APETALA2/ethylene response factor (AP2/ERF) transcription factors are involved in biological and abiotic stress response, plant development, and growth. AP2/ERF genes are classified into five families (AP2, DREB, ERF, RAV, and soloist), and most genes belong to DREB and ERF families. So far, genomic analysis of DREB and ERF family genes of various plant species has been performed, and classifications based on the homology of AP2/ERF-specific DNA binding domain, arrangement of exons and introns, and similarity of group-specific conserved motifs have been conducted. These classifications provide plausible information for the prediction of AP2/ERF gene function. In this paper, an overview of the classification, structure, evolution, and function of AP2/ERF genes is described, and the functional properties and regulatory mechanisms of ERF family genes that have been identified are summarized by group according to the functional classification of Arabidopsis ERF family genes. This shows that group-specific conserved motifs of Arabidopsis ERF family genes are closely linked with group-specific functions and regulatory mechanisms, indicating that the effective functional prediction of ERF family genes through such a classification scheme can be usefully applied to the trait improvements of various plants.

The ω5-gliadins are the major allergens in wheat-dependent excise-induced anaphylaxis (WDEIA). In this study, SDS-PAGE analysis was used to assign the ω5-gliadins (Gli-B1) alleles in thirty two Korean wheat cultivars, compared with eleven standard wheat cultivars for Gli-B1a~m alleles. These results were reconfirmed with their complementary Glu-B3 low-molecular-weight glutenin subunits alleles tightly linked with Gli-B1 locus revealed with 2-DGE in our previous study. As a result, one Gli-B1b, four Gli-B1d, two Gli-B1f, six Gli-B1m and nineteen Gli-B1h varieties were identified. This is the first report on revealing the Gli-B1 alleles in Korean wheat cultivars and represents valuable basic data on wheat allergy, relationship between gliadin and wheat quality, and development of hypo-allergenic wheat.

A wheat mutant of low-molecular-weight glutenin (LMW-GS) “Gunji-2” at Glu-B3 locus was derived among the double haploid lines. Gunji-2 was derived from F1 plants of Keumkang and Olgeuru crosses using the wheat × maize system according to the procedures of Inagaki and Mujeeb-Kazi at International Maize and Wheat Improvement Center (CIMMYT). Deletion of Glu-B3 LMW-GS proteins was found by allele specific DNA marker, one dimensional SDS-PAGE and two dimensional gel electrophoresis (2-DGE). Tandom mass spectrometry (MS/MS) was used to obtain direct evidence of LMW-GS deletion. In addition, we examined the basic agronomic traits, protein content, dough properties of mixing and bread loaf volumeof Gunji-2 and parental wheat cultivars grown for two years. This mutant will represent a valuable resource in quality test for specific allele or gene at Glu-B3 locus.

Anthocyanins, providing the bright red-orange to blue-violet colors, flavonoid-derived pigments with strong antioxidant activity that have benefits for human health. We isolated RsMYB1, which encodes an R2R3 MYB transcription factor (TF), from red radish plants (Raphanus sativus L.) that accumulate high levels of anthocyanins. RsMYB1 shows higher expression in red radish than in common white radish, in both leaves and roots, at different growth stages. regulatory genes. Transient expression of RsMYB1 in tobacco showed that RsMYB1 is a positive regulator of anthocyanin production. Also, the synergistic effect of RsMYB1 with B-Peru was larger than the effect of Arabidopsis plants stably expressing RsMYB1 produced red pigmentation throughout the plant, accompanied by up-regulation of the six structural and two regulatory genes for anthocyanin production. This broad transcriptional activation of anthocyanin biosynthetic machinery in Arabidopsis included up-regulation of TRANSPARENT TESTA 8, which encodes a bHLH-type TF. These results suggest that overexpression of RsMYB1 promotes anthocyanin production by triggering the expression of endogenous bHLH genes as potential binding partners for RsMYB1. In addition, RsMYB1-overexpressing Arabidopsis plants had a higher antioxidant capacity than did non-transgenic control plants. Taken together, RsMYB1 is an actively positive regulator for anthocyanins biosynthesis in radish plants and it might be one of the best targets for anthocyanin production by single gene manipulation being applicable in diverse plant species.

Although it is well known that low-molecular-weight glutenin subunits (LMW-GS) affects bread and noodle processing quality, the function of specific LMW-GS proteins mostly remain unclear. It is important to find a corresponding gene for a specific LMW-GS protein in order to understand the function of the specific LMW-GS protein. The objective of this study was to identify LMW-GS genes and haplotypes using well known Glu-A3, Glu-B3 and Glu-D3 gene specific primers and to interlink their protein products by proteomic approaches in a wheat variety. A total of 36 LMW-GS genes and pseudo-genes were amplified including 11 Glu-3 gene haplotypes, designated as GluA3-13K and GluA3-22K (pseudogene) at Glu-A3 loci, GluB3-33K and GluB3-43K at Glu-B3 loci and GluD3-11K, GluD3-21K, GluD3-31K, GluD3-42K, GluD3-5K, GluD3-6K and GluD3-393K (pseudogene) at Glu-D3 loci. To determine the relationship between gene haplotypes and their protein products (to identify the corresponding LMW-GS proteins), we conducted N-terminal amino acid sequencing and tandem mass spectrometry (MS/MS) analysis of the 17 LMW-GS spots separated by 2-DGE. Successfully, LMW-GS proteins of the Glu-3 gene haplotypes except pseudo-genes mentioned above were identified. This is the first report on comprehensive characterization of LMW-GS genes and their corresponding proteins and establishment of specific correspondence between each other in a single wheat cultivar. Our approach will be useful to understand the molecular basis of the LMW-GS and to study their contribution to the end-use quality of flour.

Although it is known that the composition of HMW-GSs and LMW-GSs are important factor for end-product quality as bread, noodle and cookie, it is still not clear which HMW-GSs and LMW-GSs confer specific processing properties. In this study, to investigate distinctive glutenin proteins and expression level for characteristic processing properties, we carried out qualitative and quantitative analysis of gluetenin protein in noodle and bread wheat cultivars by two-dimensional electrophoresis. Unexpectedly, five LMW-GS spots were found to be expressed at a common position in all cultivars and these spots may play something in glutenin biosynthesis. Also we found LMS-GS spots to distinguish Korean wheat cultivars mostly used as noodle and western bread wheat cultivars. These spots may contribute to characteristic processing properties. The 2DE results for each cultivar will be used as reference map or protein marker discriminating wheat cultivars, wheat and rice, imported and Korean flour. For quantitative analysis of gluetenin, we calculated relative expression level of the HMW-GS, LMW-GS and HMW-GS/ LMW-GS ratio in each cultivar by 2DE. The results presented in this study provide new insight into relation of specific glutenin proteins and end-use quality and will be useful to choose elite breeding line for improvement of wheat flour quality.

Low-molecular-weight glutenin subunits (LMW-GS) play a crucial role in the processing quality of wheat flour. They are encoded multi gene family located at the Glu-A3, Glu-B3 and Glu-D3 on the short arm of chromosome 1A, 1B and 1D respectively. Typical LMW-GSs are composed of three parts including a short N-terminal domain, a relatively short repetitive domain and a C-terminal domain. Further, typical LMW-GS sequences are divided into LMW-s, LMW-m and LMW-i types, on the basis of the first amino acid of the mature proteins (serine, methionine and isoleucine, respectively). Although it is known that the allelic variation of LMW-GSs affect the properties of dough, it is still not clear which LMW-GSs confer better bread-making quality because of the larger number of expressed subunits and their overlapping mobility with abundant gliadin proteins. Therefore, it is important to characterize LMW-GS genes and develop functional markers to identify different LMW-GS alleles for application in wheat breeding. In this review, we discuss the various aspects of LMW-GS, including their structural characteristics, the development of marker, relationship between LMW-GSs and bread wheat quality, and genetic engineering of the LMW-GSs.

이차원 전기영동 분석을 이용하여 국내 밀 32 품종의 HMW-GS 단백질 발현의 정성 및 정량적인 분석을 통해 품 HMW-GS 발현 정도를 평가하여 국내 밀 품종 육성의 초 자료로 활용하고자 수행하였다. 평균 HMW-GS 스팟 수는 11.78개였으며, Glu-A1 1.31개, Glu-B1 5.53개, 그리고 Glu-D1에서 4.94 개였다. Glu-B1과 Glu-D1에서는 subunit에 따른 단백질 스팟 수가 차이가 없기 때문에, Glu-A1에서는 1과 2* subunit을 지닌 품종이 null allele 품종에 비하여 단백질 스팟 수가 많았다. 단백질 스팟 수는 조경밀이 18개로가장 많았으며, 다홍밀은 7개로 제일 적었다. 단백질의 상대적인 발현량을 조사한 결과 평균 0.44로 대비 품종인 Chinese Spring에 비하여(1.0) 낮았고, 고분밀이 1.11로 가장 높았으며, 은파밀이 0.24로 가장 낮았다. 단백질 스팟수와 발현량을 이용한 유연관계 분석 결과, 국내 밀 품종을 6개 그룹으로 분류할 수 있었다.

To evaluate expression level of HMW-GS protein qualitatively and quantitatively, we separated glutenin fractions and conducted two-dimensional electrophoresis (2DE) in 32 cultivars of Korean wheat for the use of as the basis of wheat breeding. The average spot number of HMW-GS in all Korean wheat cultivars was 11.78 which included 1.31, 5.53 and 4.94 to Glu-A1, Glu-B1 and Glu-D1 loci, respectively. Cultivars harboring 1, 2* subunits had many spots more than ones harboring null allele in Glu-A1 loci because there is no difference of spots between Glu-B1 and Glu-D1 loci. In total spot number of HMW-GS, the highest one was Jokyung as 18 and Dahong the lowest as 7. When the Korean wheat cultivars were compared with the Chinese spring in the average relative expression level, Korean one’s were lower as 0.44. Especially, Gobun was the highest as 1.11 and Eunpa was the lowest as 0.24. Also we investigated phylogenetic relationship based on both frequency of HMW-GS spots and quantification value of each spot to all HMW-GS spots. As a result, Korean the varieties of Korean wheat could be classified into six groups.

LMW-GSs represent approximately 1/3 of the total wheat gluten fraction, which have not been widely studied, even though they are important in the context of wheat end-use quality. In this study, we report on the qualitative and quantitative analysis of LMW-GS in korean wheat cultivars by 2DE in 32 cultivars of Korean wheat for the use of the basis of wheat breeding. We firstly identified spots corresponding each of Glu-3 alleles. The 2DE results for each cultivar will be used as reference map or protein marker discriminating wheat cultivars, wheat and rice, imported and Korean flour. Unexpectedly, five LMW-GS spots were found to be expressed at a common position in hexaploid wheat cultivars, and these spots might play something in glutenin biosynthesis. Total spot numbers were expressed variously between 20 and 10, and average spot number was shown 17.12. The average number of spots in Glu-A3, Glu-B3 and Glu-D3 were 3.0, 4.56 and 2.96 respectively. When the Korean wheat cultivars were compared with the Chinese spring (1.0) in the average relative expression level, Korean one’s were lower as 0.67. Especially, Gobun was the highest as 1.32 and Baekjoong was the lowest as 0.24. Also we investigated phylogenetic relationship based on frequency of HMW-GS spots and quantification value of each spot to all LMW-GS spots. As a result, the varieties of Korean wheat could be classified into five groups.

국내 밀 품종 조경, 금강 그리고 중국 밀 품종인 Chinese spring의 genomic DNA를 주형으로 LMW-GS 특이 프라이머세트를 이용하여 3개의 새로운 LMW-GS i 타입 유전자를분리하였고 이들의 분리된 유전자는 각 각 조경 II-2, CSIII-5 그리고 금강 6-12로 명명하였다. 이들의 유추 아미노산을 분석한 결과 20개의 시그널 펩타이드, 이소루신으로 시작하는 N-말단 부분 그리고 글루타민이 많은 반복도메인 그리고 C-말단 부분으로 구성되어 있으며 조경 II-2와 CS III-5는 전형적인 LMW-GS i-type 유전자처럼 C-말단에 8개의 시스테인 잔기가 있었다. 금강 6-12는 특이하게도 하나 더 많은 9개의 시스테인 잔기가 존재하였는데 이 여분의 시스테인 잔기는7번째 시스테인 잔기의 11잔기 앞에 존재하며 TAT(타이로신)이 TGT(시스테인)로 바뀐 결과이다. LMW-i 타입 글루테닌 유전자들 간의 SNP와 InDel을 확인하기 위해서 본 연구에서 클로닝 된 조경 II-2, CS III-5 그리고 이전에 본 그룹에서 확인된 조경 HQ619933와 기존 문헌에 나와 있는 6배 체 밀 유래의 10개의 LMW-GS i 타입 유전자들과 다중염기서열 분석을 실시하였고, 이들 사이에서 15개의 SNP와 1개의 insertion이 확인되었다. 밀 품종 조경의 Glu-A3 단백질을 동정하기 위해 글루테닌을 추출 이차원전기영동을 하고 Glu-A3c 위치의 스팟을 절취하여 in-gel digestion한 후 LC-ESI MS/MS 분석을 수행한 결과 조경의 i 타입 LMW-GS 유전자 좌는 Glu-A3c로 확인되었다. LMW-i 타입 글루테닌 유전자들의 연관 관계를 분석하기 위해 본 연구 그룹에서 클로닝 한 조경 II-2, CS III-5, 금강 6-12 그리고 조경 HQ6199333와 Genebank DB의 35개의 LMW-i 타입 글루테닌 유전자의 유추 아미노산 서열을 이용하여 Phylogenic tree를 완성하였다. 이들 39개의 계통도 분석 결과 이배체 밀과 4배체 밀의 LMi 타입 글루테닌이 육배체 밀의 LMW-i 타입 글루테닌과 크게 나눠지는 것을 확인하였으며, 육배체 밀의 LMW-i 타입 글루테닌들은 Glu-A3a부터 GluA-3g까지 7개 subgroup으로 나눠지는 것을 확인하였다. 금강 6-12는 GluA-3a와 GluA-3c 사이에 존재하였고 조경 II-2와 CS III-5는 GluA-3d와 일본 연질 밀인 농림 61의 AB062878과 같은 subgroup에 존재하였고 조경 HQ6199333은 Glu-A3c subgroup에 위치하였다. LMW-i 타입 글루테닌 유전자들의 유추 아미노산 다중서열분석결과 반복 도메인은 length polymorphism은 179～149개 정도의 long 타입과 91, 51, 10, 2개의 short 타입으로 나눠지고 이것은 long 타입과 short 타입 LMW-i 타입 글루테닌 유전자를 구분 할 수 있는 마커의 근거가 된다.

Rice seed storage proteins (SSPs) are accumulated in storage organelles of the endosperm during seed maturation. The SSPs from the rice seeds consist of glutelins as a major SSP, and prolamins and globulins comprise about the rest 20 % of the SSPs. To improve the nutritional quality of rice seeds or processing properties of rice flour, we are attempting to change the composition of the SSPs in rice seeds. For this purpose, we generated many transgenic rice plants, which show the altered levels of the SSPs, by using the RNA interference (RNAi). Accumulation of glutelins was 76% reduced in the GluA-RNAi lines. The Pro-RNAi lines revealed the reduced levels of prolamins to 36%. The protein level of globulins was 61% reduced in the Glb-RNAi lines. Interestingly, an obvious reduction of glutelins, prolamins, and globulins was not examined in the GluA:Pro:Glb-RNAi lines. This suggests that a reduction of a few SSPs could be compensated by the increases of other SSPs at the protein levels. We are also attempting to generate transgenic rice plants expressing both a high-molecular-weight (HMW) glutelin subunit and a low-molecular-weight (LMW) glutelin subunit. These manipulations of rice SSPs might be an important contribution on improving the functional properties of rice seeds.

To develop a strong root-specific gene expression system, six gene promoters were investigated by using transgenic Arabidopsis and a GUS:GFP reporter gene. These promoters were initially selected from Arabidopsis genes which are specifically expressed only in roots, based on the TIGR information. The GUS activity of these promoters was measured in several tissues of Arabidopsis by using both histochemical and fluorimetric GUS assays. The results showed that the activity of these promoters was strongly detected only roots. This was also confirmed by RT-PCR analysis. Therefore, these six promoters could be used for utilization of a root-specific expression of target genes.

Although it is known that the composition of high-molecular weight glutenin subunits (HMW-GSs) and low-molecular weight glutenin subunits (LMW-GSs) are important factor for bread, noodle and cookie, it is still not clear which HMW-GSs and LMW-GSs confer improved processing properties and how those HMW-GSs and LMW-GSs interact each other. In this study, to investigate distinctive glutenin proteins for characteristic processing properties for noodle, glutenins extracted from seeds of several Korean and Chinese wheat cultivars were focused in IPG gel strip and subjected to SDS-polyacrylamide gel electrophoresis. Differential protein expression level was analyzed using image master platinum 6. Then to characterize the HMW-GSs of Korean cultivar Uri, extracted glutenin proteins were separated by two dimensional gel electrophoresis. Nine spots digested with trypsin resulting peptide fragmentation were identified by LC ESI-MS/MS and MASCOT database. We also separated HMW-GSs from wheat cultivar Uri by fast protein liquid chromatography (FPLC) withResource Phe column using gradient buffer condition with 4M Urea and 0.45M ammonium sulfate.Each single band of 1Dx 2.2, 1Bx7 and double bands of 1Dy8 and 1By12 were separated.