COMMENTARY COMMENTARY

Increasing resistant starch content in rice for better consumer health Reynante Lacsamana Ordonioa and Makoto Matsuokab,1

Rice is an excellent source of starch, which is normally hydrolyzed by enzymes in the digestive tract to be converted into glucose that cells directly use to produce energy for their metabolic functions. However, when there is less energy demand from cells, any excess calories from starch are stored in the body as glycogen or fats for later use. Therefore, long-term overeating coupled with lack of proper exercise or sedentary lifestyle could potentially lead to some health problems, such as obesity, type 2 diabetes, and other complications. As rice is eaten, the starch is usually acted upon by α-amylases starting from the mouth and mainly at the small intestine. However, there is “resistant starch” (RS), which usually comprises less than 3% of hot cooked rice (1) that can escape digestion almost entirely (2) and therefore, its calories are unavailable for cells to use. Based on the cause of enzyme resistance, RSs are categorized into five types (2, 3). Type 1 RS is starch that is physically inaccessible, such as that in whole grains. Type 2 RS is often found in raw potato and banana, and its enzyme resistance is a result of the tight packing of starch within the starch granules. Type 3 RS is retrograded starch, which forms when cooked starchy foods are cooled. Cooling allows the amylose and linear parts of amylopectin to form crystalline structure that reduces digestibility. Type 4 RS results from chemical treatment/modification of starch. Finally, type 5 RS is starch wherein the amylose component forms complexes with lipids (amylose–lipid complex), which makes it more thermally stable (4). Digestibility of cooked rice starch is usually determined by the amount of amylose in the grain. The more amylose there is, the slower is the digestion of rice and the lower is the glycemic index (5), which indicates the effect on blood sugar. However, in rice with type 5 RS, such starch may take several hours to digest or not at all, like a form of dietary fiber (2). This is because the amylose–lipid complex in type 5 RS restricts swelling of the starch granule during cooking, making it resistant to hydrolytic enzymes (4). Thus, an increase in the amount of type 5 RS could make rice safer for people with diabetes or for those who simply would like to avoid the extra calories. In PNAS, Zhou et al. (6) analyze a new indica rice mutant, b10, with the highest level of type 5 RS in rice so far. Their report

presents a new insight on the physiological mechanism for amylose–lipid complex synthesis in amyloplasts, and also on how to develop healthier alternative commercial rice with more heat-stable RS in the future. To identify a new RS locus in a rice mutant, b10, Zhou et al. (6) performed map-based cloning and reveal that a defect in soluble starch synthase IIIa (SSIIIa) is responsible for the high RS level in b10. Their genetic analysis using F2 plants indicates that such a mutation is inherited in a partially dominant manner. The authors confirmed the relationship of high RS with SSIIIa defect by complementation experiments using its wild-type genomic fragment, overexpression of its cDNA, and by knockdown of its function by RNA interference. Zhou et al. also examined the RS content of two japonica loss-of-function (ssIIIa) mutants, one of which was previously reported as flo5-1 (7). Zhou et al. (6) find that b10 had altered starch granule structure, namely, the transition from normally polyhedral starch granules in wild-type to rounded, variably sized, and irregularly surfaced ones like those in earlier ssIIIa mutants (8). This internal structure apparently influences the overall appearance of the mutant grains, which exhibit white cores (7, 8). Zhou et al. (6) discuss the mechanism of increased type 5 RS levels in the ssIIIa mutants based on the maize amyloplast carbon partitioning model proposed by Hennen-Bierwagen et al. (9). Those authors paid attention to a unique characteristic of SSIIIa, which can interact with some amylopectin-synthesizing enzymes, pyruvate orthophosphate dikinase (PPDK), and plastid ADP-glucose pyrophosphorylase (AGPase) (Fig. 1A). In such a model, PPDK acts to regulate carbon partitioning between starch and lipids by regulating plastid AGPase activity (10). Plastid AGPase competes with starch synthesis by using ADP-Glc, the same substrate for starch production, to synthesize Glc-1-P using pyrophosphate (PPi). However, the amount of PPi in the amyloplast is very low because of high pyrophosphatase activity, and therefore the production of Glc-1-P, a substrate for lipid synthesis, from ADP-Glc, could be negligible under normal conditions (Fig. 1A). However, when PPDK and plastid AGPase physically interact in a single enzyme complex, plastid

a

Plant Breeding and Biotechnology Division, Philippine Rice Research Institute, Maligaya, Science City of Munoz 3119, The Philippines; and Bioscience and Biotechnology Center, Nagoya University, Chikusa, Nagoya 464 8601, Japan Author contributions: R.L.O. and M.M. wrote the paper. The authors declare no conflict of interest. See companion article on page 12844. 1 To whom correspondence should be addressed. Email: [email protected]. b

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PPase AMP

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Fig. 1. Model for RS biosynthesis in rice amyloplasts. (A) Amylopectin-synthesizing enzymes, such as SSIIIa, SSIIa, SSIVb, BEI, BEIIb, and PUL [colored shapes; according to Crofts et al. (11)] can physically interact with each other to form multienzyme complexes. According to a study on such enzyme complex in maize by Hennen-Bierwagen et al. (9), the complex may also contain other enzymes, such as PPDK and plastid AGPase, that are considered to function in the global regulation of carbon partitioning between starch and lipid. As a result of the very low level of PPi concentration in the amyloplast because of the high activity of pyrophosphatase (PPase), PPDK could promote plastid AGPase activity by directly supplying PPi through a substrate-channeling mechanism (blue arrow), resulting in the smooth conversion of ADP-Glc to Glc-1-P and enhancing the biosynthesis of lipids. Interaction between PPDK/AGPase and SSIIIa could inhibit the activity of the former (red hooks from SSIIIa). As a result, ADP-Glc is more easily used as a substrate for amylose and amylopectin synthesis by GBSS1 and amylopectin-synthesizing enzymes, respectively. (B) When SSIIIa is defective (light gray), the interaction between PPDK/AGPase and SSIIIa is disrupted, making AGPase free to channel more ADPGlc for the synthesis of Glc-1-P, a substrate for lipid production. Mutation of SSIIIa also causes a defect in amylopectin biosynthesis (light gray). Amylose synthesis actively proceeds in the presence of a fully functioning GBSS1 in the indica b10 (ssIIIa) mutant, resulting in high levels of amylose-lipid complex or type 5 RS. BE, starch branching enzyme; PEP, phosphoenolpyruvate; PK, pyruvate kinase; SS, starch synthase.

AGPase activity is enhanced because PPDK can directly supply PPi to plastid AGPase through a substrate-channeling mechanism, therefore allowing plastid AGPase to produce Glc-1-P even with the negligible amounts of PPi present (9). According to a recent study by Crofts et al. (11), rice amylopectinsynthesizing enzymes, such as SSIIIa, SSIIa, SSIVb, BEI, BEIIb, and PUL (pullulanase), form multienzyme complexes with various partnership patterns. Although there is no direct evidence that these multienzyme complexes also contain PPDK and plastid AGPase, such an assumption may be possible considering the maize multienzyme complex consisting of SSIIIa, PPDK, plastid AGPase in rice, and some amylopectin-synthesizing enzymes mentioned above (9). Such possible multienzyme complex formation with PPDK/AGPase could provide a way to harmonize the metabolic pathways responsible for starch and lipid synthesis. In this context, Zhou et al. (6) hypothesize that the amylopectin biosynthetic enzymes in the complex can exert a constraining effect on PPDK and AGPase to control the partitioning of ADP-Glc into lipid or starch. Based on this hypothesis, a defect in SSIIIa could result in the destabilization of the multienzyme complex: hence, releasing PPDK and AGPase (Fig. 1B) and subsequently increasing Glc-1-P and lipid synthesis. At the same time, loss of SSIIIa activity diverts the carbon flow by channeling ADP-Glc for amylose synthesis by granule-bound starch synthase1 (GBSS1) encoded by the Waxy gene (Wx) (12). These two events simultaneously increase amylose and lipid contents. As mentioned above, amylose and lipids are components of type 5 RS, and thereby increase its amount. Zhou et al. (6) also reveal that increased RS content mediated by SSIIIa requires high levels of GBSS1. It is widely known that there are two types of cultivated Asian rice, indica and japonica. Grains of indica rice generally contain higher amylose than japonica and this makes a good basis for distinguishing them from each other. The type of Wx gene that they have sets them apart; that is, indica rice has the fully active wild-type allele (Wxa), whereas japonica rice has the intermediate one (Wxb). Zhou et al. (6) found that the RS level is different between indica and japonica and such difference

Ordonio and Matsuoka

depends on their Wx alleles. Some previous studies reported that higher amylose content increases RS level (2, 3), and thus it is not surprising for the Wxb allele in japonica to cause lower RS content relative to that in indica rice. Previously, Fujita et al. (13) reported that the ssIIIa mutation causes 1.4- to 1.8-fold increase in GBSS1 and 1.3fold increase in the amylose content of japonica rice, which is good in terms of being able to increase RS in japonica. Crofts et al. (12) introduced the indica Wxa allele into japonica rice carrying SSIIIa or ssIIIa. The amount of GBSS1 was increased in the japonica rice carrying SSIIIa, as expected. In the japonica rice carrying ssIIIa, the amylose content was also increased but not Wx expression, probably because the GBSS1 level was already maximal. Based on these observations, Zhou et al. (6) discuss that the interaction between SSIIIa and GBSS1 is likely to occur at the posttranslational stage. Although the work of Zhou et al. (6) greatly contributes to our understanding of the molecular mechanisms behind RS, it cannot be denied that the challenge to produce rice that is high in RS and yet more palatable and easier to cook still remains. This is because RS increases the gelatinization temperature and decreases the peak viscosity values of rice (6), which means that the rice becomes more difficult to cook and harder in texture (14). Additional research must therefore be done to develop more acceptable rice varieties in the future. Fighting diabetes and obesity are just two of the reasons why high-RS rice is gaining momentum. Still, another promise of high-RS rice is to fight colon cancer because the undigested RS is saved for the good microbial flora in the large intestine, which ferment it to produce beneficial substances (2, 3). Indeed, increasing amylose and RS may mean sacrificing palatability and going the extra mile in the cooking process (14). Still, even with this inconvenience, it is comforting to know that rice consumers are now being given more options and alternative ways to safeguard health. In this modern world, we are prone to lifestyle-related diseases more than ever, and it is high time for us to intensify our fight against them on our very own tables by increasing our intake of RS.

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Yang CZ, et al. (2006) Starch properties of mutant rice high in resistant starch. J Agric Food Chem 54(2):523–528. Sajilata MG, Singhal RS, Kulkarni PR (2006) Resistant starch—A review. Compr Rev Food Sci Food Saf 5:1–17. Birt DF, et al. (2013) Resistant starch: Promise for improving human health. Adv Nutr 4(6):587–601. Hasjim J, Ai Y, Jane J (2013) Novel applications of amylose-lipid complex as resistant starch type 5. Resistant Starch: Sources, Applications and Health Benefits, eds Shi Y, Maningat CC (John Wiley and Sons, Hoboken, NJ), 1st Ed, pp 79–94. Frei M, Siddhuraju P, Becker K (2003) Studies on the in vitro starch digestibility and the glycemic index of six different indigenous rice cultivars from the Philippines. Food Chem 83(3):395–402. Zhou H, et al. (2016) Critical roles of soluble starch synthase SSIIIa and granule-bound starch synthase Waxy in synthesizing resistant starch in rice. Proc Natl Acad Sci USA 113:12844–12849. Ryoo N, et al. (2007) Knockout of a starch synthase gene OsSSIIIa/Flo5 causes white-core floury endosperm in rice (Oryza sativa L.). Plant Cell Rep 26(7): 1083–1095. Toyosawa Y, et al. (2016) Deficiency of starch synthase IIIa and IVb alters starch granule morphology from polyhedral to spherical in rice endosperm. Plant Physiol 170(3):1255–1270. Hennen-Bierwagen TA, et al. (2009) Proteins from multiple metabolic pathways associate with starch biosynthetic enzymes in high molecular weight complexes: a model for regulation of carbon allocation in maize amyloplasts. Plant Physiol 149(3):1541–1559. Mechin ´ V, Thevenot ´ C, Le Guilloux M, Prioul J-L, Damerval C (2007) Developmental analysis of maize endosperm proteome suggests a pivotal role for pyruvate orthophosphate dikinase. Plant Physiol 143(3):1203–1219. Crofts N, et al. (2015) Amylopectin biosynthetic enzymes from developing rice seed form enzymatically active protein complexes. J Exp Bot 66(15):4469–4482. Crofts N, et al. (2012) Lack of starch synthase IIIa and high expression of granule-bound starch synthase I synergistically increase the apparent amylose content in rice endosperm. Plant Sci 193–194:62–69. Fujita N, et al. (2007) Characterization of SSIIIa-deficient mutants of rice: the function of SSIIIa and pleiotropic effects by SSIIIa deficiency in the rice endosperm. Plant Physiol 144(4):2009–2023. Kesarwani A, Chiang PY, Chen SS (2016) Rapid visco analyzer measurements of japonica rice cultivars to study interrelationship between pasting properties and farming system. Int J Agron 2016:1–6.

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Ordonio and Matsuoka