Genome-wide identification and extensive analysis of rice-endosperm preferred genes using reference expression database

홍우종,유요한,박선아,문선옥,김성렬,안진흥,정기홍 한국식물학회 2017 Journal of Plant Biology Vol.60 No.3

Studying endosperm development in crop species enables us to understand the molecular mechanisms for producing and metabolizing carbohydrates as a main energy source. A significant accumulation of genome-wide transcriptome data can enhance the performance of metaexpression data for diverse applications. Using a gene search tool in Genevestigator (https://genevestigator.com/) for anatomical samples, we first conducted a meta-anatomical expression analysis based on 2566 Affymetrix array data in rice (Oryza sativa) and intended to identify 400 endospermpreferred probes. Tissue-preferred expression patterns were confirmed by performing an additional meta-analysis of anatomical expression data comprising 219 spatial or temporal Agilent 44K array data. We then identified 299 genes that showed strong endosperm-preferred expression. The functional significance of 24 previously characterized genes was evaluated, and tissue-specific expression patterns of two genes were validated using a GUS reporter system. This second approach demonstrated that new tools are available for delivering agronomic traits in the endosperm. MapMan (mapman.gabipd.org/) analysis revealed the Metabolism and Regulation overviews associated with the process of endosperm development. In particular, the starch metabolism pathway is very closely related to that process in rice. We then constructed a regulatory network using both KEGG (www. genome.jp/ kegg/) pathway information for our candidate genes and a predicted protein–protein interaction network tool. Examination of an osbzip58-1 mutant with defects in endosperm development was combined with global transcriptome data in the network. Our results indicated that osbzip58-1 regulates the starch and sucrose metabolism pathways as well as the production of seed storage protein precursors. This new fundamental information adds to our understanding about the molecular mechanism for endosperm development in rice, and the resulting data will contribute to future studies that work to enhance the agronomic trait(s) associated with the endosperm.

아밀로스 함량이 다른 쌀 품종의 이화학적 특성

최인덕(Induck Choi) 한국식품영양과학회 2010 한국식품영양과학회지 Vol.39 No.9

일품벼에서 유래된 신형질미의 취반 및 가공 등 용도개발을 위한 목적으로 백미와 현미에 대한 성분 분석 및 물리적 특성을 분석한 결과, 원품종인 일품벼과 신형질미인 고아미2호, 백진주, 설갱의 이화학적 특성은 품종 간에 유의한 차이를 보였다. 고아미2호는 고 아밀로스 품종으로 분류되며, 난소화성 다당류의 함량도 높게 나타난 반면에, 백진주는 저아밀로스 품종으로 반 찰벼의 특성을 보였다. 지방산은 백미와 현미에서 linoleic acid와 oleic acid가 전체 지방산의 70~75%를 차지하는 양질의 기름으로서, 백미는 linoleic acid가 현미에는 oleic acid 함량이 높은 경향을 보였다. 한편, 고아미2호는 palmitic acid 함량이 가장 높아서 다른 품종과 다소 다른 지방산 조성을 나타내었다. 백미의 주요 아미노산은 aspartic acid, glutamic acid, cysteine, hydroxy lysine이고, 현미에서는 cysteine을 제외한 aspartic acid, glutamic acid, hydroxy lysine인 것으로 나타났다. 쌀 품종의 호화특성을 DSC로 분석한 결과, 호화에 필요한 흡열엔탈피는 반 찰성인 백진주가 가장 높고, 다음은 설갱, 일품, 고아미2호 순으로, 고아미2호가 가장 낮은 흡열엔탈피을 보였다. 취반미의 식미치는 일품벼가 가장 높았으나, 밥의 텍스쳐를 분석한 결과, 고아미2호의 경도가 가장 높고, 일품, 백진주, 설갱 순으로 경도가 낮아지는 것으로 분석되었다. 신형질미 중에서, 고아미2호 취반미는 푸석한 조직감을 보여 다른 품종과는 현저히 다른 특성을 나타내므로, 고아미2호는 취반용 쌀로서 이용하기 보다는 가공용 기능성 곡류 소재로서 활용 가능성이 높으며, 반면에 백진주와 설갱은 원품종인 일품과 비교했을 때 취반용과 가공용도에 모두 적합할 것이다. Rice cultivars of Goami2 (G2), Baegjinju (BJJ), and Sulgaeng (SG) with different amylose contents were developed by mutation breeding via N-methyl-N-nitrosourea (MNU) treatment to Ilpumbyeo (IP), high japonica rice. They were identified by different appearances such as grain size, color, and shape. In this experiment, the compositional and physical qualities of those cultivars were examined. The G2 rice classified as a high-amylose rice cultivar was significantly higher in its non-digestable carbohydrates contents. Linoleic and oleic acid were composed of 70~75% of all fatty acids composition regardless of milled and brown rice, except G2 rice in which palmitic acid was the major fatty acid followed by linoleic acid and oleic acid in order. Major amino acids were aspartic acid, glutamic acid, and hydroxy lysine. It was found that cysteine contents were higher in the cultivars of endosperm mutant rice. The DSC analysis revealed that enthalpy was the highest in BJJ followed by SG, IP, and G2 rice. The lowest enthalpy of G2 might be attributable to the higher amylose content. Ilpumbyeo in its cooked rice form showed the highest in Toyo value and less in hardness, but G2 was vise versa. Results of gelatinization and cooked rice properties suggest that G2 was less suitable for cooked rice, but has a potential for functional ingredients from nutritional point of view. The BJJ and SG could be used for traditional cooking as well as for processed foods.

Characteristics of Endosperm Starch of the Rice Mutant Lines Induced by Sodium Azide

Young Seop Shin,Chlul Soo Park,Yong Weon Seo,Ji Ung Jeung 한국육종학회 2009 한국육종학회지 Vol.41 No.2

Rice consumption per capta, in South Korea, has been decreased dramatically, owing to the changes of living patterns. Because of not only the major energy source of Korean people but also major income source of Korean farmers, diversifying end-use-quality of rice has been demanded. To the context, 'Suweon 472', a high yielding and early mature japonica line and released as 'Namilbyeo' to framers in 2002, was treated with a chemical mutagen, Sodium Azide to find endosperm mutant types. A total of nine endosperm mutat lines, including five waxy, one dull, two floury, and one white core type, were identified from the 3,542 mutatagen treated lines. Amylose contents, iodine reaction, disintegration in alkali solution, gelatinization in urea solution and amylogram properties of those nine endosperm mutant lines were evaluated to address the possibility as new genetic materials for diversifying rice quality of Korean japonica cultivars. All embryo mutants were clearly differentiated from their wild type, 'Suweon 472', in terms of physic-chemical properties evaluated. The endosperm mutant lines would be very useful in expanding untiliztation of rice through opening new rice markets of processed foods from Korean japonica rice.

Sugary Endosperm is Modulated by <i>Starch Branching Enzyme IIa</i> in Rice ( <i>Oryza sativa</i> L.)

Lee, Yunjoo,Choi, Min-Seon,Lee, Gileung,Jang, Su,Yoon, Mi-Ra,Kim, Backki,Piao, Rihua,Woo, Mi-Ok,Chin, Joong Hyoun,Koh, Hee-Jong Springer US 2017 Rice Vol.10 No.-

<P><B>Background</B></P><P>Starch biosynthesis is one of the most important pathways that determine both grain quality and yield in rice (<I>Oryza sativa</I> L.). Sugary endosperm, <I>sugary-1</I> (<I>sug-1</I>), is a mutant trait for starch biosynthesis. Rice plants carrying <I>sug-1</I> produce grains that accumulate water-soluble carbohydrates instead of starch, even after maturity. Although this trait enhances the diversity of grain quality, sugary endosperm rice has hardly been commercialized due to the severely wrinkled grains and subsequent problems in milling. This study was conducted to identify the genes responsible for the <I>sug-h</I> phenotype through a map-based cloning technology.</P><P><B>Results</B></P><P>We induced a mild sugary mutant, <I>sugary-h</I> (<I>sug-h</I>) through the chemical mutagenesis on the Korean <I>japonica</I> cultivar Hwacheong. Grains of the <I>sug-h</I> mutant were translucent and amber-colored, and the endosperm appeared less wrinkled than <I>sug-1</I>, whereas the soluble sugar content was fairly high. These characteristics confer greater marketability to the <I>sug-h</I> mutant. Genetic analyses indicated that the <I>sug-h</I> mutant phenotype was controlled by a complementary interaction of two recessive genes, <I>Isoamylase1</I> (<I>OsISA1</I>), which was reported previously, and <I>Starch branching enzyme IIa</I> (<I>OsBEIIa</I>), which was newly identified in this study. Complementation tests indicated that <I>OsBEIIa</I> regulated the properties of sugary endosperm<I>.</I></P><P><B>Conclusions</B></P><P>Complementary interactions between the starch biosynthesis genes <I>OsISA1</I> and <I>OsBEIIa</I> determine the mild sugary endosperm mutant, <I>sugary-h</I>, in rice. Our finding may facilitate the breeding of sugaryendosperm rice for commercial benefit.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (doi:10.1186/s12284-017-0172-3) contains supplementary material, which is available to authorized users.</P>

Transgenic rice lines expressing maize <i>C1</i> and <i>R-S</i> regulatory genes produce various flavonoids in the endosperm

Shin, Young-Mi,Park, Hee-Jin,Yim, Sun-Duck,Baek, Nam-In,Lee, Choong-Hwan,An, Gynheung,Woo, Young-Min Blackwell Publishing Ltd 2006 PLANT BIOTECHNOLOGY JOURNAL Vol.4 No.3

<P>Summary</P><P>Flavonoids, compounds that possess diverse health-promoting benefits, are lacking in the endosperm of rice. Therefore, to develop transgenic lines that produce flavonoids, we transformed a white rice cultivar, <I>Oryza sativa japonica</I> cv. Hwa-Young, with maize <I>C1</I> and <I>R-S</I> regulatory genes. Expression of these transgenes was restricted to the endosperm using the promoter of a rice prolamin gene. The pericarp of the <I>C1</I>/<I>R-S</I> homozygous lines became dark brown in accordance with their maternal genotype, whereas the endosperm turned chalky, similar to the opaque kernel phenotype. Analysis via high-performance liquid chromatography (HPLC) revealed that numerous kinds of flavonoids were produced in these transgenic kernels. To identify individual flavonoids, the number of HPLC peaks was reduced through moderate acid hydrolysis, followed by ethyl acetate partitioning. Amongst the major flavonoids, dihydroquercetin (taxifolin), dihydroisorhamnetin (3′-<I>O</I>-methyl taxifolin) and 3′-<I>O</I>-methyl quercetin were identified through liquid chromatography/mass spectrometry/mass spectrometry and nuclear magnetic resonance analyses. Fluorescence labelling with diphenylboric acid showed that the flavonoids were highly concentrated in the cells of four to five outer endosperm layers. More importantly, a high fluorescence signal was present in the cytosol of the inner endosperm layers. However, the overall signal in the inner layers was significantly lower because starch granules and protein bodies occupied most of the cytosolic space. Our estimate of the total flavonoid content in the transgenic kernels suggests that <I>C1</I>/<I>R-S</I> rice has the potential to be developed further as a novel variety that can produce various flavonoids in its endosperm.</P>

Original Article: Food Science/Microbiology : Development of Protein Biomarkers for the Authentication of Organic Rice

( Ju Young Lee ),( Jinkyu Lim ) 한국응용생명화학회 2015 Journal of Applied Biological Chemistry (J. Appl. Vol.58 No.4

The rice protein profiles of Oryza sativa L (Koshihikari) grown under organic and conventional cultivation regimes were compared on 2-D gels to develop diagnostic marker proteins for organic rice. The selected proteins, differentially expressed between organic and conventional rice, were compared with the differentially expressed proteins of another organic and conventional rice pairing, produced at a different location. In the first comparison among conventional, no-chemical, and organic rice grown in the same region, Korea, 13 proteins exhibiting differential expression in organic and conventionally grown plants were selected. Eight of the 13 proteins were down-regulated, and the 5 remaining proteins were up-regulated from conventional to organic rice. The second comparison pairing from Kyungju, revealed 12 differentially expressed proteins, with 8 down-regulated and 4 up-regulated proteins. Ten of the differentially expressed proteins that overlapped between the two comparison sets could not be clustered into any functional group using a functional annotation clustering tool. Further comparisons using another set of conventional and organic rice, belonging to a different variety of Oryza sativa L and produced in Sanchung, revealed 8 differentially expressed proteins, 5 of which were down-regulated and 3 of which were up-regulated in the organic rice. Overall, 3 differentially expressed proteins were commonly found in all three organic rice crops. These 3 proteins, along with other overlapping differentially expressed proteins, can provide a good starting point for the development of signature proteins that can be used for the authentication of organic rice with a follow-up studies with more comparison sets.

Application of two bicistronic systems involving 2A and IRES sequences to the biosynthesis of carotenoids in rice endosperm

Ha, Sun-Hwa,Liang, Ying Shi,Jung, Harin,Ahn, Mi-Jeong,Suh, Seok-Cheol,Kweon, Soon-Jong,Kim, Dong-Hern,Kim, Young-Mi,Kim, Ju-Kon Blackwell Publishing Ltd 2010 PLANT BIOTECHNOLOGY JOURNAL Vol.8 No.8

<P>Summary</P><P>Coordination of multiple transgenes is essential for metabolic engineering of biosynthetic pathways. Here, we report the utilization of two bicistronic systems involving the 2A sequence from the foot-and-mouth disease virus and the internal ribosome entry site (IRES) sequence from the crucifer-infecting tobamovirus to the biosynthesis of carotenoids in rice endosperm. Two carotenoid biosynthetic genes, phytoene synthase (<I>Psy</I>) from <I>Capsicum</I> and carotene desaturase (<I>CrtI</I>) from <I>Pantoea,</I> were linked via either the synthetic 2A sequence that was optimized for rice codons or the IRES sequence under control of the rice globulin promoter, generating <I>PAC</I> (<I><U>P</U>sy-2<U>A</U>-<U>C</U>rtI</I>) and <I>PIC</I> (<I><U>P</U>sy-<U>I</U>RES-<U>C</U>rtI</I>) constructs, respectively. The transgenic endosperm of <I>PAC</I> rice had a more intense golden color than did <I>PIC</I> rice, demonstrating that 2A was more efficient than IRES in coordinating gene expression. The 2A and IRES constructs were equally effective in driving transgene transcription. However, immunoblot analysis of CRTI, a protein encoded by the downstream open reading frame of the bicistronic constructs, revealed that 2A was ninefold more effective than IRES in driving translation. The <I>PAC</I> endosperms accumulated an average of 1.3 &mgr;g/g of total carotenoids, which was ninefold higher than was observed for <I>PIC</I> endosperms. In particular, accumulation of &bgr;-carotene was much higher in <I>PAC</I> endosperms than in <I>PIC</I> endosperms. Collectively, these results demonstrate that both 2A and IRES systems can coordinate the expression of two biosynthetic genes, with the 2A system exhibiting greater efficiency. Thus, the 2A expression system described herein is an effective new tool for multigene stacking in crop biotechnology.</P>

Physicochemical Characteristics of Starches in Rice Cultivars of Diverse Amylose Contents

Mi-Ra Yoon,AReum Chun,Sea-Kwan Oh,Ha-Cheol Hong,Im-Soo Choi,Jeong-Heui Lee,Young-Chan Cho,Yeon-Gyu Kim 한국작물학회 2012 Korean journal of crop science Vol.57 No.3

Through the sampling four rice cultivars with differing amylose contents, the relationship between the structural and gelatinization properties of endosperm starches was analyzed. These rice varieties exhibited different chain length distribution ratio within the amylopectin cluster as well as varing amylose levels. The proportion of amylopectin short chains of in Goami cutlivars was higher than the other varieties, whereas the Goami 2 which shows amylose extender mutant properties in the endosperm showed the highest proportion of long chains. In X-ray diffraction analysis of rice starches, the Goami 2 variety displayed a B-type pattern whereas the other varieties were all A-type. Among the cultivars with high and normal rice starch levels, those with the higher amylose contents showed distinctly lower swelling. Goami 2 rice was found to have the highest onset and peak gelatinization temperature from the differential scanning calorimetry results. The four rice cultivars under analysis also showed different rates of hydrolysis by amyloglucosidase. These findings suggest that the composition and chemical structure of the starch content is a major determinant of both the gelatinization and functional properties of rice.

Quantitative comparison of acidic polysaccharides in the endosperm of two major varieties of rice

Hyun, Gyu Hwan,Lim, Dong Kyu,Kwon, Sung Won The Korean Society of Analytical Science 2017 분석과학 Vol.30 No.4

Rice endosperm, the portion that remains after milling, is the part of the rice seed that is primarily consumed as a source of nutrients. There have been many studies on polysaccharides, such as hemicellulose, cellulose, and pectins, derived from the cell walls of various plant groups. It has been reported that the acidic polysaccharide fractions, which contain water-soluble pectins that have been shown to have pharmacological effects in vivo and in vitro, have common chemical structures that include galacturonic acid polymers, rhamnose, arabinose, and galactose. However, few studies have been conducted on the acidic polysaccharides contained in the endosperm of rice. In this study, we quantitatively compared the differences in the acidic polysaccharide contents from samples from two of the main varieties of rice consumed as staple foods, japonica and indica, using a colorimetric method. Rice samples were collected from 39 different regions in Korea, China, Thailand and Vietnam. Acidic polysaccharide fractions were obtained by precipitation of the alcohol-insoluble residue (AIR) and enzyme treatment of each sample. The total amount of carbohydrates and uronic acid in each acidic polysaccharide fraction were measured using the phenol-sulfuric acid method and the carbazole-sulfuric acid method, respectively. The differences in the total polysaccharide contents in the acidic polysaccharide fractions were not statistically significant (p = 0.07), but the uronic acid contents were significantly different between the two groups (p = 0.04).

Physicochemical Characteristics of Starches in Rice Cultivars of Diverse Amylose Contents

Yoon Mi-Ra,Chun Areum,Oh Sea-Kwan,Hong Ha-Cheol,Cho Young-Chan 韓國作物學會 2012 한국작물학회지 Vol.57 No.3

Through the sampling four rice cultivars with differing amylose contents, the relationship between the structural and gelatinization properties of endosperm starches was analyzed. These rice varieties exhibited different chain length distribution ratio within the amylopectin cluster as well as varing amylose levels. The proportion of amylopectin short chains of in Goami cutlivars was higher than the other varieties, whereas the Goami 2 which shows amylose extender mutant properties in the endosperm showed the highest proportion of long chains. In X-ray diffraction analysis of rice starches, the Goami 2 variety displayed a B-type pattern whereas the other varieties were all A-type. Among the cultivars with high and normal rice starch levels, those with the higher amylose contents showed distinctly lower swelling. Goami 2 rice was found to have the highest onset and peak gelatinization temperature from the differential scanning calorimetry results. The four rice cultivars under analysis also showed different rates of hydrolysis by amyloglucosidase. These findings suggest that the composition and chemical structure of the starch content is a major determinant of both the gelatinization and functional properties of rice.