New insights into amylose and amylopectin biosynthesis in rice endosperm

Journal Pre-proof New insights into amylose and amylopectin biosynthesis in rice endosperm Jihui Zhu, Wenwen Yu, Changquan Zhang, Yajun Zhu, Jianlong Xu, Enpeng Li, Robert G. Gilbert, Qiaoquan Liu

PII:

S0144-8617(19)31324-4

DOI:

https://doi.org/10.1016/j.carbpol.2019.115656

Reference:

CARP 115656

To appear in:

Carbohydrate Polymers

Received Date:

18 September 2019

Revised Date:

13 November 2019

Accepted Date:

21 November 2019

Please cite this article as: Zhu J, Yu W, Zhang C, Zhu Y, Xu J, Li E, Gilbert RG, Liu Q, New insights into amylose and amylopectin biosynthesis in rice endosperm, Carbohydrate Polymers (2019), doi: https://doi.org/10.1016/j.carbpol.2019.115656

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New insights into amylose and amylopectin biosynthesis in rice endosperm

Jihui Zhua,e, Wenwen Yub, Changquan Zhanga,c, Yajun Zhud, Jianlong Xud,

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Enpeng Lic, Robert G. Gilbertc,e*, Qiaoquan Liu a,c*

Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, Key Laboratory

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of Plant Functional Genomics of the Ministry of Education, College of Agriculture,

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Yangzhou University, Yangzhou, Jiangsu, 225009, China

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b Department of Food Science & Engineering, Jinan University, Huangpu West Avenue 601, Guangzhou, Jiangsu Province, China

Jiangsu Key Laboratory of Crop Genetics and Physiology, Joint International

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Research Laboratory of Agriculture and Agri-Product Safety, Co-Innovation Center for Modern Production Technology of Grain Crops of Jiangsu, Yangzhou University,

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Yangzhou, Jiangsu, 225009, China

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Agricultural Genomics Institute, Chinese Academy of Agricultural Sciences, Shenzhen,

China

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The University of Queensland, Centre for Nutrition and Food Sciences, Queensland

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Alliance for Agriculture and Food Innovation, Brisbane, QLD, 4072, Australia

*Corresponding authors: Prof RG Gilbert, [email protected]; Prof Q Liu,

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[email protected]

Highlights

Aim: which enzymes are involved in both amylose and amylopectin synthesis? Starch chain-length distributions from 95 different rice cultivars were measured These were fitted with biosynthesis-based models based on enzyme activities. Correlation analysis of the resulting parameters was performed. GBSS, SBE and SS are involved in both amylose and amylopectin synthesis.

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Abstract

How various isoforms of rice-starch biosynthesis enzymes interact during amylose and

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amylopectin synthesis is explored. The chain-length distributions of amylopectin and amylose from 95 varieties with different environmental and genetic backgrounds were

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obtained using size- exclusion chromatography, and fitted with biosynthesis-derived

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models based on isoforms of starch synthase (SSI-SSIV), starch branching enzyme (SBE, including SBEI and SBEII) and granule-bound starch synthase (GBSS) that are involved in amylose and amylopectin synthesis. It is usually thought that these are synthesized by separate enzymes. However, the amount of longer amylopectin chains correlated with that of shorter amylose chains, indicating that GBSS, SBE and SS affect

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both amylose and amylopectin synthesis. Further, the activity of GBSS in amylose correlated with that of SS in amylopectin. This new understanding of which enzymes are suggested by the statistics to be involved in both amylose and amylopectin synthesis could help rice breeders develop cereals with targeted properties.

Keywords: starch; biosynthesis; enzymes; amylose; amylopectin; rice

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1. Introduction Rice (Oryza sativa L.) is the largest source of food energy for half of the world’s

population (Juliano, Perez & Cuevasperez, 1993). The molecular structural features of

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rice starch are major determinants of functional properties (e.g., thermal and pasting

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properties) and also of in vitro and in vivo digestibility.

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Starch is a branched glucose polymer with (1→4)-α linear links and (1→6)-α branch points. It has two forms, amylose (Am) and amylopectin (Ap). Ap has a high

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molecular weight and a large number of short-chain branches, while Am has a lower molecular weight and a small number of long-chain branches. Starch biosynthesis is

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controlled by isoforms of five enzymes. Adenosine 5' diphosphate (ADP)

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pyrophosphorylase (AGPase) produces ADP-Glc (“Glc” = glucose) as the first step in starch biosynthesis (Okita, 1992; Okita, Nakata, Anderson, Sowokinos, Morell & Preiss, 1990). ADP- Glc is elongated by starch synthases (SSs). There are number of SS isoforms in rice, including granule bound starch synthase I (GBSSI), SSI, SSII-1, SSII2, SSII-3, SSIII-1, SSIII-2, SSIV-1, and SSIV-2 (Fujita, Satoh, Hayashi, Kodama, Itoh,

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Aihara & Nakamura, 2011; Fujita, Yoshida, Kondo, Saito, Utsumi, Tokunaga, Nishi, Satoh, Park, Jane, Miyao, Hirochika & Nakamura, 2007; Li, Sun, Xu, Chu, Mukai, Yamamoto, Ali, Rampling, Kosar-Hashemi, Rahman & Morell, 2003). Ap synthesis involves multiple isoforms of SSs, starch branching enzymes (SBEs) and starch debranching enzymes (DBEs). SBE snips (1→4)-α linked chains and then adds a snipped chain to form (1→6)-α linkage branch points. The enzymes in Am synthesis

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are mainly GBSS (Fujita, 2014; Jeon, Ryoo, Hahn, Walia & Nakamura, 2010; Satoh, Shibahara, Tokunaga, Nishi, Tasaki, Hwang, Okita, Kaneko, Fujita, Yoshida, Hosaka,

debranching enzymes (DBEs) are also involved.

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Sato, Utsumi, Ohdan & Nakamura, 2008) and one or more SBEs. Isoforms of

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It is likely that all of these enzymes (SSI-SSIV, SBEI-II and GBSS) are involved in the

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synthesis of Am and Ap, although the mechanistic details are not fully understood. An Am extender (ae) mutation of SBEIIb, which suppresses SBE, increases the number of

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Am chains (Guan, Li, Imparl-Radosevich, Preiss & Keeling, 1997; Yun & Matheson, 1993). Furthermore, SSI and SBEIIb could affect the function of mutation of SSIIa

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(Luo, Jobling, Millar, Morell & Li, 2015). GBSS mainly functions to elongate Am and

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to increase the amount of extra-long chains of Ap, while SS I – IV are believed to be only involved in Ap synthesis (Fujita, 2014; Hanashiro, Itoh, Kuratomi, Yamazaki, Igarashi, Matsugasako & Takeda, 2008; Jeon et al., 2010; Smith, 2001). GBSS has two isoforms but only the isoform GBSSI is found in the seed endosperm (Hirose & Terao, 2004). The different functional alleles of the Waxy gene that can encode GBSSI have

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been reported to have strong effects on Am content (Dian, Jiang, Chen, Liu & Wu, 2003; Hirose & Terao, 2004; Itoh, Ozaki, Okada, Hori, Takeda & Mitsui, 2003; Vrinten & Nakamura, 2000).

One possible way to obtain information on the activities of starch biosynthetic enzymes is based on the precise measurement of the chain length distributions (CLD)

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of Am and Ap. The amylopectin model developed by Wu et al. (Wu, Morell & Gilbert, 2013) assumes that Ap biosynthesis is controlled by different independent enzyme sets

in which different isoforms of SS, SBE, and DBE are involved, and has proved a useful

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way to do this. In this model, SS, SBE and DBE are considered as the three core enzyme

classes that are important in utilizing ADP-glucose to synthesize starch. The CLD of

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Ap can be accurately measured by fluorophore-assisted carbohydrate electrophoresis

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(FACE) (Morell, Samuel & O'Shea, 1998) (high-performance anion exchange chromatography, HPEAC, could also be used, but the method suffers from mass bias,

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which is laborious to correct (Wong & Jane, 1997). The fitting yields the ratio of the activities βi of SBE and SS for each enzyme set i, and the relative propagation rates hi

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of SS in these sets (Wu et al., 2013).

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Unlike Ap, the development of a generally accepted biosynthesis- based model for

analyzing Am CLDs is still limited. One of the reasons for this problem is that the Am CLD can currently only be measured by size- exclusion chromatography (SEC, a type of gel-permeation chromatography, GPC), which, however, suffers both from band broadening (unlike FACE) which distorts the shape of the CLD, and from uncertainties 5

in the assumptions and parameter values required to convert SEC elution time to degree of polymerization (DP, symbol X) (Vilaplana & Gilbert, 2010). The model developed by Nada, Zou, Li and Gilbert (2017) partially overcomes these problems, and also gives values of the two fitting parameters β and h which are analogous to those for Ap (Yu, Li, Zou, Tao, Zhu & Gilbert, 2019). Similar to that of Ap, the model assumes that Am CLDs are controlled by different individual enzyme sets including GBSS (for chain

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growth, possibly with other SSs) and SBEs (for chain stoppage).

In this study, the CLDs of extracted starches from mature grains of 95 different rice

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cultivars were measured by SEC. The CLDs of both Am and Ap were then fitted with the biosynthesis-based models developed by Wu et al. (2013) for the Ap CLD and by

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Nada et al. (2017) for that of Am. These reduce the CLDs to a number of

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biosynthetically meaningful parameters, which can then be used in appropriate statistical analyses. Correlations could provide useful information concerning the

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interactions between Am and Ap synthesis.

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Our aim is to see if enzymes that are mainly responsible for Ap synthesis (particularly SBE and its isoforms) are also involved in Am synthesis, and vice-versa,

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even though it is often thought that Ap and Am are synthesized by separate sets of enzymes. Additionally, this will show if any interactions between Ap and Am synthesis enzymes depend on the amount of Ap and Am within a particular DP range. This study could provide information of rice breeders and molecular biotechnologists to develop rice cultivars with better functional properties. 6

2. Materials and methods 2.1. Materials

95 rice cultivars (S01- S95) were planted and harvested in Hainan Province, China, in April 2017 and were provided by the Chinese Academy of Agricultural Sciences Agricultural Genomics Institute, Shenzhen, China. These samples were gathered from

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29 countries and are subdivided into five different rice populations, Indica, Temperate Japonica, Tropical Japonica, Aus (early summer rice) and Admix (cultivated with unspecified genetic background), as shown in Supplementary Table 1.

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2.2. Extraction of rice starch

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Rice kernels were dried for 3 days at 38 ℃. Following this, the grain was dehusked

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with a rice huller (Model SY88-TH, Korea), then polished with a grain polisher (Model Kett, Tokyo, Japan). The grain was immersed in sodium hydroxide solution (pH 8.0-

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8.5) overnight to protect starch from degradation. Rice was then ground into flour in a 50 mL centrifuge tube surrounded by ice, by inserting a High Speed Disperser (XHF-

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DY, Scienzt, Ningbo, China). During grinding, the XHF-DY was removed every 10 s

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to minimize heating. The whole process was repeated 6 times, and then the flour filtered through a 100-mesh and then a 200-mesh sieve.

Protease (alkaline protease, Beijing Solarbio Science & Technology Co. Ltd.,

Beijing, China) 50 mg (~20,000 U/g) solution containing sodium nitrate (0.04 g mL–1) was mixed with the flour kept at 16 h at 42℃ to remove most protein, and then was

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centrifuged and the supernatant discarded. The residue was washed three times with deionized water to remove amino acids and then twice with ethanol to remove liquids. Finally, purified starch was precipitated after centrifugation (4000 g for 10 min) and then freeze-dried. 2.3. Starch debranching

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Around 7~9 mg purified rice starch was dissolved in 1.5 mL dimethyl sulfoxide (DMSO) containing 0.5% LiBr (w/w) overnight, followed by using 6 mL absolute

ethanol to reprecipitate starch, and then centrifuging (4000 g, 10 min) with the

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supernatant being discarded. Another 6 mL absolute ethanol was used and the

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suspension centrifuged to remove remaining DMSO. The residue was mixed with 0.9 mL of warm distilled water and kept in a boiling water bath for 15 min or longer until

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starch was fully gelatinized. After cooling to room temperature, iso-amylase in acetate buffer (0.1 M, pH ~ 3.5) containing sodium azide (0.04 g/mL) was added. The mixture

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was incubated in a water bath at 37 °C for 3 h. After this, 0.1 M sodium hydroxide solution was added to neutralize the mixture to pH ~ 7, followed by incubating at 80 °C

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for at least 1 h, then freeze-dried overnight. The dried debranched starch was re-

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dissolved in DMSO/LiBr (4-6 mg/mL) and was transferred to SEC phials for further analysis.

2.4. SEC

Enzymatically debranched starches dissolved in DMSO/LiBr were analyzed using a Waters SEC-MALLS system (Wyatt Technology), equipped with differential 8

refractive index (DRI) using the method detailed elsewhere (Tao, Li, Yu, Gilbert & Li, 2019). For linear polymers, including debranched starches, there is a unique relationship between size and molecular weight, so the SEC signal can be processed to yield the weight CLD as a function of the degree of polymerization, w(logX). The relation between the CLD number distribution (the number of chains of a given degree of polymerization X following debranching), Nde(X), and the corresponding weight

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distribution is w(logX) = X2 Nde(X). The relationship between the SEC elution time, the SEC separation parameter, the hydrodynamic radius Rh of the eluted molecule, and X

Houwink equation (Vilaplana & Gilbert, 2010).

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2.5. Fitting amylose and amylopectin CLDs

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for linear polymers is found by calibration with known standards and the Mark-

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The Ap CLD was fitted with the model of Wu et al. (2013) using publicly available code (https:// sourceforge.net/projects/starchcldfit/?source = directory). It

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takes as a starting point sets of the enzymes involved in starch synthesis: the various isoforms of starch synthesis, branching and debranching enzymes. Briefly, for a given

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region (there are three amylopectin regions chosen in this study: DP 6–24, 28–58 and

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68–78), it is assumed that the CLD is primarily (but by not entirely) dominated by one or two enzyme sets, these comprising one or two of each of a starch synthase, a branching and a debranching enzyme. The final result is the ratio of activities of the starch synthesis (SS) and branching enzyme(s) (SBE) in enzyme set j (j=i, iii, v), βAp, j, and the relative amount of the overall CLD produced by that set to that produced by the

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set j (j=i, iii, v): hAp, j. DBE is also involved, but the mathematical development shows that its activity is determined by those of SS and SBE.

The Am CLD was fitted with the model of Nada et al. (2017), which takes band broadening into account. The fits were implemented with a new app for this purpose, again using publicly available code (http://github.com/snada88/AmyloseBroadening/).

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Two amylose regions are used in this study (DP 100–500, 500–1000); the Am fitting parameters, βAm, j (j=i, ii) and hAm, j (j=i, ii), have the same meaning as those for amylopectin. 2.6. Statistical analysis

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Pearson correlations were calculated with SPSS 20.0 (Statistical Graphics Corp.,

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Princeton, NJ).

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3. Results 3.1. Starch molecular structure analysis

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The w(logX) of 95 cultivated rices were obtained and fitted to the models

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(Supplementary Figures 1 and 2 and Supplementary Table 2). Fig. 1a shows 20 typical weight distributions of the debranched rice starches. All rice samples have w(logX)

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distributions which are similar to those commonly seen, e.g. in (Tao et al., 2019). DPs less than the minimum near X= 100 are assumed to be Ap chains, and those above this to be Am chains.

For Ap chains, the first peak in the SEC CLD (DP 10 ~ 20) corresponds to chains confined to a single lamella chains, and is assumed to be predominantly but not 10

exclusively produced by enzyme set i, and slightly higher DPs to set ii (DP ~20). The bump over DP 30~ 40, for chains crossing into a second lamella, is predominantly controlled by enzyme sets iii and iv. The rest of the features in chains spanning more than two lamellae (DP> 40) arise mainly from enzyme sets v and vi (Yu, Tan, Zou, Hu, Fox, Gidley & Gilbert, 2017). As shown in Fig. 1a, for all rice samples, the shapes around the first peak are similar for all samples, whilst significant differences in the

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second peak (DP ~40) are observed.

Figure 1. SEC weight CLDs, w(logX), of debranched starches (a) whole DP region

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from typical samples, normalized to the maximum of the Ap component and (b)

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an enlargement of the Am region.

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Figure 2. SEC weight CLDs, w(logX), of debranched starches in the Am region

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from typical samples, divided into four sub-populations.

The weight distribution of Am CLD (DP ≳ 100) is shown in Fig. 1a and enlarged

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in Fig. 1b. The genetic background of Tropical Japonica and Temperate Japonica are similar, and thus we group them into traditional Japonica to compare our results

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efficiently. The four main sub-population groups of the cultivated rice are shown in Fig. 2. One can distinguish three features with different DP ranges for Am, similar to previous studies (Yu, Tao & Gilbert, 2018). The first feature is for DP ≲ 200, the second one over DP 200~ 400 and the last one for DP ≳ 1000. For some samples, one can only distinguish one or two features. 12

Similar to the Ap CLD fitting method, different features of Am CLDs are assumed to be controlled by the activities of the different isoforms of Am biosynthesis enzymes (Nada et al., 2017). SBE can snip the longer chains which arise from an SS elongating shorter glycosidic chains. Seeing a third peak for some rice samples may possibly arise because of the higher enzyme activity of an isoform of GBSS, but this is just a hypothesis. We note that the values of β are in vivo measurements of enzyme activity;

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while an in vitro activity measurement could show the same trends, our in vivo

measurements from β are in fact the best way of obtaining information on activities in

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the plant.

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3.2. Fitting amylose and amylopectin CLDs to biosynthetic models

For Ap fitting (Wu et al., 2013), six parameters, hAp, i, βAp, i, hAp,iii, βAp,iii, hAp,v and βAp,v ,

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are given by the model; here, the DP ranges of the three regions (i, iii, v) are chosen as 6 ~24, 28 ~58 and 68 ~78, respectively. The Am fitting used the step-by-step description

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of Tao et al. (2019). The Am CLDs of the rice samples were divided into two rather than three regions, whence the fitting of two Am region yielded four parameters, hAm,i,

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βAm,i, hAm,ii, and βAm,ii.

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Domesticated rice varieties can be classified into two major sub-population (Indica and Japonica) or varietal groups, and different populations have different networks of alleles controlling numbers of variety traits (Sweeney & McCouch, 2007; Zhao, Wright, Kimball, Eizenga, McClung, Kovach, Tyagi, Ali, Tung, Reynolds, Bustamante & McCouch, 2010). For a better comparison of the differentiation of the rice accessions, 13

the averages of the hAp,j, hAm,j, βAp,j and βAm,j were calculated by dividing the samples BD into different sub-population, as shown in Table 1. The value of the parameters in different sub-population groups are close except those of hAp,v and hAm,i. Because of the significant differences in the hAp,v and hAm,i, one sample, G649(S80), which had a relatively high hAp,v and hAm,i but a lower hAm,ii , was selected to compare the differences bettwen individual samples. The fitted CLDs of amylopectin and amylose are shown in

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Fig. 3. All values of G649 are given in Table 1. A sample C602 (S18) with relatively lower hAp,v and hAm,i but with a higher hAm,ii , was selected to compare with sample

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G649, as shown in Fig. 4.

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Figure 3. Fitting results for Ap (a) and Am (b) CLD for G649 (S80).

Figure 4. Fitting results of Ap (a) and Am (b) CLD for C602(S18).

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Significant differences can be found in both hAm, i and hAm, ii among four subpopulation groups (fittings of typical samples G649 and C602 are shown in Figs. 3 and 4). As the fitted results and Table 1 show, more amylose with longer chains (higher hAm, ii)

is found in both sub-populations Aus and Admix, while a larger amount of shorter

Am chains (higher hAm, i) was seen in both sub-populations Indica and Aus; a typical

chains (higher hAp,v) is found in sample G649. 3.3. Correlations between Ap and Am CLD parameters

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sample is shown as the Indica group member G649 in Fig. 3. More Ap with longer

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The correlations between the Ap CLD and Am parameters CLD of 95 samples

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were calculated by SPSS 20.0 (Statistical Graphics Corp., Princeton, NJ) and shown in Table 2. There are a number of correlations between Am and Ap fitting parameters.

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With β values (larger values of β in a given region imply more smaller chains), a significant negative correlation was found between βAp,iii and βAm,i. For the values of h

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for amylose (proportional to the amount of amylose in a given region), it is seen that the correlation between hAp,v and hAm,i is highly significant. A significant positive

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correlation was found between hAp,i and hAm,ii. In addition, a strong positive correlation

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was seen between hAp,v and βAm,ii, and βAp,iii had a significant negative correlation with hAm,i.

4. Discussion A rich diversity in the CLDs was seen in the 95 cultivated rices (all of which are given in the Supplementary Information), especially in the amylose CLDs, which show 15

the same trends in 4 different populations (Fig. 2). Similar rice CLD diversity was also seen in previous work (Tao et al., 2019).

The synthesis of Ap is mainly controlled by different isoforms of SS, SBE and debranching enzyme (DBE), while GBSSI dominates Am biosynthesis. As reported elsewhere, SBE takes part in Am synthesis (Nishi, Nakamura, Tanaka & Satoh, 2001;

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Wang, White, Pollak & Jane, 1993). There are very few studies providing direct and/or indirect evidence to see if these enzymes catalyze the synthesis together, and if an enzyme set which takes part in the synthesis of Ap can also catalyze Am biosynthesis.

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In this study, a strong significant positive correlation (r= 0.896, p <0.01, Table 2)

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between hAp, v (long Ap chains, DP 68~78) and hAm,i (short Am chains, DP 100~500) was observed. A larger value of hAp and hAm represents a higher relative amount of both

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Ap and Am branches, which possibly results from a higher ratio of the enzyme activities of SBE to that of SS, and SBE to that of GBSS respectively (Nada et al., 2017; Wu et

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al., 2013). This means that for rice starches, the amount of Ap with long chain lengths

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(DP>68) significantly positively correlates with the amount of Am with short chain lengths. Our hypothesis is that the SBE set that is involved in the synthases of long Ap

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branches is also responsible for short Am synthesis. This suggests that the SBE isoforms determining the synthesis of long Ap branches could also snip long Am chains into shorter chains.

This hypothesis is supported by a recent report that the double homozygous mutations of SBE IIa and IIb could contribute a higher Am content with distinct 16

molecular structure, compared to the single homozygous mutations of SBE IIa in wheat (Li, Dhital, Slade, Yu, Gilbert & Gidley, 2019). Furthermore, the SS in the Ap enzyme set v, which is responsible for the synthesis of long Ap chains, is probably responsible for the synthesis of long chains of Am, as suggested by the significant positive correlation between hAp,v and βAm,ii (Table 2). SSIIIa, an isoform of SS, can elongate the

provides relatively more shorter chains in a given region.

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long chains connecting Ap clusters (Fujita et al., 2007). A larger value of βAp and βAm

In the biosynthesis models used to fit the CLDs the models (Nada et al., 2017; Wu

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et al., 2013), βAp is the ratio of activities of SBE to that of SS, while βAm is the ratio of the activities of SBE to that of GBSS. As shown in Table 2, a negative correlation

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between βAp,iii and βAm,ii is seen, indicating that the correlation between the ratio of

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activities of SBE to that of SS significantly and negatively correlate with the ratio of enzyme activities of SBE to that of GBSS. This also suggests that the enzyme activity

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of SS is significantly negatively correlated with that of GBSS. Consistent with this supposition, the expression of SSI and GBSSI gene can be improved when SSIIIa is

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deficient (Fujita, 2014; Fujita et al., 2011; Fujita et al., 2007). In addition, a mutation

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of the rice genome has been found that can elevate the proportion of amylose at the expense of reduced short-chain amylopectin (Butardo, Anacleto, Parween, Samson, de Guzman, Alhambra, Misra & Sreenivasulu, 2017).

It is seen that hAp,i significantly positively correlates with hAm, ii (Table 2). This suggests that more short chains in Ap region 1 can supply more substrates for the 17

synthesis of Am. However, as shown in Table 2, neither hAp, i nor βAp, i have significant correlations with other Am fitting parameters. This suggests that enzyme set i (active in Ap region 1) does participate in any Am biosynthesis. The positive correlation (Table 2) between hAp,i and hAm,ii might be because the isoforms of SBE in Ap enzyme set i cannot snip the long chains in Am region 1. However, these inferences require more direct evidence, although SS has been reported to affect some properties of the grain

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(McMaugh, Thistleton, Anschaw, Luo, Konik-Rose, Wang, Huang, Larroque, Regina, Jobling, Morell & Li, 2014).

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Fig. 5 gives typical CLDs useful for explaining the possible mechanism beneath the

synthesis of rice Ap and Am. A larger amount of longer Ap chains (those in Ap region

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3) corresponds to a significantly lower amount of long Am branches. It is therefore

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Am chains by SBE.

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likely that the accumulation of long Ap branches is because of the snipping of longer

Figure 5. SEC weight CLDs, w(logX), of debranched G649 and C602, normalized to the maxima of the amylopectin component. 18

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Figure 6. Suggested mechanism of amylopectin and amylose synthesis. The superscript number followed by enzymes indicates that these enzymes are mainly

active in amylopectin region 1 (SS1, SBE1), amylopectin region 2 (SS2, SBE2),

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amylopectin region 3 (SS3, SBE3), amylose region 1 (GBSS4), and amylose region

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2 (GBSS5). The arrows mean that SBE snips the longer chains of amylose into shorter chains.

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Fig. 6 gives a hypothesis assuming that the isoforms of each biosynthesis enzyme

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dominating a given region form an enzyme set controlling the synthesis of amylopectin/amylose in those regions.

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5. Conclusion

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The relationship among the three main biosynthesis enzymes of starch (SBE, SS, GBSS) have been analyzed for 95 rice cultivars. The results showed that different enzyme sets of SBE and SS in amylopectin are also involved in the synthesis of rice amylose, whilst amylopectin enzyme set i is alone responsible for the synthesis of the short chains of amylopectin (DP 6~24). These short chains can also be the initial

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substrates for amylose synthesis. Furthermore, not only can amylopectin enzyme set iii catalyze the synthesis of intermediate-length amylopectin chains (DP 28~58), but it can also affect the synthesis of short and long amylose chains. Additionally, it is likely that the enzyme which is responsible for the synthesis of long amylopectin chains is also involved in the synthesis of long amylose chains. As some of the longer amylopectin (Ap set v) and shorter amylose (Am set i) chains share the enzyme set, the GBSS could

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provide more longer amylopectin chains (Crofts, Itoh, Abe, Miura, Oitome, Bao & Fujita, 2019).

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It is suggested that while part of the amorphous starch layer is amylose, one side is mainly amylopectin (Fig. 6). This inference is consistent with the report about the

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alternating amorphous and crystalline lamellae of growth-ring structure of a starch

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granule (Imberty, Chanzy, Perez, Buleon & Tran, 1988; Imberty & Perez, 1988; Pfister & Zeeman, 2016; Smith, 2001).

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This study provides useful information for rice breeders for cultivating rice with

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improved quality by understanding the correlations between starch biosynthesis enzymes: how a change in one enzyme which dominates, say, certain aspects of

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amylopectin biosynthesis, can also affect amylose characteristics. Methods that could alter the enzyme activities of starch synthases, thereby changing the CLD of rice starch, could be used for the production of rice with desirable eating quality and digestion rate (Alhambra, de Guzman, Dhital, Bonto, Dizon, Israel, Hurtada, Butardo & Sreenivasulu, 2019; Regina, Kosar-Hashemi, Ling, Li, Rahman & Morell, 2010; Shrestha, Blazek, 20

Flanagan, Dhital, Larroque, Morell, Gilbert & Gidley, 2015) (Li & Gilbert, 2018; Tanaka, Takahashi, Kato, Sawazaki, Akasaka, Fujita, Kumamaru, Saito, Shirouchi & Sato, 2018).

Author contribution

JZ was involved with the project concept, carried out the experimental work and data

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fitting, and wrote the manuscript. RGG and QL were involved with the project concept and discussion, and with manuscript preparation. WY was involved with the project concept and discussion, advice on experimental techniques, and with manuscript CZ, YZ and JX helped with data collection and processing.

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preparation.

EL was

Acknowledgements

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involved with advice on experimental techniques, and with manuscript preparation.

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We gratefully acknowledge the support of grants from the National Natural Science

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Foundation of China (31825019, 3151101138) and from the Jiangsu Province Government (2017 Jiangsu Innovation and Entrepreneurship Talents Program,

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BE2018357 and PAPD). A project funded by the Priority Academic Program of Jiangsu Higher Education Institutions.

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Table 1 Fitting parameters for Am and Ap CLDs. Region 1

Ap Region 2

Am Region 3

Region 1

Region 2

(DP 6~24)

(DP 28~58)

(DP 68~78)

(DP 100~500)

(DP 500~1000)

βAp,i hAp,i i × hAp,iii βAp,iii hAp,v βAp,v hAm,i βAm,i× hAm,ii × βAm,ii× 2 2 × 10 10 × 102 × 102 × 102 × 102 × 102 102 102 102 10.1 ± 0.2

2.6 ± 0.2

4.3 ± 0.2

Japonica

100 ± 6

10.2 ± 0.3

2.5 ± 0.2

4.5 ± 0.2

Aus

104 ± 4

10.2 ± 0.3

2.6 ± 0.1

4.2 ± 0.2

Admix

104 ± 4

10.1 ± 0.2

2.7 ± 0.2

4.3 ± 0.3

G649 C602

94

10.1

2.8

4.2

0.12 ± 0.08 0.09 ± 0.05 0.15 ± 0.08 0.11 ± 0.06 0.28

101

10.6

2.6

4.6

0.12

1.5 ± 0.6

8.7 ± 7.7

124 ± 40

11.8 ± 6.4

30 ± 17

1.4 ± 0.4

5.4 ± 5.0

136 ± 35

10.3 ± 4.9

28 ± 18

1.3 ± 0.5

10.6 ± 9.0

128 ± 18

14.6 ± 7.1

33 ± 14

1.4 ± 0.2

7.1 ± 5.7

114 ± 61

14.3 ± 7.9

22 ± 12

1.5

26.1

132

7.6

58

2.0

6.0

121

22.8

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104 ± 6

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Indica

βAp,v -0.044 0.024 -0.058 -0.284**

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βAp,iii -0.226* -0.422** -0.542** -.0258*

hAp,i 0.036 -0.071 0.006 0.485**

hAp,iii 0.276** 0.283** 0.355** 0.251*

hAp,v 0.168 0.695** 0.896** -0.161

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βAm,i βAm,ii hAm,i hAm,ii

βAp,i 0.019 -0.054 0.001 -0.139

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Table 2 Correlation between the parameters of Ap and Am.

* correlation is significant at the 0.05 level (2-tailed),** correlation is significant at the

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0.01 level (2-tailed).

26