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Keywords:

  • high-amylose rice;
  • resistant starch;
  • starch branching enzyme;
  • dietary fibre;
  • diabetes

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

A high-amylose rice with 64.8% amylose content (AC) was developed by transgenic inhibition of two isoforms of starch branching enzyme (SBE), SBEI and SBEIIb, in an indica rice cultivar. The expression of SBEI and SBEIIb was completely inhibited in the transgenic line, whereas the expression of granule-bound starch synthase was normal. Compared with wild-type rice, drastic reductions in both SBEs in the transgenic rice increased apparent AC in flour from 27.2% to 64.8%, resistant starch (RS) content from 0% to 14.6% and total dietary fibre (TDF) from 6.8% to 15.2%. Elevated AC increased the proportion of long unit chains in amylopectin and increased onset gelatinization temperature and resistance to alkaline digestion; however, kernel weight was decreased. A rat feeding trial indicated that consumption of high-amylose rice decreased body weight gain significantly (< 0.01); increased faecal mass, faecal moisture and short-chain fatty acids; and lowered the faecal pH. An acute oral rice tolerance test revealed that the high-amylose rice had a positive effect on lowering the blood glucose response in diabetic Zucker fatty rats. This novel rice with its high AC, RS and TDF offers potential benefits for its use in foods and in industrial applications.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

A cereal grain with a high amylose content (AC) is a source of resistant starch (RS) (Jiang et al., 2010), which is not digested in the small intestines but passes to the large bowel for fermentation (Asp, 1992). The effects of RS in increasing faecal mass, protecting against colorectal cancer and elevating large-bowel short-chain fatty acids (SCFA) have been reported in pigs (Nofrarías et al., 2007; Regmi et al., 2011), rats (Liu and Xu, 2008; Brites et al., 2011) and humans (Phillips et al., 1995; Hasjim et al., 2010). Furthermore, high-amylose starch assists in preventing the development of non-reversible insulin resistance (Hoebler et al., 1999; Fuentes-Zaragoza et al., 2011) and depresses plasma total lipid, cholesterol and triacylglycerol concentrations (Cheng and Lai, 2000; Lopez et al., 2001). Although the unique functional properties of a high-amylose diet are gaining acceptance as a desirable outcome for consumers, the range of crops that are high in AC is limited. As the amylose-extender (ae) mutant with high AC was first exploited in maize in 1946 (Whistler, 1984), more scientists have focused attention on breeding high-amylose crops. Schwall et al. (2000) produced a very high-amylose potato by simultaneously inhibiting starch branching enzymes (SBEs) A and B. Starch from these lines had an apparent AC of 60%–89%. Morell et al. (2003) reported that the loss of a soluble starch synthase (SSS), SSSIIa, in barley leads to a novel high-amylose barley (71.7% of AC). A high-amylose wheat (74.4% of AC) has been developed by RNA interference (RNAi), and the increased level of RS in that wheat has the potential to improve human health (Regina et al., 2006).

Rice, rich in starch, is a major diet component for more than half of the world’s population. In Asia, over 2000 million people obtain 60–70% of their daily calories from rice and its processed products (FAO, 2004). Normal or waxy rice is an important source for food and industrial applications, but both are poor sources of RS.

In rice, amylose synthesis is mainly controlled by the Waxy (Wx) gene, which encodes the granule-bound starch synthase (GBSS). Based on the level of GBSS accumulating during the process of grain filling in non-waxy rice cultivars, two alleles, Wxa and Wxb, were identified (Sano, 1984). Compared with the Wxb allele, the Wxa allele enhances the level of GBSS, resulting in high AC in the grain (Cai et al., 1998). The Wxa allele is mainly present in indica rice, whereas the Wxb allele is predominant in japonica rice (Tian et al., 2009). SBEs are responsible for the production of the α-1,6-glycosidic linkages in starch (Martin and Smith, 1995). The SBEs catalyse the cleavage of an α-1,4-linked glucan chain and subsequent transfer the part of that chain to the 6-hydroxyl along another chain; thus, SBEs play an important role in determining the structure of amylopectin. At least three isoforms of SBEs—SBEI, SBEIIa and SBEIIb—have been identified in rice endosperm (Mizuno et al., 1992; Nakamura et al., 1992). SBEI was reported to play a role in the formation of long chains of amylopectin, whereas SBEIIb generates short chains (Nishi et al., 2001; Nakamura, 2002; Satoh et al., 2003). Chain-length distribution analysis of a SBEIIa-deficient mutant of rice (Nakamura, 2002; Satoh et al., 2003) suggests that SBEIIa supports partially, but not predominantly, the function of SBEI and SBEIIb. Structural and cDNA sequences of SBEI and SBEIIb genes have been cloned and sequenced (Kawasaki et al., 1993).

In this study, after testing the effect on AC by transgenic inhibition of different SBEs’ activities, we produced transgenic indica rice containing high amylose and high RS content. The RS-enriched rice improved the indices of gut health responses in normal rats, in particular the glycaemic response in diabetic rats. This is the first report of a high-amylose rice (approximately 65% AC) with health benefits.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Effects on amylose elevation by transgenic inhibition of different SBEs

To test the effects of inhibition of different SBE(s) on increasing the amylose level, we constructed three RNAi or antisense vectors, p13dsSBE1, p13dsSBE3 and p13aSBE13 (Figure S1c). Constructs of p13dsSBE1 and p13dsSBE3 were generated by cloning partial cDNAs of the rice SBEI and SBEIIb genes in inverted repeats, respectively, whereas the p13aSBE13 clone was constructed by putting the fragments of both cDNAs together in an antisense orientation (Figure S1a–b). An endosperm-specific rice glutelin Gt1 promoter was used to drive the expression of target hairpin-RNA or antisense molecules to inhibit the activity of SBEI, SBEIIb or both in transgenic rice seeds.

These constructs were first tested for proof-of-concept in two easily transformed japonica rice cultivars, Wu-xiang-jing (WXJ) and Guang-ling-xiang-nuo (GLXN, glutinous rice), with intermediate (16.7%) and very low (1.5%) AC, respectively. More than 30 independent transgenic plants were obtained for each set of the constructs and rice varieties, and three stable transgenic rice plant lines in T2 generation were randomly chosen as representative lines for each construct/variety set (Figure 1a, d). Protein blotting analyses showed that the expression of SBEI (or SBEIIb) was undetectable in all three p13dsSBE1 (or p13dsSBE3)-derived RNAi lines, which showed no effect on SBEIIb (or SBEI) expression (Figure 1b–c,e–f); however, both SBEI and SBEIIb proteins were nearly completely inhibited in all three p13aSBE13-derived antisense transgenic lines.

image

Figure 1.  Molecular and expression analyses of transgenic plant lines. Lanes 1–4 are transgenic lines, and lanes WXJ and GLXN are wild type (WT). Panels (a), (b), (c) and (g) show the transgenic plants derived from japonica cv Wu-xiang-9915 (WXJ), and panels (d), (e), (f) and (h) represent transformants derived from the waxy japonica cv Guang-ling-xiang-nuo (GLXN). (a,d) PCR analysis of total DNA from different transgenic rice lines and their WT. (b,e) Western blot analysis of total proteins from mature seeds with polyclonal antibodies against rice SBEI. (c,f) Western blot analysis of total proteins from mature seeds with polyclonal antibodies against rice SBEIIb. (g,h) Amylose contents of mature grains in transgenic plant lines and their WTs.

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Comparing the grain ACs among the three homozygous transgenic lines and their untransformed controls showed different alteration in AC in the three types of transgenic lines (Figure 1g–h). Inhibition of SBEI had no effect on AC. A substantial increase in AC was noted in the SBEIIb RNAi transgenic rice seeds, but the increase in antisense RNAs of both SBEs transgenic grains was much more significant (< 0.01), indicating that simultaneous manipulation of two SBE genes was possible to dramatically increase the AC of rice grains.

Generation of high-amylose rice by simultaneous inhibition of two SBEs in indica rice

To obtain transgenic rice with high AC, the double antisense construct aSBE13 was introduced into an indica rice variety Te-qing (TQ), which contains the Wxa allele and a relatively high amylose level (27.2%, Figure 2e). Four independent homozygous transgenic lines in T2 generation were selected. The result from Southern blot analysis showed that one or two copies of the double antisense construct were inserted into the genome of the selected transgenic rice (Figure 2a). RT-PCR results revealed that the expression of both SBEI and SBEIIb genes was inhibited or down-regulated dramatically in the developing seeds of the transgenic plants (Figure S2). These inhibitions were further confirmed at the protein level by protein blotting analyses (Figure 2b–c). Expression analyses also implied little or no impact of the introduced p13aSBE13 construct on the expression of GBSS (Figure 2d), or on other starch biosynthesis–related genes in the transgenic rice grains (data not shown).

image

Figure 2.  Molecular and expression analyses of transgenic indica plant (lanes 1–4) and their wild-type Te-qing (TQ, lane WT). (a) Southern blotting analysis of genomic DNA extracted from plant leaf tissues and digested with Bam HI. (b, c) Western blotting analysis of total protein extracts from mature seeds with polyclonal antibodies against rice SBEI and SBEIIb, respectively. (d) SDS-PAGE analysis of GBSS in rice mature seeds. The gel was stained with Coomassie Brilliant Blue G-250. (e) Amylose contents and resistant starch contents of mature grains in transgenic plant lines (HA1, HA2) and their wild type.

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The AC of these transgenic indica plants was greatly increased compared with their wild type (WT). The maximum increase occurred in the transgenic line TQ-aSBE13-4, in which the apparent AC was 64.8% as measured by the iodometric blue-colour method, which was more than twofold higher than the 27.2% amylose in the WT (Figure 2e). A different assay for amylose based on the ConA precipitation method showed that the AC in transgenic grains was 55.6%, but only 22.5% in WT seeds (Zhu et al., 2011). Two transgenic lines with highest AC, TQ-aSBE13-3 and TQ-aSBE13-4, were renamed HA1 and HA2, respectively, and were used for further experiments.

In addition to exploring the effect of the p13aSBE13 transgene on grain AC, the major agronomic traits of HA1 and HA2 in T4 and T5 generations and their WT were carefully investigated and compared in two successive seasons’ field trials approved by the Chinese government. Total 300–400 kg of high-amylose rice was harvested every year. Results indicated minimal effects on the traits of agronomic importance, except for kernel appearance and weight of the high-amylose rice (Table S1). The kernel weight of high-amylose rice was significantly decreased (< 0.01), mainly because of the reduced thickness (Table S1). Furthermore, the brown kernels of the high-amylose rice (Figure 3d) appeared much more opaque compared with those of the WT (Figure 3a).

image

Figure 3.  Thermal properties and grain and starch granule morphology of the high-amylose rice HA2 (d–f) and its wild type (a–c). (a, d) The appearance of brown rice of wild-type Te-qing (a) and transgenic line HA2 (d). (b, e) Morphology of starch granules from wild-type Te-qing (b) and transgenic line HA2 (e) using scanning electron microscopy. (c, f) Polished grains from wild-type Te-qing (c) and transgenic line HA2 (f) soaked in 5% potassium hydroxide solution for 16 h. (g) Rapid viscosity profiles of rice flour. (h) The gelatinization of the flours was determined by differential scanning calorimeter (DSC) at 33.3% solid concentration.

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High-amylose rice with a high level of RS and total dietary fibre

In maize, as the amount of amylose in the starch granule increases, a corresponding elevation occurs in total dietary fibre (TDF) and RS content (Li et al., 2008; Jiang et al., 2010). In grains of HA1 and HA2, the RS content was 10.8% and 14.6%, respectively, measured by the AOAC Method 2002.02, whereas little or no RS was detected in the WT grain (Figure 2e). When the Englyst assay method was used, the RS content in flour was 33.4% in HA2 and 18.3% in the WT. The TDF in flour was 15.2% in HA2, which was 2.2 times that in the WT (6.8%). The same trends also were observed in isolated rice starches, although the levels of RS and TDF differed between flour and starch (Zhu et al., 2011). These results, as determined by different methods, indicated that RS and TDF were significantly higher in the high-amylose transgenic rice seeds than in normal seeds.

High-amylose rice starch highly resistant to alkali digestion and gelatinization

To understand the gelatinization properties of the high-amylose rice starch, the polished whole grains were soaked in 5% aqueous potassium hydroxide solution for 16 h. As shown in Figure 3c, the kernels of the WT rice were almost disintegrated, and their shapes were barely visible in the alkaline medium. In contrast, the kernels of HA2 (Figure 3f) retained their intact shapes, although some transparency developed after overnight gelatinization. Moreover, the concentration of urea solution for the onset of gelatinization was much higher in HA2 starch than in the WT starch (Figure S3a–b). When the supernatant from starch solubilized by 4 m urea solution was stained with iodine, the observed absorption curve from 425 to 725 nm was much lower in HA2 transgenic rice than in the WT rice (Figure S3c), suggesting that the HA2 starch granules were less soluble than those of the WT. The pasting curves of the HA1 and HA2 flours, which were measured by a Rapid Visco Analyser (RVA), showed essentially no viscosity development during heating, whereas the WT flour gave a normal pasting curve at 10% solids (Figure 3g). All of these results indicate that HA2 rice presents a strong resistance to alkaline gelatinization and swelling of its granules.

Figure 3h shows the differential scanning calorimeter (DSC) thermal properties of flours from HA2 and WT. Because the gelatinization peak in HA2 overlapped with the peak of the amylose–lipid complex, we extrapolated some of the thermal properties for that sample as shown in Figure 3h. To was the onset, Tp1 was the peak, and Tc1 was the conclusion temperature of gelatinization; Tp2 and Tc2 were the peak and conclusion temperatures of the melting of the amylose–lipid complex. No difference was found in the thermal properties of the amylose–lipid complexes between the two samples (Table S2); however, HA2 rice showed a higher onset, peak and especially conclusion gelatinization temperatures than those of the WT. In addition, HA2 exhibited a lower enthalpy than that of the WT rice, presumably because of its lower degree of crystallinity in the HA2 granules compared with the WT (Zhu et al., 2011). The same trends also were observed in the retrograded samples produced after gelatinization (Table S2).

Starch granule and fine amylopectin structure in high-amylose rice endosperm

Figure 3b,e shows the morphology of starch granules from the endosperm of the HA2 and the WT as revealed by scanning electron microscope (SEM). Isolated starch granules from the WT were polygonal in shape with sharp angles and edges. The surfaces of the granules were smooth and flat or slightly concave with no structural debris, but the HA2 starch granules were heterogeneous in size and shape and could be grouped into two populations. One group had smaller sizes and irregular sausage or elongated shapes and filamentous structures, and the other more numerous populations had large, voluminous bodies with roughly spherical or ellipsoidal profiles.

The HA2 and the WT rice were debranched by isoamylase, and their unit chains were examined by gel permeation chromatography (GPC) (Zhu et al., 2011) and high-performance anion-exchange chromatograph (HPAEC) (Figure 4). The amylopectin/amylose ratio and AC could be estimated from GPC; AC is 44.48% in HA2 and only 20.22% in the WT (Table S3). The results from either GPC or HPAEC analysis revealed that HA2 rice contained more long chains than the WT rice, but fewer A and B chains.

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Figure 4.  Difference plots of chain-length distributions of isoamylase-debranched HA2 and WT starches as determined by high-performance anion-exchange chromatograph. Differences were calculated by subtracting chain-length distributions of isoamylase-debranched WT from HA2.

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Improvement in indices of animal health in rats by high-amylose rice meal

A 4-week feeding trial was performed with Sprague–Dawley rats to understand the nutritional effects of high-amylose rice grain. Initial body weights did not differ between the groups fed diets with HA2 transgenic rice or regular WT rice (Table 1). During the feeding period, the two types of rice diets had no significant effect on food intake by the rats; however, rats in the HA2 group had a lower final body weight than those in the WT rice group (Table 1). Each rat fed with the HA2 rice diet gained only 48.9 g of body weight during the 4-week feeding, whereas individuals fed the WT rice diet gained 108 g.

Table 1.   Effect of high-amylose rice meal in Sprague–Dawley rats fed experimental diets for 4 weeks†
 HA2‡WT
  1. WT, wild type.

  2. †Data are expressed as means of eight rats’ samples with replicate ± standard deviation.

  3. ‡There is significantly difference at *< 0.05 or **< 0.01 level when compared with that of WT.

  4. §Food efficiency ratio: weight gain (g/rat)/food intake (g/rat).

  5. ¶Blood biochemical indices in rats fasting for 16 h after 4 weeks feeding

Body weight (g/rat)
 Initial247.7 ± 7.9244.0 ± 5.1
 Final296.6 ± 36.2*352.0 ± 23.8
 Gain48.9 ± 34.7**108.0 ± 22.6
Total diet intake in 4 weeks (g/rat)349.8 ± 23.1364.7 ± 33.4
Food efficiency ratio§  0.140**0.296
Short-chain fatty acids in faecal (μmole/g dry faeces)
 Acetic acid123.7 ± 29.2**63.2 ± 18.6
 Propionic acid82.1 ± 18.7**14.0 ± 5.7
 Butyric acid17.8 ± 6.5**5.9 ± 3.2
 Total223.6**83.0
Faecal output (g/day/rat)
 Fresh matter4.99 ± 1.52*3.06 ± 0.69
 Dry matter3.88 ± 0.82*2.43 ± 0.61
Faecal pH6.37 ± 0.33**7.41 ± 0.17
Organ weights (g/wet weight)
 Heart0.96 ± 0.081.02 ± 0.10
 Spleen2.05 ± 0.682.27 ± 0.45
 Kidney1.91 ± 0.242.07 ± 0.35
 Liver9.68 ± 1.009.83 ± 1.12
 Caecum3.21 ± 1.262.81 ± 0.59
Colon length (cm)11.60 ± 3.1913.17 ± 2.51
Biochemical index¶
 Plasma glucose (mm)6.59 ± 1.045.87 ± 0.32
 Insulin (uIU/ml)28.30 ± 5.4222.91 ± 5.78
 Potassium (mm)6.20 ± 0.77**4.73 ± 0.30
 Triglycerides (mm)0.68 ± 0.290.93 ± 0.26
 Total cholesterol (mm)1.21 ± 0.121.34 ± 0.08

During the 2 weeks of the metabolic cage phase, wet and dry faecal productions were found to be much higher in the treatment with the HA2 rice diet than those in the WT group. Each rat fed with the HA2 rice excreted, on average, 3.88 g/day of faecal mass, whereas each rat fed with the WT rice averaged only 2.43 g/day (Table 1). As expected, the rats consuming the HA2 rice excreted more total SCFAs than those fed the WT rice (Table 1). The concentrations of individual acids, acetate, propionate and butyrate, were all significantly higher (< 0.01) in the faeces produced by rats fed the HA2 rice compared with those of the controls, and that increase in faecal acids also resulted in a relatively lower faecal pH value (pH 6.37) (< 0.01) in HA2 rice-fed rats than in the control group (pH 7.41) (Table 1). No significant difference was measured in biochemical indicators in the blood of rats fed the two diets, except the level of potassium was elevated in the blood of the HA2 group (Table 1). No abnormal tissue changes were found in the tissue section slides for rats fed the HA2 rice (data not shown).

Blood glucose response in diabetic Zucker fatty rats fed the high-amylose rice starch

Zucker diabetic fatty rats (ZDF, type 2 diabetes) were directly fed with HA2 rice starch, and those rats exhibited substantial lower plasma glucose levels compared with those fed the WT rice (Figure 5). Blood glucose levels differed significantly at 0.5, 1 and 1.5 h, indicating that the HA2 rice starch lowered blood glucose in Zucker rats. We also fed HA2 rice to normal SD rats and to streptozotocin-induced rats (type 1 diabetes), and neither showed a major difference in lowering blood glucose within 3 h compared with the WT rice (data not shown).

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Figure 5. In vivo acute oral rice tolerance test (ORTT) in diabetic rats. Male Zucker diabetic fatty rats (n = 6) were fed with purified rice starch, which was suspended in water, from either high resistant starch transgenic rice (HA2) or its wild-type rice (WT) (4.8 g/kg body weight). Blood was collected at 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6 and 7 h after feeding, and blood glucose level was determined using blood glucose strips (ONE TOUCH System).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Effect of inhibition of different SBE(s) on amylose content

SBEs are responsible for the synthesis of α-1,6-glucosidic linkages by catalysing the cleavage and transfer of α-1,4-linked glucan chains to branch 6-hydroxyl groups, thus producing branches in amylopectin. Reduction in SBEs’ activity reduces the frequency of branch points in the amylopectin fraction and increases the AC (Morell et al., 2004). Results from the present study showed that the AC in transgenic lines with p13dsSBE1 (SBEI RNAi) showed no difference compared with the WT, whereas the AC was increased in transgenic lines with p13dsSBE3 (SBEIIb RNAi), especially with p13aSBE13 (antisense for both SBEI and SBEIIb). Our results suggest that different SBEs play different roles in starch biosynthesis in plants. In rice endosperm, SBEI is the major enzyme, which accounts for about 60-70% of the total SBEs’ activity, and SBEIIa and SBEIIb contribute equally to the rest of the SBEs’ activity (Yamanouchi and Nakamura, 1992). In potato tubers, Jobling et al. (1999) reported that SBEA, the minor form of SBE, has a major impact on starch structure. SBEA can complement the activity of SBEB, and these two SBEs interact with each other. AC was increased to 77–78% when both SBEA and SBEB were inhibited to below 1% of the wild-type activities (Schwall et al., 2000). Safford et al. (1998) reported no significant differences in AC or amylopectin branch length profiles of transgenic tuber starches in potato with anti-SBEB, whereas starches from plants with anti-SBEA showed an apparent increased AC of 38% versus 30% in controls (Jobling et al., 1999). In contrast to potato and rice, inhibition of single SBEA in maize ae mutant and pea r mutant produced very high-amylose starches in these plants (Shannon and Garwood, 1984; Wang et al., 1998), suggesting that the roles of SBEs are different in different plants.

In this study, AC was increased to about 65% by the inhibition of both SBEI and SBEIIb. Further research is needed to determine whether AC can be further increased in rice by inhibiting SBEIIa as well as SBEI and SBEIIb. In addition, it is worthwhile to investigate whether alternative approaches such as using a mutagen (e.g. ethyl methane sulphonate), targeting induced local lesions in genomes technique, inhibiting other enzymes (e.g. soluble starch synthases), enhancing GBSS, or a combination of inhibiting soluble starch synthesis and enhancing GBSS can lead to a very high-amylose starch in rice.

Starch makes up approximately 80%–90% of the dry weight of rice grains, in which amylopectin is normally one of the main components. Reducing or shutting down the biosynthesis of amylopectin is likely to affect the level of starch accumulating in endosperm and thus affects kernel weight. In this study, the kernel weight of high-amylose rice was significantly decreased (approximately 38%, < 0.01). The appearance of the transgenic kernel was opaque and smaller than the control (Figure 3a,d). The width and thickness of the transgenic kernel were significantly decreased (< 0.01), thus reducing the kernel weight (Table S1). SEM revealed that the starch granule in the transgenic rice had more irregular shapes, so they packed less tightly in the endosperm and created air spaces, which decreased the kernel weight. The dry weights of rice kernels at different filling stages from HA2 rice and WT rice were determined (Figure S4). The kernel weights of the HA2 rice and the WT differed after on the eighth day after flowering (DAF). In addition, HA2 rice kernels displayed growth stagnation at 12 DAF, whereas the kernel weight for the WT increased sharply from 8 to 12 DAF and a little more from 12 to 20 DAF. Therefore, the kernel weight difference between HA2 rice and the WT mainly depended on difference in the growth on the 8–20 DAF.

Effects of increased AC on RS and enzyme resistance

In addition to the increase in AC, RS and TDF levels were significantly increased (< 0.01) in high-amylose rice (Figure 2e) (Zhu et al., 2011). Our GPC (Zhu et al., 2011) and HPAEC results (Figure 4) showed more long chains in the high-amylose rice amylopectin. Those long chains have the ability to form more stable double helices with reduced enzyme susceptibility, resulting in higher RS and TDF than in the WT amylopectin.

High-amylose rice showed more resistance to enzyme hydrolysis than the WT, not only because of its higher proportion of amylose and long amylopectin chains, but also because of its semicompound starch granules (Wei et al., 2010; Zhu et al., 2011). Compound starch granules consist of many individual granules held together by unknown forces and have less specific surface area than individual granules. Thus, compound granules bind less amylase than individual granules, which restricts hydrolysis.

High-amylose rice also displayed higher thermal resistance in DSC results than the WT. The DSC endotherm of HA2 rice starch was broader than the WT and showed a higher onset, peak and conclusion gelatinization temperatures. This agreed with results for maize starches with different ACs (Shi et al., 1998). High-amylose rice showed higher resistance on the pasting property and alkaline gelatinization, which confirmed the RS characteristic.

Potential health benefits of high-amylose rice

The feeding test on rats showed that the final body weight of the HA2 group was significantly lower (P < 0.05) than that of the WT group, even though total food intake did not differ. Those results indicate that high-amylose rice, rich in RS, may play a role in effective weight management. RS can help control body weight because it is a functional dietary fibre that can deliver some of the benefits of insoluble and soluble fibre (Premavalli et al., 2006; Bassaganya-Riera et al., 2011; Fuentes-Zaragoza et al., 2011). An increase in either soluble or insoluble fibre intake appears to increase postmeal satiety and decrease subsequent hunger (Howarth et al., 2001). In our feeding study on high-amylose rice, we found no significant effect of HA2 feeding on organ weight or the length of the colon (Table 1). Kim et al. (2003) also reported that the length of the small intestines, caecum, colon and rectum and the tissue weight of the caecum were not affected by feeding RSs from corn or rice. Notably, the caecum volume with its contents was larger in the HA2 group than in the WT group. The weight of the caecum with its contents for the HA2 group was 3.21 ± 1.26 g versus 2.81 ± 0.59 g for the WT group, which might be because RS escapes digestion in the small intestine and passes directly into the caecum where bacterial fermentation begins to occur, which enlarges the caecum volume. This phenomenon, of course, corroborated the definition of RS. No abnormal tissue changes were found in organ tissue section slides for rats fed the HA2 rice, indicating that high-amylose rice has no effect on animal organs.

Rats fed with HA2 and WT for 4 weeks showed no statistical significance in resting levels of plasma glucose, insulin, triglycerides or cholesterol, but the HA2 group showed elevated potassium. The plasma glucose in rats fed with HA2 was somewhat elevated compared with the rats fed with WT, presumably because of the slow release feature of RS as it was still being metabolized 16 h after consumption, whereas the WT rice starch was digested quickly (Aparicio-Saguilán et al., 2007). A positive correlation between the insulin and glucose levels was observed, which is also called the glucose/insulin response. Potassium absorption was significantly greater (< 0.01) in rats fed with HA2 than in the WT. Several explanations for this effect can be proposed. Lopez et al. (2001), who found that the apparent Ca, Mg, Zn, Fe and Cu absorptions in rats were enhanced by raw potato starch and high-amylose cornstarch, indicated that an increase in the exchange area (enlargement of caecum and longer transit time) and the elevation of the caecal blood flow could be a reason. Moreover, the reduced pH in the caecum increases the solubility of these minerals. Schulz et al. (1993) also suggested that native RS raised Ca and Mg absorption because it tended to enhance the solubility of these minerals in ileal and caecal digests. Many reports in the literature indicate that the consumption of RS significantly reduces blood triglycerides and total cholesterol (Brites et al., 2011); however, the present study showed no statistical difference between rats fed with HA2 and WT, although a decreasing trend was found in the HA2 group. Significance might be found if more HA2 was added to the diet or if the rats were fed for a longer time.

RS helps to keep colon tissue healthy by producing protective compounds called SCFA, primarily composed of acetic, propionic and butyric acids, which reduce intestinal pH, encourage the growth of healthy bacteria in the bowel and discourage the growth of potentially harmful bacteria (Fuentes-Zaragoza et al., 2011; Regmi et al., 2011). The formation of butyrate seems to be especially beneficial, because it is the primary energy source for colon cells and also provides protection against colorectal cancer (Scharlau et al., 2009). In this study, the HA2 group produced significantly more SCFA (< 0.01) and faecal matter (< 0.05) and resulted in lower pH (< 0.01) than the WT group. The butyrate level was doubled compared with the WT group. Those data indicate that a diet containing high-amylose rice improves gut health in rats.

Published research has shown that consumption of RS by humans resulted in a decreased glycaemic response in healthy individuals (Vonk et al., 2000) and diabetics (Giacco et al., 1998). High-amylose rice, HA2, in this study seems effective on the glucose response in type-2 diabetes, but no effect was found on normal SD rats or type-1 diabetic rats within 3 h.

In conclusion, rice with approximately 65% AC was developed; the high-amylose rice had high RS and TDF content. For the first time, we demonstrated that the diet containing the high-amylose rice had significant positive effects on animal health, such as a significantly reduced glycaemic response, an increased faecal output, increased faecal moisture, increased SCFA concentrations and decreased faecal pH. In addition, rats fed the WT rice gained twice as much weight in 4 weeks as those fed the HA2 diet, even though feed intake was equal. The relative weights of organs were not affected. This novel rice with its high AC, RS and TDF offers potential benefits for its use in foods and in medical and industrial applications.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant materials

Three elite rice cultivars from China were used in this study, including one indica, Te-qing (TQ), and two japonica, Wu-xiang-jing 14 (WXJ) and Guang-ling-xiang-nuo (GLXN). They carry the Wxa, Wxb and wx alleles at the Wx locus (Sano, 1984), which are responsible for high, intermediate and very low AC in rice endosperm, respectively. All the rice cultivars were planted in the paddy field at the campus of Yangzhou University (Yangzhou, Jiangsu Province, China), and the immature and mature seeds were collected for rice tissue culture and transformation.

Construction of RNAi and antisense vectors and rice transformation

The detailed procedure for the construction of three RNAi or antisense vectors (p13dsSBE1, p13dsSBE3 and p13aSBE13) is included in supplementary materials and methods. Rice callus derived from either immature or mature embryos was used as explants for Agrobacterium-mediated transformation according to our previous published procedure (Liu et al., 1998). Stably transformed plants were regenerated after screening for hygromycin resistance, and T0 transgenic plants were transferred into soil for molecular identification. Selected homozygous transgenic lines in T2 or later generations and their WTs were propagated for composition analysis, field and animal trials. Both PCR and Southern blot analyses (see supplementary materials and methods) were carried out to confirm the integration of the transgene(s).

RNA and protein expression analyses

Total RNA was isolated from developing rice seeds at 12 DAF by a cold-phenol method (Liu et al., 2003). The isolated RNAs were purified by treatment with DNase I and the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. For reverse transcriptase PCR (RT-PCR), an aliquot of each RNA sample was used for reverse transcription using the SuperScriptTM First-Strand Synthesis System with random hexamers (Invitrogen, Carlsbad, CA). PCR amplification was performed according to a routine condition, and the real-time RT-PCR was carried out according to our previous procedure (Li et al., 2010). The primers are listed in Table S4. The Wx proteins and seed total protein extracts containing SBEs were prepared from mature rice grains as described previously (Yamagata et al., 1982; Sano, 1985) and separated by SDS-PAGE. The Wx proteins in the SDS-PAGE gels were visualized after staining with the Coomassie Brilliant Blue. The SBEI and SBEIIb proteins were electrophoretically transferred onto a nitrocellulose membrane, then probed with rabbit polyclonal antibodies against SBEI or SBEIIb (Liu et al., unpublished data). A goat anti-rabbit IgG conjugated to alkaline phosphatase was used in the protein blotting to detect the specific SBEI or SBEIIb signals.

Measurement of grain composition

Mature rice grains were milled and polished after being harvested, air-dried and stored at room temperature for 3 months. The seeds were harvested from transgenic plants of WXJ and GLXN in T5 generation or of TQ in T4 generation. The polished rice was ground into flour using a mill (FOSS 1093 Cyclotec Sample Mill, Höganäε, Sweden) fitted with a screen having 0.5 mm opening. Apparent AC was determined by a colorimetric iodometric method according to Juliano (1971). RS content in grain was determined by AOAC Method 2002.02 (McCleary et al., 2002), using an assay kit from Megazyme International Ltd. (Wicklow, Ireland).

Measurement of gelatinization properties

The gelatinization of rice flour in urea solutions was measured as described by Nishi et al. (2001), whereas the solubility of starch granules in 4 m urea solution was determined by the method of Satoh et al. (2003). The thermal properties were measured by DSC (DSC Q100, TA Instruments, New Castle, DE) at 67% water content (Zhu et al., 2010). The pasting properties of rice flours were tested at 10% solids with a RVA (Model Super 3D; Newport Scientific, Narrabeen, Australia) following AACC Method 61-02 (AACC International, 1995).

Molecular structure of starch

Rice starches were isolated from polished rice by an alkaline protease method with slight modifications (Zhu et al., 2010). Purified rice starches were debranched by isoamylase (EC3.2.1.68; Hayashibara Biochemical Laboratories, Inc., Okayama, Japan) as previously described (Zhu et al., 2011).

The chain-length distribution of debranched starch was quantitatively analysed using a HPAEC (Dionex ICS-3000; Dionex Corp., Sunnyvate, CA) equipped with a pulsed amperometric detector, a guard column, a CarboPacTM PA1 analytical column and an AS-DV autosampler. The eluents were prepared as described previously (Shi and Seib, 1992). Eluent A was 150 mm NaOH, and eluent B was 150 mm NaOH containing 500 mm sodium acetate. The gradient programme was as follows: 40% of eluent B at 0 min, 50% at 2 min, 60% at 10 min and 80% at 40 min. The separations were carried out at 25 °C with a flow rate of 1 mL/min. The concentration of debranched starch was 2 mg/mL in 1 m NaOH solution. Maltohexaose and maltoheptaose (Sigma-Aldrich, Inc., St. Louis, MO) were used as standards.

Scanning electron microscope

The starch granules were coated with gold palladium using a sputter coater (Denton Vacuum, LLC, Moorestown, NJ) and viewed at 4000× resolution with a SEM (S-3500N; Hitachi Science Systems, Ltd., Tokyo, Japan) operating at an accelerating voltage of 20 kV.

Animal feeding experimentation

The grains of WT or transgenic rice lines HA2 (HA2) in T5 generation were milled and ground and subsequently used as diet preparation. The ingredients of the diets were modified from the American Institute of Nutrition (AIN)-93G formulation recommended by the AIN (1993) and formulated to contain the same level of total starch, total protein and other ingredients, except for the starch source (Table S5). Maize starch and casein were used to adjust total starch and total protein of each diet to the same level.

With a licence (SCXK 2002-0045) from the Chinese government, 16 Sprague–Dawley (SD) rats, half male and half female with a body weight of around 200 g, were individually housed in the metabolic cages with stainless steel mesh bottoms, allowing separate collection of faeces and urine. The cages were kept in a room of controlled temperature (24 ± 2 °C) and humidity (50 ± 10%) with a 12-h light/dark cycle. After acclimation to the normal diet for 3 days, eight rats were randomly selected for each diet group, and each animal had free access to water and diets during the whole 4 weeks of the experiment. The intake of diet and gain of body weight by each rat were measured every day. Faeces were collected for the last 2 weeks of the experimental period, freeze-dried, then ground with a mortar and pestle and stored at −20 °C until analysis. To determine the pH of faeces, the ground dry faecal matter was weighed and diluted with ten volumes of 0.9% NaCl solution. The slurry was vortexed vigorously until dispersed completely, and then, the pH was measured with a compact DELTA 320 pH meter. Measurement of the SCFA content was taken as described by Phillips et al. (1995). Pure acetic, butyric and propionic acids (analytical reagent grade 99%) were used as standards in the analysis.

Acute oral rice tolerance test (ORTT) in diabetic rats

Male Zucker diabetic fatty rats (ZDF, n = 6) were used to study the acute ORTT. The rats were obtained from the Animal Services Centre of the Chinese University of Hong Kong and acclimatized at 23 ± 1 °C with a 12-h light/dark cycle. Food and water intake were given ad libitum. The animals were maintained on a high-fat 5008 Purina diet (Purina, St Louis, MO) for 20 weeks. Diabetes was confirmed by a 2-h oral glucose tolerance test, in which blood glucose level attained 11.1 mm. To begin ORTT, ZDF rats were fasted for 16 h prior to feeding, then fed a slurry of rice starch suspended in water, where the starch was from either high RS transgenic rice (HA2) or its WT rice (4.8 g/kg body weight). Blood was collected at 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6 and 7 h after feeding, and blood glucose level was determined using blood glucose strips (ONE TOUCH System, Johnson & Johnson, Milpitas, CA). All procedures were conducted in accordance with the guidelines set by the Animal Services Centre of the Chinese University of Hong Kong.

Statistical analysis

For the characterization of the samples, at least two replicate measurements were taken, unless otherwise specified. All data were reported as the mean ± standard deviation (mean ± SD). The results were analysed by using analysis of one-way variance (ANOVA) (SPSS version 13.0, SPSS Inc., Chicago, IL), and the Student’s t test was used to examine the differences. Results with a corresponding probability value of < 0.05 and < 0.01 were considered to be statistically significant and very significant, respectively.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

This study was supported by the Ministry of Science and Technology (2012CB944803), the National Natural Science Foundation (Grant Nos. 31071383), the Priority Academic Program Development from Jiangsu Government and the National Special Program for Transgenic Research (2011ZX08001-006) of China. This is contribution no. 12-021-J from the Kansas Agricultural Experiment Station.

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Figure S1 The constructs of T-DNA region used for producing transgenic rice.

Figure S2 Expression analyses of SBE genes in developing seeds of transgenic indica rice and its wild-type Te-qing.

Figure S3 Gelatinization of starch from (a) wild-type (WT) and (b) the high-amylose rice (HA2) in various concentrations (0–8 m) of urea solution. (c), Absorbance spectra of resolved starches, stained with I2/KI solution, from the wild-type (WT) and high-amylose rice (HA2) in 4 m urea solution.

Figure S4 Dry weight of caryopsis at different filling stages for high-amylose rice (HA1, HA2) and their wild type, Te-qing (WT).

Table S1 The main agronomic traits of two transgenic rice lines HA1 and HA2 and their wild type (WT).

Table S2 Gelatinization temperatures and enthalpies of flours from high-amylose rice HA2 and wild type (WT) determined by differential scanning calorimeter (DSC) at 33.3% solids concentration.

Table S3 Degree of polymerization (DP), ratio of amylopectin to amylose and amylose content revealed by GPC.

Table S4 Primers used for RT-PCR analysis in this study.

Table S5 Composition of experimental diets.

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PBI_667_sm_TableS1-S5.doc105KSupporting info item

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