Maziah, M.

Maznah, I.

Nyo-Nyo, M.

Siti, N.A.A.

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47SOLUBLE STARCH SYNTHASE IIa (SSIIa) ENZYME EXPRESSION IN ENDOSPERM AND ALKALI DISINTEGRATIONMalays. Appl. Biol. (2011) 40(1): 47-50* To whom correspondence should be addressed.Research NoteSOLUBLE STARCH SYNTHASE IIa (SSIIa) ENZYME EXPRESSIONIN ENDOSPERM AND ALKALI DISINTEGRATION IN SEEDS OFMYANMAR RICE CULTIVARSNYO-NYO, M.1, MAZIAH, M.1,2,3*, MAZNAH, I.1 and SITI, N.A.A.21Institute of Bioscience, 2Institute of Tropical Agriculture,3Faculty of Biotechnology and Biomolecular Sciences,Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia*E-mail: maziahm@biotech.upm.edu.myRice (Oryza sativa L.) is grown all over the worldand consumed by human beings worldwide. It is themost important crop in Myanmar because it is themain staple food of the entire nation. Starch is theend product of carbon fixation in photosynthesis andis accumulated in storage organs as an energysource. It comprises 90% of the total dry matter inthe rice grain and can mainly affect grain quality.Starch biosynthesis in cereal plants is catalyzed byfour classes of enzymes, namely; ADP-glucosepyrophosphorylase (AGPase), soluble starchsynthase (SSS), starch-branching enzyme (BE), andstarch-debranching enzyme (DBE) (Smith et al.,1997; Myers et al., 2000; Nakamura, 2002; Fujitaet al., 2006). Starch synthase proteins from rice andother plant species can be grouped into five classes,soluble starch synthase I (SSI), soluble starchsynthase II (SSII), soluble starch synthase III (SSIII),soluble starch synthase IV (SSIV) and granule-boundstarch synthase (GBSS). One gene for SSI, threegenes for SSII (SSIIa, SSIIb, SSIIc) and, two genesfor SSIII (SSIIIa, SSIIIb), SSIV (SSIVa, SSIVb) andGBSS can be found in rice (Hirose and Terao, 2004).The physiochemical property of cooked rice ismainly determined by the amylose content.However, amylopectin structure also greatlyinfluences the physical properties of starch and theirfunctionalities. Nakamura et al., (2002) suggestedthat SSI is mainly involved in the synthesis of shortchain of degree of polymerization (DP) with ≤12amylopectin in rice endosperm. SSIIa gene isresponsible for the structural difference betweenamylopectin of japonica type and indica type riceand the amount of SSIIa in japonica rice cultivarsare lower than that in indica (Umemoto et al., 2002).They further suggested that SSIIa is a candidate genefor controlling differences in alkali disintegration ofrice grains. However, the information on correlationbetween SSIIa enzyme activity and alkalidisintegration in Myanmar local rice cultivars is notavailable. Therefore, a study was carried out todetect SSIIa enzyme expression in Myanmar ricecultivars in comparison with japonica and indicatypes of rice cultivars.Rice cultivars Nanjing 11 (indica),Yamasenishiki (japonica), MMR1, MMR2, MMR3,MMR4 and MMR5 were used in these experiments.Immature endosperms of rice grain were used toextract the enzymes. Native-Page gel electrophoresismethod was carried out as reported by Okamoto etal., (2002). It was performed on slab gel systemprepared with 7.5% (w/v) resolving gel containing0.4% (w/v) rabbit liver glycogen (TypeIII; Sigma).After electrophoresis, the gels were incubated with10ml buffer of 100mM N,N-bis (2-hydorozyethyl)glycine (Bicine) (pH8.5), 500mM tri sodium citrate(pH8.5), 1mM EDTA, 100mlL-1 glycerol, 2mM DTTfor 15 min twice on ice by gentle shaking andanother 24 hours at 27°C in 10ml buffer of 100mMN,N-bis (2-hydorozyethyl) glycine (Bicine) (pH8.5),500mM sodium citrate (pH8.5), 1mM EDTA,100mlL-1 glycerol, 2mM DTT either in the presenceor in the absence of 1mM ADP-glucose.  SSI andSSIIa enzymes activities in the gels were detectedin iodine buffer containing 1% potassium iodide (KI)and 0.2% iodine (I2).Three grains of polished rice were used for eachcultivar in alkali test. They were soaked in 10ml of1.7% potassium hydroxide (KOH) in a petri dish (6x3 cm, 1 cm deep) for 24 hours at room temperature.Alkali disintegration for each grain was scoredaccording to Little et al., (1958) and repeated threetimes.The SSI activity in the developing endospermwas detected in all cultivars (Fig.1a). The SSIIaactivity was detected in Nanjing 11 (Fig.1b) but not48 SOLUBLE STARCH SYNTHASE IIa (SSIIa) ENZYME EXPRESSION IN ENDOSPERM AND ALKALI DISINTEGRATIONin Yamsenishiki, a japonica type. This may be dueto reduced activity of SSIIa or functional capacityrelative to the activity of this enzyme in indica typerice. The expression of SSIIa of Myanmar ricecultivars in endosperm was absent in this experimentcompared to Nanjing 11cultivar.Alkali spreading value of Nanjing 11 cultivarshowed that it is resistant to disintegration whileYamasenishiki was very sensitive in 1.7% KOHsolution. Myanmar rice cultivars (MMR1, MMR2,MMR3, MMR4 and MMR5) can be seen asintermediate sensitive to alkali solution (Fig.2).Nanjing 11 showed alkali disintegration score of 3while Yamasenishiki cultivar showed susceptibilityin alkali disintegration score (7.8) and Myanmar ricecultivars seemed to have intermediate alkalidisintegration score of 4-6 (Fig.3).SSII plays a role in synthesizing of intermediatechains of amylopectin from the analysis of the SSIIdeletion mutant of Chlamydomonas (Fontaine et al.,1993). In the case of rice, SSIIa in japonica andindica rice causes the phenotypic difference betweenthe two rice cultivars in amylopectin structure. Itmight be different either in the amount of SSIIa orin the functional capacity of SSIIa in the two typesof rice (Umemoto et al., 2002). Umemoto and co-Fig. 1. Native-PAGE activity staining for starch synthase activity in soluble  fraction.SSI activity in rice endosperm in all cultivars.SSIIa activity in Nanjing 11 cultivar.Fig. 2. Different disintegration of starch granules in 1.7% KOH solution in a) Nanjing 11; b) Myanmar cultivar;c) Yamasenishiki.49SOLUBLE STARCH SYNTHASE IIa (SSIIa) ENZYME EXPRESSION IN ENDOSPERM AND ALKALI DISINTEGRATIONworkers (2004) provided strong evidence that therole of SSIIa in amylopectin biosynthesis wasdistinct and could not be substituted by SSIIb orSSIIc, or that SSIIa was the only SSII enzymeexpressed in developing rice endosperm.This study shows that SSI activity is expressedin all but SSIIa activity is lacking in Myanmar ricecultivars. It can be generally assumed that SSIIaenzyme expression level was reduced in theirendosperm. Okamoto et al., (2009) also suggestedthat SSIIa activity is usually very weak in zymogramanalysis compared to SSI, sometimes only faint bandcan be detected even with long chain of amylopectinin rice cultivars (Umemoto et al., 2004). It mightalso be a reflection of chain length distribution ofamylopectin in these Myanmar rice cultivars. Furtherstudies are needed in order to confirm whether thereis SSIIa activity in related chain length distributionof amylopectin in Myanmar rice cultivars.ACKNOWLEDGEMENTSWe thank Dr. Takyuki Umemoto, Senior Researcher,National Institute of Crop Science, Tsukuba, Japanfor sharing Native-Page gel protocol. We alsoacknowledge U Khin Soe, Director General,Department of Agricultural Research (DAR),Naypyidaw, Myanmar and Datuk Dr. Othman Omar,Principal Research Officer, Rice and Industrial CropResearch Centre, MARDI, Penang, Malaysia forprouding rice in this experiment. This study wasfinancially supported by the Organization forWomen in Science for the Developing World(OWSDW) formerly known as Third WorldOrganization for Women in Science (TWOWS).REFERENCESFontaine, T., D’Julst, C., Maddelein, M.L., Routier,F., Pepin, T.M., Decq, A., Wieruzeski, J.M.,Delrue, B., Koornhuyse, N.V.D., Bossu, J.P.,Fournet, B. & Ball, S. 1993. Toward anunderstanding of the biogenesis of the starchgranule: evidence that Chlamydomonas solublestarch synthase II controls the synthesis ofintermediate size glucan of amylopectin. Journalof Biological Chemistry.  268: 16223-16230.Fujita, N., Yoshida, M., Asakura, N., Ohdan, T.,Miyao, A., Hirochika, H. & Nakamura, Y. 2006.Function and Characterization Starch ofSynthase I Using Mutants in Rice. PlantPhysiology. 140: 1070-1084.Hirose, T. & Terao, T. 2004. A comprehensiveexpression analysis of the starch synthase genefamily in rice (Oryza sativa L.). Planta. 220: 9-16.Little, R.R., Hilder, G.B. & Dawson, E.H. 1958.Differential effect of dilute alkali on 25 varietiesof milled white rice. Cereal Chemistry. 35: 111-126.Myers, A.M., Morell, M.K., James, M.G. & Ball,S.G. 2000. Recent progress towardsunderstanding biosynthesis of the amylopectincrystal. Plant Physiology. 122: 989-997.Nakamura, Y. 2002. Towards a better understandingof the metabolic system for amylopectinFig. 3. Distribution of alkali disintegration in 1.7 % KOH solution at room temperature for differentrice cultivars.50 SOLUBLE STARCH SYNTHASE IIa (SSIIa) ENZYME EXPRESSION IN ENDOSPERM AND ALKALI DISINTEGRATIONbiosynthesis in plants: rice endosperm as amodel tissue. Plant and Cell Physiology. 43:718-725.Nakamura, Y., Sakurai, A., Inaba, Y., Kimura, K.,Iwasawa, N. & Nagamine, T. 2002. The finestructure of amylopectin in endosperm fromAsian cultivated rice can be largely classifiedinto two classes. Starch/Stãrke. 54: 117-131.Okamoto, K., Kobayashi, K., Hirasawa, H. &Umemoto, T. 2002. Structural differences inamylopectin affect waxy rice processing. PlantProduction Science.  5: 45-50.Okamoto, K., Hirasawa, H. & Umemoto, T. 2009.Screening and characterization of cultivar withM-type amylopectin in Japanese upland rice.Breeding Science. 59: 179-186.Smith, A.M., Denyer, K. & Martin, C. 1997. Thesynthesis of starch granule. Annual Reviews.Plant Physiology and   Plant Molecular Biology.48: 67-87.Umemoto, T., Yano, M., Satoh, H., Shomura, A. &Nakamura, Y. 2002. Mapping of a generesponsible for the difference in amylopectinstructure between japonica-type and indica-typerice varieties. Theoretical Applied Genetics. 104:1-8.Umemoto, T., Aoki, N., Lin, H., Nakamura, Y.,Inouchi, N., Sato, Y., Yano, M., Hirabayashi, H.& Maruyama, S. 2004. Natural variation in ricestarch synthase IIa affects enzyme and starchproperties. Functional  Plant Biology. 31: 671-684.

Soluble starch synthase IIa (SSIIa) enzyme expression in endosperm and alkali disintegration in seeds of Myanmar rice cultivars

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