Sequencing of Protein from a Single Spot of a 2-D Gel Pattern: N-Terminal Sequence of a Major Wheat LMW-Glutenin Subunit

Kasarda, Donald D.; Tao, Hongwen; Evans, P. K.; Adalsteins, A. Elva; Yuen, Sylvia

doi:10.1093/jxb/39.7.899

Cited by 42 publications

(12 citation statements)

References 12 publications

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“…The blots were stained with Amido black ( 13), and pieces of blots containing the pl 6.3 and 6.5 polypeptides were excised for sequencing. Sequence analysis was performed using an Applied Biosystems model 477A Pulsed Liquid Phase amino acid sequencer equipped with a model 120A on-line phenylthiohydantoin amino acid analyzer as described previously (17,32 Antibodies to wheat germin were a generous gift from Dr. Byron Lane, University of Toronto.…”

Section: Amino Terminal Sequencingmentioning

confidence: 99%

Germin-Like Polypeptides Increase in Barley Roots during Salt Stress

Hurkman

Tao²,

Tanaka³

1991

Plant Physiol.

104

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The 26 kilodalton, isoelectric point 6.3 and 6.5 (Gsl and Gs2) polypeptides that increase in barley (Hordeum vulgare L.) roots during salt stress were isolated and identified. Both Gsl and Gs2 had high sequence similarity to germin, a protein that increases significantly in germinating wheat seeds. Like germin, Gsl and Gs2 were resistant to proteases and were glycosylated. lmmunoblots were probed with antibodies to Gs1 and Gs2 to determine the distribution of these polypeptides among organs and cell-free fractions. Gsl and Gs2 were present in roots and coleoptiles, but absent from leaves. In roots, Gsl and Gs2 were present in the mature region, but not the tip. Gsl and Gs2 increased in roots, but decreased in coleoptiles in response to salt stress. Gsl and Gs2 were distributed among the soluble, microsomal, and cell wall fractions of roots, but the majority of Gsl and Gs2 was present in the soluble fraction. Although Gsl and Gs2 were heat stable, their synthesis was not affected by abscisic acid treatment. Gs2 accumulated during abscisic acid treatment, whereas Gsl did not. However, a 25.5 kilodalton, isoelectric point 6.1 polypeptide that was immunologically related to Gsl did accumulate with abscisic acid treatment.Salt stress alters the levels of a number of polypeptides and translatable mRNAs in barley roots (10, 1 1, 13-15). The most obvious changes in protein synthesis are increases in polypeptides with molecular masses of 26 and 27 kD and pIs2 of 6.3 and 6.5 (10,11,13,14). Evidence from studies on length of NaCl treatment, concentration of NaCl, recovery from salt stress, effect of other stresses, and analysis of different cvs indicates that these polypeptides have a specific role in barley plants during salt stress. The increase in the synthesis of the pl 6.3 and 6.5 polypeptides occurs within a short time after exposure of plants to salt (1 1, 27) and at NaCl concentrations as low as 50 mM (13). The synthesis of the pI 6.3 and 6.5 polypeptides decreases when salt-treated seedlings are transferred to nutrient solution without NaCl (13). The increase in the synthesis of the pl 6.3 and 6.5 polypeptides is a specific response to salt stress; synthesis does not increase in response to water deficit (14) or heat shock (10,13 synthesis of these polypeptides increases in CM 72, a salttolerant cv, but not in Prato, a salt-sensitive cv (1 1).The pl 6.3 and 6.5 polypeptides were isolated using preparative two dimensional gels to obtain clues to their identities and functions. N-terminal amino acid sequences were determined for the pl 6.3 and 6.5 polypeptides, and sequence comparisons were used to identify these polypeptides. Antibodies were raised against the pl 6.3 and 6.5 polypeptides and used to probe blots of polyacrylamide gels to determine their distribution among barley organs and cell-free fractions. Because the levels of osmotin (28, 31), rab2 1 (24), and salT (1) respond to salt stress and ABA, the effect of ABA on the levels of the pl 6.3 and 6.5 polypeptides was also examined. MATERIALS AND ...

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Section: Amino Terminal Sequencingmentioning

confidence: 99%

Germin-Like Polypeptides Increase in Barley Roots during Salt Stress

Hurkman

Tao²,

Tanaka³

1991

Plant Physiol.

104

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“…Due to the difficulties in separating individual gluten proteins by chromatographic methods [4], quantitative two-dimensional gel electrophoresis (2-DE) has become a method of choice for identifying proteins that respond to environmental cues. To date, a number of studies have used 2-DE to discover gluten protein responses to high temperature, fertilizer, or drought [5-11] and have reported changes in as few as three [8] to as many as 107 2-DE spots [11].…”

Section: Introductionmentioning

confidence: 99%

Comparative proteomic analysis of the effect of temperature and fertilizer on gliadin and glutenin accumulation in the developing endosperm and flour from Triticum aestivum L. cv. Butte 86

et al. 2013

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BackgroundFlour quality is largely determined by the gluten proteins, a complex mixture of proteins consisting of high molecular weight-glutenin subunits (HMW-GS), low molecular weight-glutenin subunits (LMW-GS), and α-, γ-, and ω-gliadins. Detailed proteomic analyses of the effects of fertilizer and high temperature on individual gliadin and glutenin protein levels are needed to determine how these environmental factors influence flour quality.ResultsWheat plants (Triticum aestivum L. cv. Butte 86) were grown in greenhouses under moderate and high temperature regimens with and without post-anthesis fertilizer. Quantitative two-dimensional gel electrophoresis was used to construct accumulation profiles in developing endosperm for the entire complement of gluten proteins identified previously by tandem mass spectrometry. Amounts of individual gliadins and glutenins were also determined in flour produced under each of the regimens. Under all environmental regimens, most HMW-GS, LMW-GS, γ- and ω-gliadins accumulated rapidly during early stages of grain development and leveled off during middle stages of development. A subset of LMW-GS showed a second distinct profile, accumulating throughout development, while α-gliadins showed a variety of accumulation profiles. In flour, fourteen distinct gluten proteins responded similarly to fertilizer, high temperature, and high temperature plus fertilizer. The majority of HMW-GS and ω-gliadins and some α-gliadins increased while two LMW-GS and a minor γ-gliadin decreased. Fertilizer did not influence gluten protein accumulation under high temperature conditions. Additionally, the effects of fertilizer and high temperature were not additive; very few changes were observed when plants that received fertilizer were subjected to high temperature.ConclusionsAlthough post-anthesis temperature and fertilizer have very different effects on grain development and yield, the two treatments elicit surprisingly similar effects on the accumulation of gluten proteins. The similarity of the responses to the different treatments is likely due to source-sink activities of nitrogen reserves in the wheat plant. Because each protein that showed a response in this study is linked to a gene sequence, the work sets the stage for transgenic studies that will better elucidate the roles of specific proteins in flour quality and in the response to the environment.

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“…On the basis of N-terminal amino acid sequences, they can be divided into LMW-m, LMW-s, and LMW-i types (Kasarda et al 1988;Tao and Kasarda 1989;Lew et al 1992;Cloutier et al 2001). The LMW-s type have been found to be the predominant type by N-terminal sequencing of proteins and belongs to the B group of LMW glutenins (Lew et al 1992;Masci et al 2002).…”

Section: Introductionmentioning

confidence: 99%

Characterization of low-molecular-weight glutenin genes in Aegilops tauschii

et al. 2004

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This paper reports the characterization of the low-molecular-weight (LMW) glutenin gene family of Aegilops tauschii (syn. Triticum tauschii), the D-genome donor of hexaploid wheat. By analysis of bacterial artificial chromosome (BAC) clones positive for hybridization with an LMW glutenin probe, seven unique LMW glutenin genes were identified. These genes were sequenced, including their untranslated 3' and 5' flanking regions. The deduced amino acid sequences of the genes revealed four putative active genes and three pseudogenes. All these genes had a very high level of similarity to LMW glutenins characterized in hexaploid wheat. The predicted molecular weights of the mature proteins were between 32.2 kDa and 39.6 kDa, and the predicted isoelectric points of the proteins were between 7.53 and 8.06. All the deduced proteins were of the LMW-m type. The organization of the seven LMW glutenin genes appears to be interspersed over at least several hundred kilo base pairs, as indicated by the presence of only one gene or pseudogene per BAC clone. Southern blot analysis of genomic DNA of Ae. tauschii and the BAC clones containing the seven LMW glutenin genes indicated that the BAC clones contained all LMW glutenin-hybridizing bands present in the genome. Two-dimensional gel electrophoresis of an LMW glutenin extract from Ae. tauschii was conducted and showed the presence of at least 11 distinct proteins. Further analysis indicated that some of the observed proteins were modified gliadins. These results suggest that the actual number of typical LMW glutenins may in fact be much lower than previously thought, with a number of modified gliadins also being present in the polymeric fraction.

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Sequencing of Protein from a Single Spot of a 2-D Gel Pattern: N-Terminal Sequence of a Major Wheat LMW-Glutenin Subunit

Cited by 42 publications

References 12 publications

Germin-Like Polypeptides Increase in Barley Roots during Salt Stress

Germin-Like Polypeptides Increase in Barley Roots during Salt Stress

Comparative proteomic analysis of the effect of temperature and fertilizer on gliadin and glutenin accumulation in the developing endosperm and flour from Triticum aestivum L. cv. Butte 86

Characterization of low-molecular-weight glutenin genes in Aegilops tauschii

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