Negatively Charged Lipids Are Essential for Functional and Structural Switch of Human 2-Cys Peroxiredoxin II

The function of ubiquitous 2-Cys peroxiredoxins (Prxs) can be converted alternatively from peroxidases to molecular chaperones. This conversion has been reported to occur by the formation of high-molecular-weight (HMW) complexes upon overoxidation of or ATP/ADP binding to 2-Cys Prxs, but its mechanism is not well understood. Here, we show that upon binding to phosphatidylserine or phosphatidylglycerol dimeric human 2-Cys PrxII (hPrxII) is assembled to trefoil-shaped small oligomers (possibly hexamers) with full chaperone and null peroxidase activities. Spherical HMW complexes are formed, only when phosphatidylserine or phosphatidylglycerol is bound to overoxidized or ATP/ADP-bound hPrxII. The spherical HMW complexes are lipid vesicles covered with trefoil-shaped oligomers arranged in a hexagonal lattice pattern. Thus, these lipids with a net negative charge, which can be supplied by increased membrane trafficking under oxidative stress, are essential for the structural and functional switch of hPrxII and possibly most 2-Cys Prxs.     

Locus-specific primers for LMW glutenin genes on each of the group 1 chromosomes of hexaploid wheat

To reveal the chromosomal location of three known low-molecular-weight (LMW) glutenin genes in wheat, we designed and used three sets of sequence-specific primers in polymerase chain reactions (PCR) on Chinese Spring and its derived group 1 aneuploid nullisomic-tetrasomic stocks. Two sets proved to be chromosome specific and amplified sequences from the Glu-A3 and Glu-D3 loci, respectively. The third set was apparently composed of conserved sequences as it produced PCR products in each of the aneuploids. Two of these products were cloned, and their sequences differed from the known LMW glutenin genes at several positions. Again, primer sets specific for these sequences were designed. One set was directed to the Glu-A3 locus, the second set resulted in two PCR products differing in length, one of which was located on chromosome 1B and the other on 1D. Primer sets constructed for the latter two sequences were specific for the Glu-B3 and Glu-D3 loci, respectively. Hence, primer sets specific for each of the three homoeologous chromosomes of the group 1 (1A, 1B, 1D) are available. In addition, these locus-specific primers were assayed for their ability to distinguish among wheat cultivars. PCR products amplified with one of the Glu-A3-specific primer sets showed length polymorphisms in various wheat varieties. Varieties carrying the 1RS.1BL translocated chromosomes could be recognized by the absence of a PCR product when the Glu-B3 primer set was used. These results suggest that PCR with locus-specific primers can be useful in the molecular genetic analysis of hexaploid wheat.     

中国特有小麦Gli-1、Gli-2和Glu-1位点的遗传多样性(英文)

运用APAGE和SDS_PAGE方法 ,研究了 32份中国特有小麦Gli_1、Gli_2和Glu_1位点的遗传多样性。在 1 4份云南铁壳麦 (Triticumaestivumssp .yunnaneseKing)中 ,共出现 8种醇溶蛋白带型和 3种高分子谷蛋白带型。在 9份西藏半野生小麦 (T .aestivumssp .tibetanumShao )中 ,发现 9种醇溶蛋白带型和 4种高分子谷蛋白带型。在 9份新疆稻麦 (T .petropavlovskyiUdacz.etMigusch .)中 ,观察到 9种醇溶蛋白带型和 5种高分子谷蛋白带型 ,其中 1份新疆稻麦 (稻麦 2 )具有Glu_D1编码的新亚基 2 .1 1 0 .1。在这 3种中国特有小麦群体中 ,Gli_1位点分别检测出 1 0、1 4和1 1个等位基因 ;Gli_2位点各具有 1 1、1 4和 1 2个等位基因 ;Glu_1位点也分别出现 5、6和 8个等位基因。云南铁壳麦、西藏半野生小麦和新疆稻麦群体内的Nei’s遗传变异系数分别为 0 .3798、0 .56 2 5和 0 .56 93。这些结果说明 ,与云南铁壳麦相比 ,西藏半野生小麦和新疆稻麦群体内的遗传变异相对较大。     

High-molecular-weight glutenin subunits in the Cylindropyrum and Vertebrata section of the Aegilops genus and identification of subunits related to those encoded by the Dx alleles of common wheat

The high-molecular-weight (HMW) glute-nin subunit composition of seven species from the Cylindropyrum and Vertebrata sections of the Aegilops genus was studied using SDS-PAGE and Western blot analysis. Two subunits were detected in Ae. caudata and three in Ae. cylindrica. In both species, subunits showing electrophoretic mobility similar to that of 1Dx2 were present. Western blot analysis using a monoclonal antibody (IFRN 1602) specific for the 1Ax and 1Dx subunits of bread wheat showed that the 1Dx-like subunit of Ae. caudata gave only a weak reaction. This indicates that Ae. caudata expresses subunits which are more distantly related to the 1Dx subunits. Two subunits were detected in each of the 60 accessions of Ae. tauschii, including several 1D^tx subunits showing different electrophoretic mobilities from those of the 1Dx subunits commonly found in bread wheat. All of the 1D^tx subunits reacted strongly with IFRN 1602, confirming their close relationship to the 1Dx subunits of bread wheat. Three subunits were found in Ae. crassa (6 x), four in Ae. ventricosa and Ae. juvenalis and five in Ae. vavilovii. In these four species, the subunits that showed electrophoretic mobility similar, or close, to that of 1Dx2 all reacted with IFRN 1602. In addition, Ae. ventricosa contained a subunit showing electrophoretic mobility slower than that of 1Dx2.2, which also reacted with IFRN 1602. These results suggest that the D-genome component in the multiploid Aegilops species express at least one HMW glutenin subunit that is structurally related to the 1Dx subunits of bread wheat. Received: 5 November 1999 / Accepted: 12 February 2000     

Silencing of HMW glutenins in transgenic wheat expressing extra HMW subunits

Wheat HMW glutenin subunit genes 1Ax1 and 1Dx5 were introduced, and either expressed or overexpressed, into a commercial wheat cultivar that already expresses five subunits. Six independent transgenic events were obtained and characterized by SDS-PAGE and Southern analysis. The 1Dx5 gene was overexpressed in two events without changes in the other endosperm proteins. Overexpression of 1Dx5 increased the contribution of HMW glutenin subunits to total protein up to 22%. Two events express the 1Ax1 subunit transgene with associated silencing of the 1Ax2* endogenous subunit. In the SDS-PAGE one of them shows a new HMW glutenin band of an apparent M_r lower than that of the 1Dx5 subunit. Southern analysis of the four events confirmed transformation and suggest that the transgenes are present in a low copy number. Silencing of all the HMW glutenin subunits was observed in two different events of transgenic wheat expressing the 1Ax1 subunit transgene and overexpressing the Dx5 gene. Transgenes and expression patterns were stably transmitted to the progenies in all the events except one where in some of the segregating T₂ seeds the silencing of all HMW glutenin subunits was reverted associated with a drastic lost of transgenes from a high to a low copy number. The revertant T₂ seeds expressed the five endogenous subunits plus the 1Ax1 transgene. Received: 16 June 1999 / Accepted: 29 July 1999     

高分子量麦谷蛋白１４和１５亚基的纯化、Ｎ—末端序列及部分生化特性研究

用ＳＤＳ－ＰＡＧＥ制备电泳技术结合一种新的凝胶中蛋白质显色方法,对普通小麦（Triticum aestivum)小偃六号的高分子量麦谷蛋白１４和１５亚基进行了有效的分离纯化,将其转印于ＰＶＤＦ膜上测定了Ｎ－端的氨基酸顺序,通过比较了发现它们与已知序列的其他的高分子是麦谷蛋白亚基高度同源。用两种双向电泳技术确定了它们的等电点（ＰＩ）属于碱性范围。     

Immunochemical Assay for Quality and Immunological Homology of Storage Proteins in Different Closely Related Genera and Species of Wheat

An immunochemical assay using polyclonal and monoclonal antibodies of high-molecular-weight glutenin subunit (HMW-GS) in Triticum aestivum L. was carried out to determine the quality of wheat and to investigate the immunological homology of the storage proteins in cereal endosperms in different closely related wheat genera and species. The results showed that correlation between the antigen-antibody reaction and the wheat quality varied with the type of antibodies used and the quality. The correlation coeffecient was slightly higher when the polyclonal antibodies were used than monoclonal antibodies were used. The correlation coeffecient was high between the antibody binding and the protein content, and wet/dry gluten coment, with Zeleny sedimentation value, while that between antibody binding and bread characters was lower. The highest correlation coeffecient between the polyclonal antibody binding and the protein content in grains, wet and dry gluten contents, bread volume and bread ratio volume was 0. 762 0, 0. 894 2, 0. 887 3, 0.610 3, 0.459 8 and 0.474 4 respectively, while the highest correlation coeffecient between the monoclonal antibody binding and the above parameters was 0. 783 7, 0. 774 5, 0.782 2, 0. 684 1, 0. 687 3 and 0. 598 2 respectively. The immunological homologies between I-IMW-GS 1Dyl0 in common wheat and endosperm storage protein in wheat grains of different genera and species were noticed. The cross-reac-tion among Triticum aestivum L., Secale cereale, T. spelta L., Aegilops squearrosa L. and T. Turgidtan L. was stronger than that among other cereals.     

小麦高分子量麦谷蛋白亚基1Dx5启动子驱动外源基因的表达

将小麦高分子量麦谷蛋白亚基(HMW-GS)基因的胚乳组织特异性表达启动子驱动的外源突变型1Dx5基因和gus基因导入小麦中.对其转基因植株连续3代的跟踪研究表明,突变型1Dx5基因的重复序列导致其表达蛋白分子量增大,并影响其它1Bx17 1By18亚基基因的表达.组织化学分析观察到gus基因在1Dx5基因启动子驱动下的表达表现出胚乳组织特异性,在开花2周后开始表达,表达量呈持续上升,至腊熟期达到最高,其次为籽粒成熟期.     

Study of high molecular weight wheat glutenin subunit 1Dx5 by 13C and 1H solid-state NMR spectroscopy. I. Role of covalent crosslinking

This work describes a carbon and proton solid-state NMR study of the hydration of a high molecular weight wheat glutenin subunit, 1Dx5. The effect of the presence of disulfide bonds on the hydration behavior of the subunit is investigated by a comparison of the unalkylated and alkylated forms of the protein. Hydration induces partial plasticization of the protein so that some segments become more mobile than others. The 13C cross-polarization and magic-angle spinning (MAS) spectra of the samples in the dry state and at two hydration levels (approximately 40 and approximately 65% D2O) were used to monitor the protein fraction resisting plasticization (trains). Conversely, 13C single pulse excitation and 1H-MAS experiments were used to gain information on the more plasticized segments (loops). The molecular motion of the two protein dynamic populations was further characterized by 13C T1 and 1H T(1rho), T2, and T1 relaxation times. The results suggest that hydration leads to the formation of a network held by a cooperative action of hydrogen bonded glutamines and some hydrophobic interactions. The looser protein segments are suggested to be glycine- and glutamine-rich segments. The primary structure is therefore expected to significantly determine the proportion of trains and loops in the network. The presence of disulfide bonds was observed to promote easier plasticization of the protein and the formation of a more mobile network, probably involving a higher number of loops and/or larger loops.     

Sequence similarity between allelic Glu-B3 genes related to quality properties of durum wheat

 Low-molecular-weight glutenin subunits (LMW-GSs) are wheat endosperm proteins mostly encoded by genes located at the Glu-3 loci. These proteins are of particular interest in durum wheat because a correlation between LMW-GSs encoded by genes at the Glu-B3 locus and the pasta-making quality of durum wheat semolina has been shown. We isolated and characterized two allelic lmw-gs genes located at the Glu-B3 locus and present in durum wheat lines displaying different qualitative properties. The clones pLMW1CL and λLMW3.1 were found to contain allelic sequences encoding LMW-GSs belonging to the good and poor quality-related groups named LMW-2 and LMW-1, respectively. The LMW-GSs specified by these genes have very large repetitive domains which are composed of repeats regularly distributed along the domain. The main difference between these two proteins is an insertion of 13 amino acids within the repetitive domain which, by itself, seems insufficient to explain the qualitative differences between LMW-2 and LMW-1. These results further support the hypothesis that the greater amount of LMW-2, rather than sequence peculiarities, accounts for the better quality observed in durum wheat cultivars possessing these subunits. The characterization of the complete primary structure of these alleles, other than providing information for an understanding of the structure-function relationship among LMW-GSs and furnishing basic material for wheat engineering, should also assist in our understanding of the evolutionary relationship between the different lmw-gs genes. Received: 8 May 1998 / Accepted: 5 August 1998