Variation in Free Amino Acid Profile Among Some Rice (Oryza Sativa L.) Cultivars

Breeding Science 60: 46–54 (2010)

Variation in free amino acid profile among some rice (Oryza sativa L.) cultivars

Joseph Sherman Kamara, Shogo Konishi, Tsuneo Sasanuma and Toshinori Abe*

Department of Bioresources Engineering Faculty of Agriculture, Yamagata University, 1-23 Wakaba-machi, Tsuruoka, Yamagata, 997- 8555, Japan

The free amino acid (FAA) concentration of rice is becoming an increasingly important grain quality factor because of its apparent influence on the organoleptic acceptability of cooked rice. To determine the variability of this character among rice cultivars the FAA profiles of 49 cultivars were determined using Pico-Tag method. Among these 13 cultivars were selected to determine variation in FAA accumulation pattern after 24-hour germination treatment. The results show significant variation in the concentrations of total as well as individual FAAs among cultivars. There were also significant differences between indica- and japonica- type cultivars in the concentrations of some FAAs. The ratio of the total concentration of aspartate-derived to glutamate-derived FAAs (A/G ratio) evaluated for the japonica group (0.68) was significantly lower than that for the indica group (1.07). This suggests that typically, japonica-type rice grains tend to accumulate more Glu-derived than Asp-derived FAAs. Other results show a decline in the A/G ratios of both groups in response to germination treatment, with the indica group exhibiting a more rapid response. These results appear to suggest key differences in the FAA accumulation patterns of japonica- and indica-type rice grains especially with respect to the contents of aspartate-derived and glutamate-derived amino acids.

Key Words: rice grain quality, free amino acid, indica, japonica, glultamate-derived FAAs, aspartate-derived FAAs.

Introduction FAA accumulation is a complicated process involving a complex of biochemical networks and control mechanisms, Polished rice grains consist mainly of carbohydrates and most of which remain clouded in mystery. However, many protein with minor fractions of lipids, ash, fibre, sugars and attempts are being made to shed light on these intricacies free amino acids. Although too small to be of major nutri- through studies adopting physiological (Schaeffer and tional significance, FAAs contribute significantly to the Sharpe 1997), biochemical (Lee et al. 2001), genetic (Shi et overall acceptability of rice grains by serving as sensory ac- al. 1996, Wang et al. 2008) and environmental (Masato et tive flavor agents in cooked rice. The concentrations of cer- al. 1999) approaches. The general trend of opinions emerg- tain free amino acids (and soluble sugars) have been linked ing from these studies tends towards the conclusion that directly to the taste scores of rice (Tran et al. 2004, 2005). although significantly affected by environmental factors like This apparent relationship between the palatability of temperature and soil fertility status the process of FAA accu- cooked rice and the FAA profile of rice grains has generated mulation in rice grains is under the influence of tight genetic a growing interest in research on the physiological basis of and developmental controls. For instance, during the early to FAA accumulation in rice grains. intermediate stages of grain development, especially around The accumulation of FAAs in rice grains may occur the period of starch accumulation, the FAA pool increases through various possible means. These include the effect of rapidly and declines at maturity (Tamaki and Ebata 1989). differences between the rates of translocation of amino acids This late stage decline leaves only a tiny amount of FAAs in from synthetic sites and the rate of their subsequent incorpo- the grain at maturity, the qualitative and quantitative proper- ration into grain proteins, the relative activity of proteolytic ties of which constitute the FAA pool of the grain. disassembly of enzyme systems, amino acid recycling, or After maturation the next amino acid accumulation event more importantly, the de novo synthesis of amino acids from in the rice grain occurs during germination. Anoxic stress the metabolic intermediates. This implies that the de facto imposed by grain submergence (a general requirement for concentration and relative composition of individual amino germination) induces concurrent changes in both nitrogen acids in the FAA pool depend on the contributions (additive and carbon metabolism (Reggiani and Bertini 2003). The or subtractive) of each source or sink. It is thus apparent that production of germinated brown rice (GBR), also known as GABA rice is one recent application that takes advantage of Communicated by D.S. Brar the FAA accumulation ability of germinating rice grains Received September 25, 2009. Accepted January 15, 2010. (Ito and Ishikawa 2004). Rice grains are subjected to *Corresponding author (e-mail: [email protected]) germination treatment for a number of hours to induce an Variation in free amino acid profile among rice cultivars 47 amino acid accumulation event alongside other biochemical The dried pellet was dissolved in 1 ml of a 1 : 1 solution of changes. In addition to improved cooking, eating and func- 1% triflouroacetic (TFA) and acetonitrile prior to separation tional properties that result from the treatment, the main in a C-18 Sep-Pak® (Waters Corp., Milford, MA, USA) col- objective of GBR production is to tap from the massive umn that had been activated with two passes of approxi- turnover of GABA and other sensory-active amino acids mately 5 ml each of methanol, water and 1% TFA solution associated with the process (Ohtsubo et al. 2004, Morita et respectively. The eluate was dried and the resulting al. 2007). Besides culinary advantages, many potential moisture-free pellet was washed in 20 μl of a solution con- health benefits have also been ascribed to GBR (Okada et al. taining ethanol, MilliQ water and triethylamine (TEA) in the 2000, Kayahara et al. 2003, Ito and Ishikawa 2004). These ratio 2 : 2 : 1 respectively, and dried. This pellet was then advantages bear the potential to popularize and stimulate dissolved in 20 μl of the derivatization (labeling) solution research interest in GBR as major rice product of the future. containing ethanol, water, TEA and phenylisothiocyanate The declining rice consumption pattern that accompanies (PITC also known as Edman’s reagent) in the ratio 7 : 1 : 1 : 1 economic growth in traditional rice eating countries has in- respectively, and labeling was allowed to proceed at room spired the need for more innovative ways of making rice temperature for 20 minutes. Finally, the derivatized sample available to such populations (Suzuki et al. 2003, Juliano was dried and then re-dissolved in the sample diluent (95% 2005, Pingali 2006). In the near future there will be in- 0.05 M sodium phosphate buffer: 5% acetonitrile) and fil- creased demands for more palatable rice and rice products tered through a 0.20 μm Millex®–LG syringe-driven filter with known health benefits. It is therefore important to iden- (Millipore, Tokyo, Japan) into sample vials for subsequent tify cultivars with the appropriate quality traits to be used as amino acid analysis by the PicoTag method as described in materials in breeding for eating quality and rice product de- Wang et al. 1998). velopment; taking full advantage of the large phenotypic di- versity among rice cultivars (Toledo and Burlingame 2006). Germination treatment This study was conducted to compare various rice cultivars Germination treatment was conducted for 13 cultivars, 7 with the purpose of determining the levels and pattern of japonica- and 6 indica-type cultivars to compare their free variability in the free amino contents of polished grains and amino acid accumulation capacity after 24 hours. One gram the FAA accumulation capacity of brown rice under germi- of dehulled grains per sample (in three replicates) was used nation treatment. in the germination treatment. Prior to treatment, all grain samples were surface-sterilized by shaking in an Erlenmeyer Materials and Methods flask containing about 20 ml of 99.8% ethanol for 10 to 15 seconds followed immediately by rinsing five times in dis- Plant materials tilled water. The sterilized grains were then transferred to a Polished rice grains of 49 rice cultivars selected from Petri dish containing distilled water and kept in a preheated three rice ecotypes (japonica, 30 cultivars; javanica, 5 culti- germination chamber at 35°C for 24 hours. The treatment vars; and indica, 14 cultivars, as listed in Table 1.) were was terminated by rapid cooling in liquid nitrogen and sam- evaluated for free amino acid content. Thirteen of these, in- ples were stored at minus 80°C until time of analysis. cluding 7 japonica- and 6 indica-types were selected and subjected to germination treatment. These grains were sam- Statistical analysis pled at random from rice plants cultivated in the same paddy Treatment means were calculated from duplicate or trip- field under similar environmental conditions, in the univer- licate samples. All statistical analyses were performed with sity plot at the Faculty of Agriculture, Yamagata University, the use of suitable packages in the R statistical software Tsuruoka, Japan. (http://www.r-project.org/) complemented by basic functions in Microsoft Excel 2003. These analyses including Determination of free amino acid content analysis of variance (ANOVA), correlation, and linear dis- Powdered samples were used for the extraction of free criminant analysis were performed on treatment means. amino acids from polished (PR) and brown rice (BR), where as in the case of samples used for germination treatment, Results whole grains of germinated brown rice (GBR) were used for the analysis. In each case 200 mg of the sample was weighed Free amino acid concentration of polished rice grain samples out and homogenized for one minute in a mortar containing The free amino acid (FAA) concentrations of 49 cultivars 1.5 ml 80% alcohol solution. The homogenate was carefully were compared for 20 protein and two non-protein amino transferred to an eppendorf tube and allowed to mix on a acids (i.e., γ-aminobutyric acid, GABA and α-aminobutyric microtube mixer for one hour. Tubes from the mixer were acid, AABA;). This result is summarized by rice grain type centrifuged for 10 minutes at 16,800 g (15,000 rpm in a micro- in Table 2. Total free amino acid concentration was general- centrifuge). The supernatant was collected and freeze-dried ly low for all cultivars and the concentrations of individual in a work station unit consisting of a centrifugal concentra- amino acids showed a wide range. Among FAAs with very tor, rotary vacuum pump and freeze dryer connected in line. low levels were Lys, GABA, Cys, Gly and Met while those 48 Kamara, Konishi, Sasanuma and Abe

Table 1. List of rice cultivars used in the study Description Accession Cultivar Name Ecotype Origin/Source 1000 grain Number Type Appearance of polished grain weight (g) Akenohoshi 00011612 japonica Hiroshima, Japan Medium 21.7 White belly Asahi† 00007432 japonica Kyoto, Japan Medium 21.0 White belly Dewasansan† — japonica Japan Medium 29.3 White core Fujiminori 00006154 japonica Aomori, Japan Medium 22.0 Translucent Fukuhibiki — japonica Japan Medium 24.0 Translucent Fukunishiki† 00006740 japonica Akita, Japan Medium 23.3 Slight white belly Gohyakumangoku† 00016519 japonica Niigata, Japan Medium 29.0 White core Haenuki — japonica Japan Medium 21.7 Translucent Joushu 00006524 japonica Yamagata, Japan Medium 19.7 Translucent Kameji 00010833 japonica Shimane, Japan Medium 22.3 Translucent Kamenoo 00008335 japonica Yamagata, Japan Medium 23.3 White belly Kiyonishiki 00006746 japonica Akita, Japan Medium 24.7 Translucent Koganenishiki 00011735 japonica Kanagawa, Japan Medium 23.3 Translucent Kojonishiki — japonica Japan Medium 23.7 Slight white belly Koshihikari† 00016480 japonica Fukui, Japan Medium 22.3 Translucent Koshijiwase 00016326 japonica Niigata, Japan Medium 21.3 Slight white core Mezuromochi — japonica Japan Medium 22.0 Waxy Milky Queen — japonica Japan Medium 22.0 Translucent Nourin-22 00010768 japonica Hyogo, Japan Medium 19.7 Translucent Reimei 00006615 japonica Aomori, Japan Medium 23.0 Translucent Riku-132 00006513 japonica Japan Medium 25.7 Slight white belly Sarry Queen — japonica Japan Medium 26.3 Translucent Sasanishiki 00005938 japonica Japan Medium 22.0 Translucent Senitsu 00008042 japonica Japan Medium 16.8 Translucent Senshu 00012678 japonica China Medium 25.3 White bellied Snowpearl — japonica Japan Medium 23.7 Milky white Somewake 00007500 japonica Japan Medium 25.3 White belly Tamanishiki† 00007964 japonica Ibaraki, Japan Medium 23.6 Slight white belly Yamadanishiki† 00007684 japonica Hyogo, Japan Medium 28.3 White core Yoneshiro — japonica Japan Medium 25.0 Translucent Arditane 00015634 javanica Italy Medium 22.7 Translucent Lomelto 00015680 javanica Italy Medium 34.0 White-core/belly Rinatto-7616 00015622 javanica Italy Medium 25.3 Slight white belly S-82 00015635 javanica Italy Long 29.0 Slight white belly Stripe 136 00016276 javanica France Medium 25.7 Slight white belly Akamai† 00010614 indica Tohoku, Japan Medium 19.0 Red Ambar† 00016464 indica — Long 16.7 Light brown British Honduras Creole (BHC)† 00016133 indica Honduras Long 23.3 Slight white core Gaiya Dhan Tosar (GDT)† 00015064 indica Nepal Short 18.0 Small, red, twisted, White-bellied Had Saduri 00015404 indica Iran Long 17.0 Translucent IR-8 00013561 indica Philippines Long 21.0 White bellied IR-50 — indica Philippines Long 16.3 Slight white core Kinandan Puti 00013639 indica Philippines Long 25.3 Light brown Milyang-22 00012535 indica Rep. Korea Medium 22.7 Translucent Modan 00014032 indica India Medium 25.7 Red Panbira† 00014126 indica India Medium 19.0 Pale red Taichung Native-1 (TN-1)† 00013443 indica Taiwan Medium 21.3 White bellied Toitsu — indica Thailand Medium 23.7 White bellied Zenith† 00008262 indica U.S.A. Medium 19.0 Translucent Cultivars marked with “†” were selected for germination treatment. “—” implies that accession numbers were not acquired. at relatively higher levels include Asn, Ala and Tyr. There Within the japonica group for instance, the cultivar Asahi were however significant variations among individual culti- showed the highest total FAA content with 59.1 mg/100 g vars, and in some cases, within the same ecotype grouping. followed by Yamadanishiki (53.9mg/100g), Kameji Variation in free amino acid profile among rice cultivars 49

Table 2. Free amino acid concentration (mg/100g dry weight basis) in polished rice grains

Free amino acid concentration (mg/100g d.b.) Cultivar Type Asp Glu Asn Ser Gln Gly His Arg GABA Thr Ala Pro AABA Tyr Val Met Cys Ile Leu Phe Trp Lys Total Japonica Mean 0.8 1.9 2.1 1.4 2.1 0.6 0.9 1.1 0.5 0.5 2.9 1.7 1.1 2.9 1.9 0.6 0.5 0.8 1.9 0.7 0.9 0.4 28.1 SD 0.6 1.1 1.1 0.9 3.4 0.4 0.4 0.4 0.3 0.3 1.3 0.5 0.3 1.6 0.6 0.4 0.1 0.3 1.0 0.2 0.4 0.3 10.2 Range 3.1 4.2 4.3 4.4 13.8 2.2 1.6 1.5 1.3 1.5 5.3 1.7 1.1 6.6 2.5 1.7 0.5 1.8 4.5 0.7 1.5 1.2 42.7 Javanica Mean 0.6 1.3 1.5 1.4 0.5 0.6 0.9 1.0 0.7 0.5 2.3 1.5 0.7 2.7 1.4 0.5 0.4 0.8 1.5 0.6 0.9 0.5 22.6 SD 0.4 0.4 0.3 1.0 0.2 0.6 0.5 0.5 0.3 0.3 1.0 0.7 0.3 2.7 0.8 0.2 0.2 0.3 0.8 0.1 0.4 0.1 9.3 Range 0.9 0.9 0.6 2.5 0.4 1.4 1.1 1.0 0.8 0.7 2.5 1.7 0.8 5.8 1.8 0.5 0.5 0.7 1.9 0.3 0.8 0.3 22.9 Indica Mean 0.7 1.7 2.6 0.7 1.0 0.4 0.7 0.8 0.4 0.6 1.5 1.2 0.9 2.8 1.2 0.4 0.4 0.9 1.6 0.7 0.9 0.5 22.3 SD 0.3 0.7 1.3 0.3 0.6 0.3 0.4 0.4 0.2 0.3 0.9 1.0 0.3 3.6 0.7 0.1 0.1 0.6 1.5 0.4 0.8 0.4 12.5 Range 1.0 2.8 4.0 1.4 2.1 1.0 1.4 1.2 0.5 1.0 3.1 3.9 1.0 11.6 2.5 0.5 0.4 2.0 5.3 1.3 2.8 1.2 47.2 Group Comparisons ns ns ns ** * ** * ** ns ns ** ** * ns ** * ** ns ns ns ns ns ns P 0.000 0.039 0.009 0.029 0.004 0.000 0.035 0.019 0.002 0.028 0.001 The symbols “**” and “*” mean significantly different between japonica and indica groups at α levels of = 1% and 5% respectively. Only amino acids that were found to be significantly different between japonica and indica cultivar groups are assigned probability values.

(40.7 mg/100 g), Koshihikari (38.0 mg/100 g) and Senitsu individual concentrations of most amino acids increased sig- (35.3 mg/100 g). In all of these cultivars, there was a tenden- nificantly under germination treatment. The relative changes cy toward higher than average concentrations for Glu and in total and individual FAAs due to the treatment were deter- Asn. Besides these, Asahi and Yamadanishiki by far exceed- mined by expressing the FAA concentration in GBR as a ra- ed the rest of the group in Gln at 14.1 and 13.0 mg/100 g re- tio of that in BR. Table 3 shows these values for both total spectively. and individual FAAs concentrations in cultivars observed. Although the group mean for total FAA in the javanica The most rapidly increasing FAAs were GABA (17.8 folds) group is numerically closer to that of the indica group than and Met (10.2 folds). No apparent change was observed for the japonica group, mean concentrations for various amino Asn (1.0) AABA (1.0) and Cys (1.1). Analysis of variance acids seem to place it in-between; being closer to japonica in suggested that differences in values for relative changes some instances or to indica in others. This group was not were highly significant among cultivars. Although the tested due to its small sample size. However, within the means for total relative change in the indica and japonica group, the cultivar Stripe-136 emerged with the highest total groups were numerically different, this difference was not FAA (37.3 mg/100 g). statistically significant. However, the result suggests that Some atypical values were also observed in the concen- Ala and Met accumulated at significantly faster rates in the trations of certain FAAs among cultivars in the indica group. indica group than in the japonica group over the duration of For instance, the Nepalese cultivar, Gaiyan Dhan Tosar the treatment. (GDT) was found to contain 59.9 mg/100 g total FAA (al- most three times the group mean and the highest observed in Relationships among amino acids in grain samples of differ- the study). Other exceptional cultivars observed in the indica ent cultivar groups group were IR-8 which led the rest of the group in free Asp To examine the degree of interrelatedness among amino and Ile, Milyang-23 which contained the highest Thr level in acids in polished rice grains correlation coefficients were de- the group, as well as Modan and Ambar which also had rel- termined for all amino acids and some amino acid combina- atively high levels of free Asn. tions including total aspartate-derived (Asp, Asn, Thr, Lys, Results from one-way analysis of variance (ANOVA) for Ile and Met) and total glutamate derived (Glu, Gln, His, Arg, both total FAA and individual amino acids confirmed the Pro, and GABA) FAAs. The analysis show that most free significance of variations observed among cultivars. Group amino acids in polished rice grains were significantly corre- comparisons performed by Student’s t-test for indica and lated (Table 4). A strong positive correlation was also ob- japonica cultivars also show that the concentrations of up to served between the total concentration of aspartate-derived 11 amino acids were significantly different between the two and that of glutamate-derived FAAs. groups. An interesting observation was made from a comparison of the combination of total concentrations aspartate-derived Changes in free amino acids due to germination treatment and glutamate-derived FAAs (Fig. 3). The scatter patterns The free amino acid content of BR and GBR were deter- and slopes of the linear fits appeared to be distinctly different mined so that the changes in concentrations of total and indi- for indica (0.72) and japonica (1.78) samples (Fig. 2). Fur- vidual FAAs could be observed. The result from this com- ther, the total concentration of aspartate-derived free amino parison is summarized in Figure 1 As expected, the total and acids was expressed as a ratio of the total concentration of 50 Kamara, Konishi, Sasanuma and Abe

Fig. 1. Comparison between the free amino acid (FAA) profiles of brown rice (BR) and germinated brown (GBR) for 13 rice cultivars. The heights of columns represent the total FAA concentration detected in the grains of the various rice cultivars as labeled. The concentrations of individual amino acids are represented by the different colours, as identified in the legend (above). glutamate-derived amino acids for each cultivar to deter- vars showing uniquely high or uniquely low values in cer- mine a ratio, referred to here as the A/G ratio The mean A/G tain amino acids. However, although FAA profiles varied for the japonica group (0.68) was found to be significantly among rice cultivars, the interrelatedness among individual less than that for the indica group (1.07). A resubstitution FAAs was depicted in the pattern of correlation among plot of discriminant scores calculated for all indica and FAAs in polished rice grains (Table 4). japonica cultivars using Fisher’s linear discriminant analysis Two of the high FAA accumulating cultivars (Asahi and also appears to suggest that the two grain types could be dis- Yamadanishiki) are known late maturing cultivars. It is criminated on the basis of free amino profiles (Fig. 3). A/G probably not a mere coincidence that both of these cultivars ratios were also compared for BR and GBR. The mean had relatively very high levels of free Gln, a major form in values for brown rice appear to be very similar to those for which nitrogen is transported in plants. This implies the like- polished rice in both indica and japonica groups. However, lihood that other agronomic,characteristics, especially those after germination treatment mean A/G ratios seem to decline that relate to translocation efficiency, might help explain the in both grain types. It was also observed that the indica variation observed in FAA profiles among cultivars. N’tanos group showed a larger decline from a mean of 1.1 (in brown and Koutroubas (2002) made similar observations while ob- rice) to 0.5 (in germinated brown rice) unlike the japonica serving nitrogen accumulation in rice plants under Mediter- group which changed only slightly from 0.6 to 0.4. ranean conditions. Also, considering that the indica plant materials were all grown under temperate conditions, there Discussion is a slight chance that the observed differences in FAA profiles might be reflective of some form of adaptive response The study revealed that the total FAA concentrations of pol- to stress since temperature stress is an important factor in ished rice grain samples were generally low in all cultivars protein synthesis and hence, FAA accumulation. Also, it observed in the present study. The relatively higher concen- was noted that the cultivar GDT showed relatively very high trations of Glu, Gln, Asn, Tyr and Ala, relatively lower con- levels of Pro in both brown and polished grains. Since Pro is centrations of Lys and Met are consistent with other reports the precursor for the synthesis of 2-acetyl pyrolline (2-AP), on FAA concentration in the rice endosperm (Matsuzaki et the source of the popular popcorn aroma in aromatic rices al. 1992). A wide variation was also noted among cultivars (Wongpornchai et al. 2003, Itani et al. 2004, Champagne in the concentrations of individual FAAs, with some culti- 2008), this characteristic might be related to the aromatic Variation in free amino acid profile among rice cultivars 51 groups respectively. indica ificantly different among the 14 cul- and japonica -level of 5%) between α rs. All amino acids were highly sign ntration in GBR ÷ concentration BR) .46 1.29 1.25 0.51 1.67 0.40 0.99 1.40 1.48 1.39 1.17 nd significant difference (at ation treatment for 14 rice cultiva ation due to germination treatment: (conce ent no significant difference a e to germination treatment centration after a 24-hour germin Relative change in concentr ino acid concentration du ange in free amino acid con Asp Glu Asn Ser Gln Gly His Arg GABA Thr Ala Pro AABA Tyr Val Met Cys Ile Leu Phe Trp Lys Total ) 2.7 2.2 1.0 2.0 4.6 1.5 4.8) 2.5 3.7 12.8 2.5 4.2 0.9 2.2 3.6 2.6 7.7 0.4 3.0 3.0 8.5 3.4 4.5 7.2 23.4 0.9 6.6 3.6 4.5 6.8 4.5 5.6 1.6 5.9 5.0 3.7 5.5 13.6 2.8 1.2 5.3 8.3 10.4 7.3 6.3 4.4 P 0.021 0.033 SD 0.9 0.3 0.4 0.8 3.1 0.9 1.5SD 0.7 1.8 7.7 1.2 1.1 0.6 0.6 3.3 0.5 4.5 0.2 2.1 0.9 4.8 0.7 2.7 15.1 1.3 5.7 0.6 2.1 1.0 3.7 3.0 1.6 1.6 4.6 1.2 4.2 0.9 6.6 0.7 0.9 5.1 5.7 11.2 4.3 4.8 2.7 SD 1.4 0.8 0.5 2.3 4.0 1.7 3.8 2.0 12.4 4.0 1.9 2.6 1.2 3.2 3.0 5.5 0.7 3.5 4.3 7.7 3.0 3.5 2.0 LSD 0.55 0.33 0.25 1.03 2.23 0.77 2.52 ns 4.49 2.11 0.47 0 indica CV (%) 34.9 12.9 38.1 38.6 67.3 61.3 31.6 26.2 60.1 27.1CV (%) 47.2 25.6 47.5 20.3 62.9 38.9 90.3 28.5 59.1 20.8 71.8 18.5 56.2 65.6 59.1 28.4 64.4 44.3 85.6 28.0 47.3 20.6 81.7 24.8 103.2 25.3 91.9 76.7 48.4 73.8 96.0 69.5 107.7 59.1 76.6 62.6 CV (%) 44.6 35.5 47.5 83.7 66.3 78.4 58.3 59.8 70.2 74.3 57.3 74.1 125.4 80.1 67.9 53.8 69.3 79.4 58.1 98.6 46.1 70.5 57.3 japonica Relative changes in free am -level of 1% except Arg. The notations, “ns” and “*” repres Mean ( 3. α

Overall (Mean) 3.2 2.3 1.0 2.8 6.0 2.2 6.5 3.4 17.7 5.3 3.2 3.5 1.0 3.9 4.4 10.2 1.1 4.4 7.5 7.9 6.5 4.9 3.5 Mean ( Table 1 Asahi2 Dewasansan3 Fukunishiki4 Gohyakumangoku5 Koshihikari 2.5 4.76 Tamanishiki 2.4 2.7 1.9 2.67 Yamadanishiki 2.1 2.0 0.9 1.4 2.6 1.0 1.7 2.3 2.1 1.7 3.5 1.9 1.5 2.5 2.4 6.3 2.1 7.9 0.6 1.7 8.5 0.8 1.3 0.8 3.4 1.9 1.1 1.0 1.9 4.0 1.0 6.6 4.8 5.6 3.5 1.4 1.8 1.5 2.4 1.6 3.0 3.3 19.1 1.0 4.2 0.9 2.9 20.1 8.4 4.5 6.8 4.2 4.3 2.2 6.4 2.9 2.9 3.2 1.5 1.7 6.2 2.8 23.1 1.5 3.4 3.1 8.5 3.6 2.5 4.6 3.0 0.7 0.4 3.3 1.9 0.5 2.4 0.4 3.8 3.0 2.3 2.3 2.9 2.2 4.0 4.4 3.7 2.0 0.2 3.8 0.3 3.7 8.9 7.1 0.6 2.0 7.6 3.9 8.0 2.0 0.9 1.9 2.8 0.4 3.1 0.7 3.7 3.5 2.3 7.6 4.7 6.6 4.9 5.8 8.4 4.7 10.5 0.5 0.5 8.9 7.2 6.0 1.4 6.5 2.4 3.6 7.2 5.8 4.3 2.3 6.8 3.8 7.9 5.7 2.4 3.3 2.1 5.0 3.9 5.5 4.0 4.1 2.7 3.2 2.7 4.6 6.0 3.2 7.8 3.3 4.7 3.2 2.3 2.5 1.9 1 Akamai2 Amber3 GDT4 Panbira5 TN1 5.76 Zenith 2.0 4.0 1.4 1.7 6.1 2.5 10.0 0.3 3.8 1.3 14.6Group comparisons 2.6 0.9 3.4 1.4 0.4 7.1 2.9 3.5 1.8 2.4 ns 1.8 8.2 0.6 1.4 1.0 5.9 4.1 ns 7.0 3.6 6.3 3.0 2.2 45.7 1.6 11.9 ns 17.8 2.2 5.9 3.7 5.6 2.3 27.0 ns 8.6 3.1 7.6 10.2 1.6 11.8 4.1 17.2 ns 12.6 7.7 2.6 1.7 3.2 5.9 35.0 2.4 3.4 ns 13.7 12.2 2.5 5.3 2.9 13.7 3.5 ns 6.8 0.7 22.3 4.8 2.2 2.2 3.9 ns 2.3 2.5 4.2 0.9 4.8 4.2 15.3 4.0 ns 1.0 1.6 2.6 18.4 0.4 6.3 6.0 ns 32.7 1.9 3.3 3.5 15.5 0.3 4.7 8.9 9.8 * 15.9 5.2 13.5 3.5 0.6 0.5 20.7 9.5 ns 1.4 4.6 1.4 2.1 2.0 4.6 4.7 ns 2.6 5.1 4.9 10.7 6.5 ns 3.5 3.3 8.1 8.6 3.8 3.8 3.7 ns 9.8 7.3 1.9 2.7 4.5 7.0 5.9 * 2.3 3.6 5.0 4.5 ns 4.4 ns ns ns ns ns ns Cultivar Values represent the relative ch tivars 52 Kamara, Konishi, Sasanuma and Abe +

G His Total

Gln A

+ Total

FAA Total 0.36 and 0.-46) are significant at − 0.28, − Met) and total glutamate-derived (Glu

Ile

Lys

0.04 1.00 − Thr

e 0.28, 0.36 and 0.46 (or below Asn

ations in Table 2. All values abov tration of total aspartate-derived (Asp 0.05 0.44 0.65 0.59 0.66 0.71 0.21 0.60 0.28 1.00 0.11 0.250.07 0.45 0.320.02 1.00 0.64 0.560.11 0.28 0.74 0.210.22 1.00 0.62 0.38 0.46 0.62 0.31 0.42 1.00 0.16 0.54 0.49 0.51 1.00 0.66 0.23 1.00 − − − − − − 0.07 0.27 0.36 0.75 0.38 0.54 0.71 0.41 0.36 0.47 0.52 1.00 0.07 0.17 − − − 1 0.04 0.46 0.27 0.46 0.25 0.26 0.37 0.21 .40 0.12.54 0.45 0.08 0.40 0.31 0.58 0.60 0.27 0.66 0.31 0.28 0.49 0.45 0.50 0.71 0.05 0.41 0.68 0.24 0.27 0.52 0.68 0.36 0.20 0.67 0.31 0.20 0.75 0.06 1.00 0.91 0.73 1.00 0.04 0.34 1.00 rice grains based on Spearman’s correlation coefficient − .34 0.72 0.15 0.28 0.71 0.83 0.43 0.69 0.84 0.42 0.46 0.57 0.64 0.77 0.48 0.28 1.00 0.01 0.25 1.00 0.38 − − 48) using free amino acid concentr

3 0.33 0.65 0.06 0.25 0.63 1.00 =

.75 0.17 0.64 0.43 0.32 1.00 0.01 1.00 − 0.05 0.250.08 0.44 0.10 0.46 0.19 0.29 0.34 0.05 0.22 0.38 0.04 0.48 0.30 0.49 0.19 0.56 0.24 0.42 0.23 0.05 0.22 0.51 0.12 0.24 0.25 0.67 0.07 0.46 0.41 1.00 0.21 0.55 1.00 0.13 0.03 0.31 0.02 0.18 0.71 0.74 0.14 − − − − − ce. Total A and total G refer the concen 0.05 0.00 0.02 0.20 from 50 cultivars (i.e., df − − Asp Glu Asn Ser Gln Gly His Arg GABA Thr Ala Pro AABA Tyr Val Met Cys Ile Leu Phe Trp Lys GABA) FAAs.

Correlation matrix of free amino acid concentrations in +

Pro

TrpLysTotal FAA 0.75 0.09 0.84Total G 0.14 0.04 0.58 0.08 0.06Correlations were computed 0.76 0.15 0.23 0.87 0.70 0.06 0.91 0.73 0.55 0 0.80 0.92 0.72 0.15 0 AspGluAsnSer 1.00 Gln 0.85 1.00 Gly 0.60 0.74His 0.67 1.00 0.83 0.70 0.81 0.37 0.55 0.42 1.00 0.65 0.03 0.73 0.35 0.08 1.00 0.90 0.62 1.00 LeuPhe 0.15 0.31 0.52 0.18 0.75Total A 0.23 0.63 0.06 0.44 0.78 0.27 0.45 0.86 0.60 0.46 0.91 0.66 0.32 0.58 0.10 0.53 0.60 0.55 0.00 0 ArgGABA 0.36 0.15 0.42 0.00 0.30 0.03 0.44 0.26 0.24 0.40 0.64 1.00 Thr 0.31 0.36 0.29 0.50 0.31 0.55 AlaProAABATyr 0.36Val 0.46 0.23 0.45 0.26Met 0.27 0.65 0.69 0.18 0.48Cys 0.32 0.42 0.06 0.61Ile 0.36 0.50 0 0.02 0.37 0.24 0.39 0.63 0.04 0.6 0.26 0.54 0.36 0.11 0.59 0.24 0.40 0.53 0.13 0.63 0.53 0.30 0.19 0.46 0.17 0.58 0.45 0.31 0.43 0.46 0.53 0.16 0.64 0.53 0.29 0.84 0.43 0.31 0.03 0.51 0.04 0.3 Arg Table 0.05, 0.01 and 0.001 levels of significan Variation in free amino acid profile among rice cultivars 53

amino acids in their respective endosperms. Rice researchers have identified many fundamental differences between indica and japonica-type rices (Wang et al. 1998, N’tanos and Koutroubas 2002, Lon et al. 2004, Namai et al. 2009). The difference observed in the present study might therefore fit within the broader framework of the indica-japonica dichotomy. Besides suggesting a key difference between indica and japonica rice grain types in FAA metabolism, the precise physiological significance of the A/G ratio in the rice grain is unknown and remains open to various speculations. One such speculation, for instance, could be that generally the ratio appears to indicate that synthetic enzymes of the glutamate pathway are more active in japonica type grains than in the indica types, while those of the aspartate pathway Fig. 2. Scatterplot of total glutamate-derived against aspartate-derived are more active in the indica types. Such a speculation could free amino acid concentration for indica () and japonica () type have major implications for the selection and diversification rice grains. The trendlines represent the respective approximate linear of the rice crop. fits with corresponding regression equations and coefficients of deter- The mobilization of endosperm reserves under germina- mination (R2). The notations “*” and “**” on the values of the co- tion treatment is responsible for the surge in FAAs observed efficient of determination, denote significant and highly significant for all cultivars. While GABA concentration increased correlations respectively. rapidly in the glutamate pathway, Met also increased rapidly in the Asp pathway in an apparent competition (Lea and Leegood 1999); although not as fast as GABA. Rapid GABA accumulation has long been associated with germination response (Ohtsubo et al. 2004). However, the observation that free Met also increases very rapidly during germination treatment does not seem to have been reported for rice at the time this manuscript was compiled. Anzala et al. (2006) reported that in maize the aspartate pathway be- comes active under germination conditions due to the upregu- lation of both monofunctional aspartate kinase that favours the Lys branch and bifunctional aspartate kinase homoserine dehydrogenase which favours the Met/Thr branch of the aspartic pathway. Also, Gallardo et al. (2002) reported a strongly elevated level of methionine synthase protein (which catalyses the last step in Met synthesis in plants) in Arabidopsis seeds after imbibing water for 1-day, implying Fig. 3. Resubstitution plot of discriminant scores calculated using two a potential increase in Met under such conditions. It seems linear discriminant functions derived from the concentrations of 22 likely that enzymes of the aspartate pathway, especially those free amino acids in 45 rice cultivars using Fisher’s linear discriminant that participate in the Met/Thr branch, might also be en- analysis. Solid ( ) and open ( ) circles are used to denote points oc- hanced in germinating rice grains; a phenomenon which could cupied by indica and japonica type cultivars respectively. help explain the rapid rate of accumulation of Met in all rice grains observed. This additional information in rice might have implications for both the nutritional significance of GBR property of this cultivar. in human diets and the physiology of rice grain germination. It has been shown that typically indica type rice grains Generally, the variations in FAA accumulation rates tend to have higher levels of protein than their japonica among cultivars under germination treatment could be con- counterparts (Chen et al. 2006). This might also have sidered in relation to cultivar-specific germination response. implications for FAA accumulation and might help explain A lower rate of accumulation might imply delayed germina- the apparent difference between indica and japonica in the tion response as opposed to a deficiency in FAA accumula- rates of accumulation of up to 11 FAAs. Since A/G ratios tion capability. It is however impossible to determine the represent the relative abundance of Asp-derived and Glu- cause-effect relationships from these results. That is, for ex- derived FAAs in the grains, the difference observed between ample, whether it was the rapid germination response of the the two grain types appears to indicate key differences in the cultivar Akamai that led to a rapid accumulation of FAAs or manner in which the two rice ecotypes accumulate these free vice versa. 54 Kamara, Konishi, Sasanuma and Abe

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