type starch granules, which exhibit different starch properties, such as crystal type,

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compared with high-RS rice from normal cultivars (Shu, et al., 2007).

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The starch granule morphology of transgenic high-amylose rice materials are

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also different from that of normal rice and is characterised as fused or aggregated

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(Butardo, et al., 2011; C. Wei, F. Qin, L. Zhu, et al., 2010). Elongated starch granules

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are found in both the intact grains and the isolated starch of high-amylose maize

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(Hongxin Jiang, Horner, Pepper, Blanco, Campbell, & Jane, 2010) and rice (C. Wei, F.

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Qin, W. Zhou, et al., 2010), whereas only a few elongated starch granules are

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observed in the intact grains of MR4, and none are found in the isolated starch. This

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finding make sense because the elongated starch granules in intact rice grains of MR4

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are, in fact, compacted granules of several small starch granules, which were wrapped

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together with a membrane-like structure during grain development, and are thus

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different from the fused elongated starch granules found in high-amylose rice and

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maize. The outer layer can be destroyed after its isolation with alkaline or amylase to

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release the individual starch granules.

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4.3 RS and starch molecular weight

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Starch is a type of carbohydrate polymer that is composed of polysaccharides

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containing amylose with linear chains and amylopectins with branched chains. The

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number of amylose and amylopectin polymers confers a certain molecular weight

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distribution. Mw and Mn reflect the average molecular weight of polymers with

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different chain lengths according to their molecular size and number. During the

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formation and growth of starch, amylopectin is synthesised from small linear chains

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or short-chain glucans to obtain a branching structure with a different polymerisation

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(Zeeman, Kossmann, & Smith, 2010). The depolymerisation of amylopectin chains in

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the starch granules can decrease M w and Mn of native and modified corn starch (Chan,

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Leh, Bhat, Senan, Williams, & Karim, 2011). The negative correlation between RS

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and Mn and Mw (Figure 3C) indicated that the high-RS rice mutants contained a

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higher number of short fragments with different molecular weights than those found

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in normal rice and thus exhibited a different molecular mass distribution from that

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found for normal rice. Pd is determined by the Mw:Mn ratio and the value is related to

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RS positively (Figure 3C), the higher Pd of MR4 indicates that the starch in MR4

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contains a higher percentage of molecular fractions with low molecular weights

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compared with MR7. These findings are consistent with our previous studies, which

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showed that RS is positively correlated to the portion of short amylopectin chains and

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negatively correlated to the portion of long amylopectin chains (Shu, et al., 2007).

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4.3 RS and starch enzymes 13

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Many studies have indicated that the activities of the enzymes participating in the

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growth of rice grains peak during the earlier stages and then start to decrease (Chen, et

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al., 2001; Hirose & Terao, 2004). Our results were consisted with the previous reports

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(Figure 4).

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AGP is the first enzyme to synthesise the precursor for starch formation and

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elongates glucose polymers through the formation of α-1,4-bonds by other enzymes,

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such as SS (Hannah, 2007). This enzyme catalyzes the first committing step in this

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pathway, and its allosteric properties are essential for the control of the rates of starch

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biosynthesis (Jeon et al., 2010). An enhanced AGP activity can increase the seed

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weight. For example, rice overexpressing E. coli AGPase exhibited an up to 11%

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increase in seed weight (Sakulsingharoj, et al., 2004). The lower activities of AGP in

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MR1 and MR4 indicates that starch synthesis throughout the grain filling process in

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high-RS rice is not as effective as the synthesis of starch in low-RS common rice.

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There were five types and 10 isoforms of SSS in rice with different expression

335

developmental profiles (Hirose & Terao, 2004). The SSS isoforms expressed mainly

336

in the medium stages, such as OsSSIIIa, OsSSIIa, and OsGBSSI (Ohdan, et al., 2005),

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might affect the RS content more significantly than the other SSS isoforms because

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there were significant differences among MR1, MR4 and MR7 at that stages (Figure

339

4). The loss or reduction of the activity of one SSS isoform can induce changes to the

340

activities of the other SSS isoforms and is at least partially compensated by the other

341

SSS enzymes (Zhang, et al., 2011). This finding might explain why there were no

342

significant differences between the three mutants during the grain filling process and

343

might also explain the weak correlations between the RS content and the SSS

344

activities during development process (Figure 1, Figure 4).

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BE is mainly responsible for the synthesis of the amylopectin molecules, and 14

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there are three classes of starch branching enzymes (BEI, BEIIa, and BEIIb) in rice

347

(Satoh, et al., 2003). The combined transgenic inhibition of BEI and BEIIb in indica

348

rice enhanced the RS content from 0% to 14.7% due to the markedly increased AAC

349

(Zhu, et al., 2012). BEIIb is responsible for the formation of RS in ‘Jiangtangdao 1’

350

and explains 60.4% of the RS variation (Heazlewood, et al., 2012). Though the

351

activity of BE in MR4 and MR1 was lower than that in MR7, while no obvious

352

correlation between the activity of BE and RS, which indicates that the mutation

353

affected the activity of BE and this change in the BE activity resulted in the higher

354

AAC and RS content partly in the high-RS mutants MR1 and MR4. In contrast, other

355

enzymes were also influenced in the high RS mutants and exhibited differences from

356

the high amylose ae mutant. Other factors in addition the AAC might also play

357

important roles in the enhancement of the RS content (Shu, et al., 2007), and this

358

finding was also observed in ami-BEIIb and hp-BEIIb lines (Butardo, et al., 2011).

359

DBE hydrolyses the α-1,6-glucosic linkages of polyglucans, and two classes of

360

DBE, namely isoamylase (ISA) and pullulanase (PUL), have been characterised in

361

rice. Amylopectin and phytoglycogen are hypothesised to be formed simultaneously

362

depending on the rate and/or the specificity of DBE (Castro, Dumas, Chiou,

363

Fitzgerald, & Gilbert, 2005). With lower or absent DBE activity, the pre-amylopectin

364

would continue to elongate and branch to yield phytoglycogen (Fujita et al., 2009).

365

Higher activities of DBE would change the amylopectin chain length profile and

366

increase the number of short-chain amylopectins, which results in the higher Pd

367

(Table 2) and RS contents (Table 1) found in MR4 and MR1.

368

The physiochemical properties of starch were determined mainly by the amylose

369

content and the structure of amylopectin. The formation of RS in rice is a complex

370

process that is regulated by an interaction network of several enzymes. Four types of 15

371

enzymes, namely AGP, SSS, BE, and DBE, were found to be somewhat correlated

372

with the RS content in the developing grains, although the correlations were not

373

significant (Figure 1). GBSS is the major enzyme responsible for amylose, a lack or

374

loss of the other enzymes, such as BEIIb, can also result in a high amylose content

375

(Butardo, et al., 2011; Nishi, Nakamura, Tanaka, & Satoh, 2001; C. Wei, F. Qin, W.

376

Zhou, et al., 2010). The production of very high amylose content in rice might require

377

the modification of the expression of different combinations of target isoforms

378

(Butardo, et al., 2011; Zeeman, et al., 2010). Semicrystalline amylopectin is

379

synthesised exclusively by different isoforms of SSS and BEs. In addition to amylose,

380

the fine structure of amylopectin plays an important role on the RS content (Shu, et al.,

381

2007). The fine structure of amylopectin was also the result of the interplays of

382

several enzymes (J. S. Jeon, N. Ryoo, T. R. Hahn, H. Walia, & Y. Nakamura, 2010).

383

The changed activities of synthetic enzymes in different rice mutants could be

384

indirectly reflected by the different morphologies of the starch granules and the

385

different molecular weight and distribution of starch, which resulted in different

386

physiochemical properties. When combined the activities of four enzymes with RS,

387

AAC and reducing sugars, all the materials were clustered almost according to

388

different developing stages (Figure 1A), while materials were clustered almost

389

according to different cultivars when combined RS and starch molecular weight

390

together (Figure 3A). That might indicate the different RS contents among

391

development stages were mainly due to the changes of the activities of four enzymes,

392

while the different RS contents among MR1, MR4 and MR4 were most possible

393

resulted from different starch molecular structure. AGP putatively participates in RS

394

regulation by controlling the total starch, whereas SSS and BE most likely regulate

395

RS synthesis by influencing the amylose content and the amylopectin structure, and

16

396

DBE is inferred to regulate RS by modifying the amylopectin structure. In addition,

397

the starch synthesis might be mainly regulated by structural trimming at late stage,

398

and the structure of starch is very important to the RS content, as described previously

399

(Shu, et al., 2007). Further studies are needed to fully understanding how changes in

400

the enzyme activities affect the initial formation of starch granules and RS.

401 402

Acknowledgements

403

The authors gratefully acknowledge Dr. Yayu Qiu for helping GPC analysis. The

404

current research was supported by the Chinese Ministry of Science and Technology

405

(2011ZX08001, 2013CBA01400), National Science Foundation of China (No.

406

30800675), and Zhejiang Provincial Rice Breeding Program (2012C12901).

407 408

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510

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511

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512

126-128.

513

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514

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515

diabetic rats. Plant biotechnology journal, 10, 353-362.

516 517 518

21

519

Figure Captions

520

Figure 1: Score plot (A) and loading plot (B) of principal components 1 and 2 (PC1

521

and PC2), describing the variation in all of the parameters except for molecular

522

weight of MR1, MR4 and MR7. Arrows indicate main trends in correlations between

523

the different measured variables in the three materials. Variables in opposite showed

524

negative relationship and in orthogonal directions varied independently.

525 526

Figure 2: SEM of starch granules of MR4 and MR7 in different stages. MR4 (A: 5d,

527

C: 10d, E: 15d, G: 20d, I: 25d, K: isolated starch form mature grains), MR7 (B: 5d, D:

528

10d, F: 15d, H: 20d, J: 25d, L: Isolated starch grains from mature grains);

529 530

Figure 3: Score plot (A) and loading plot (B) of starch molecular weight and RS in

531

MR1, MR4 and MR7 by PCA. Only the first two main factors were released; PC1

532

explained the majority variances among all variables. Arrows indicate main trends in

533

correlations between the different measured variables in the three materials. Variables

534

in opposite showed negative relationship and in orthogonal directions varied

535

independently.

536 537

Figure 4: The dynamic activities of AGP (A), SSS (B), BE (C) and DBE (D) in MR7,

538

MR1 and MR4 at different developing stages

539 540 541 542 543 544 545 22

546

Table 1 AAC, RS, weight of 1000 grains and reducing sugars in 3 rice samples during

547

different developmental stages1 (To be continued)

548

DAF(d)

AAC (%) MR4

MR1

RS (%) MR7

MR4

MR1

MR7

MR4

5

18.0±0.2aC 11.8±0.1aB

3.8±0.3aA

3.30±.07aC

3.21±0.08aB

0.17±0.04aA 5.61±0.11

10

29.5±0.7bC 21.4±0.1bB

6.4±0.3bA

7.70±0.03bC

4.21±0.11bB

0.18±0.05aA 12.99±0.2

15

34.9±0.1cC 27.5±0.0cB

12.1±0.7dA 8.40±0.20bcC 5.50±0.17dB

0.62±0.07bA 18.53±0.3

20

31.1±1.3bC 27.8±0.8cB

14.8±0.5eA 8.89±0.41bcC 4.80±0.10cB

0.83±0.01cA 22.79±0.2

25

35.0±0.4cC 28.2±0.1cdB 10.6±0.2cA 9.04±0.29cC

30

36.5±0.8cC 29.0±0.5dB

4.50±0.13bcB 0.80±0.02cA 20.89±1.1

12.9±0.5dA 8.45±0.17bcC 4.57±0.08bcB 0.85±0.05cA 21.84±0.8

549

1:

550

different stages in the same materials at 0.05 level; the same capital letters indicated

551

there were no significant differences among the different materials in the same

552

develop stages at 0.05 level.

the same lower case indicated there were no significant differences among the

553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 23

572

Table 1 (continued)

573

DAF(d)

Reducing Sugars (%) MR4

MR1

MR7

5

0.315±0.005aB 0.315±0.008aB

0.187±0.000aA

10

0.074±0.001bA 0.134±0.006bC 0.084±0.007bB

15

0.048±0.000cC 0.038±0.001cB

20

0.027±0.001dB 0.023±0.001dA 0.021±0.000dA

25

0.021±0.001eA 0.021±0.000dA 0.020±0.000dA

30

0.021±0.001eA 0.023±0.002dA 0.020±0.000dA

0.024±0cA

574

1:

575

different stages in the same materials at 0.05 level; the same capital letters indicated

576

there were no significant differences among the different materials in the same

577

develop stages at 0.05 level.

the same lower case indicated there were no significant differences among the

578 579 580 581 582 583 584 585 586 587 588 589 24

590

Table 2 Parameters of starch molecular weight of MR7, MR1 and MR4 in different

591

development stages* Mn

Mw

Mp

Pd

%Area Mz+1

Mz

MR7

10d

1866698 2064986

2285769 1.106224

76.19

2322564

2210293

MR7

15d

1995718 2193526

2408200 1.099116

81.58

2445954

2335902

MR7

20d

2006377 2171825

2367086 1.082461

83.58

2395570

2296485

MR7

25d

1973104 2108561

2339384 1.068651

89.23

2342596

2232424

MR1

10d

1351465 1699606

2155783 1.257603

73.55

2058453

1918890

MR1

15d

1657886 1851727

2186357 1.116921

75.64

2133406

2009810

MR1

20d

1801488 2000604

2266182 1.110529

78.16

2254067

2145412

MR1

25d

1419450 1935166

2450493 1.363321

89.96

2416035

2240845

MR4

10d

1655087 1831572

2163622 1.106631

62.39

2077355

1970157

MR4

15d

1847751 2005599

2240611

1.085427

61.21

2223797

2127750

MR4

20d

1023030 1559479

2280604 1.524373

77.56

2160679

1938295

MR4

25d

1069098 1593395

2363860 1.49041

86.18

2239509

1995279

592

*Mw: weight-average molecular weight; Mn: number-average molecular weight; Mp:

593

Peak molecular weight; Pd: Polydispersity (Mw/Mn); Mz: z-average molecular weight;

594

Mz+1: z+1 average molecular weight

595 596 597 598 599

25

600

601

602 603

Figure 1

604 605 606 607 608 26

609

A

B

610

C

D

611

E

F

27

612

G

613

I

J

614

K

L

615

H

Figure 2

616 617 618 619

28

620

621 622

Figure 3

623

29

-

-

Activity of AGP (nmol.grain .min )

c

3.2

A

b

2.4 2.0

a

b

b

b

a a

a

b

a

1.6

a a

1.2 5

10

15

20

25

Days after flower (d)

-

-

Activity of SSS (nmol.grain .min )

624

625

b

c

2.8

RS1 RS4 R7

8 7 6 5

B

b

c

ab

a b

RS1 RS4 R7

a a a

a

a a a

4

a a a

3 5

10

15

20

25

Days after flower (d)

626

30

-

-

Activity of rSBE (U.grain .min )

0.30

b b b

0.25

C a

a

b

0.20 0.15

a a

a a

a

MR1 MR4 MR7

0.10

b ab a

0.05 5

10

15

20

25

Day after flower (d)

-1

-1

Activity of rDBE (nmol.grain .min )

627

628 629

a

80

MR1 MR4 MR7

70

b

D

a c

60

a

c b

50

a

40

b

30 a a

20 10 5

10

15

20

25

Days after flower (d) Figure 4

630 631 632 633

31

634

Highlights

635

(1) RS content was increased rapidly during maturation of mutant grains; (2) Grain

636

weight and AAC played positive effects on RS contents; (3) Small, round and

637

irregular starch granules is characteristics of high RS rice grains; (4) Mn and Mw had

638

negative correlations to RS; (5) Activities of ADP, BE, SS, and SDB all contributed to

639

the RS formation.

640 641

32