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Choun-Sea Lin, Nien-Tzu Liu, De-Chih Liao, Jau-Song Yu, Chuang-Hwei Tsao, Chao-Hsiung Lin, Chih-Wen Sun, Wann-Neng Jane, Hsing Sheng Tsay, Jeremy Jian-Wei Chen, Erh-Min Lai, Na-Sheng Lin, Wei-Chin Chang, and Chung-Chih Lin

under light conditions and resolved by two-dimensional-gel electrophoresis. About 250 protein spots were detected on two-dimensional-gels within the pH range of 4 to 7 and a MW range of 17 to 76 kDa ( Fig. 3 ). Three protein spots, with pI values and MW

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Ben-Hong Wu, Ning Niu, Ji-Hu Li, and Shao-Hua Li

may be regulated by this ratio. The aim of the present study was to assess the effect of different LF treatments on protein variation in grape berry skin using two-dimensional gel electrophoresis (2-DE). Materials and Methods Plant materials and

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Akiko Watari, Toshio Hanada, Hisayo Yamane, Tomoya Esumi, Ryutaro Tao, Hideaki Yaegaki, Masami Yamaguchi, Kenji Beppu, and Ikuo Kataoka

cDNA quantities of S-RNase and SFB were normalized using the ACTIN cDNA values and compared as relative amounts of their transcripts. Two-dimensional polyacrylamide gel electrophoresis analysis of pistil proteins. Acetone powders were

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Ren-jun Feng, Li-li Zhang, Jing-yi Wang, Jin-mei Luo, Ming Peng, Jun-feng Qi, Yin-don Zhang, and Li-fang Lu

Corporation, Kyoto, Japan). Two-dimensional gel electrophoresis (2-DE). The 2-DE was carried out as previously described ( Zhang et al., 2012 ). Protein concentration was normalized to 1 g·L −1 in rehydration buffer [8 mol·L −1 urea, 4% (w/v) CHAPS, 10% (v

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Carole L. Bassett

Levels of the large subunit (LSU) of rubisco (ribulose 1,5 bisphosphate carboxylase/oxygenase) were measured in vegetative and floral organs of `Violet' Japanese morning glory (Ipomoea [Pharbitis] nil Roth). Identification of the LSU in polypeptides separated by two-dimensional gel electrophoresis allowed estimation of the relative abundance of this polypeptide in the organs examined. Further quantitation was achieved by immunoblotting protein extracts either alone or in combination with various amounts of extracts from other organs. The amount of LSU decreased in the order leaves > cotyledons > sepals > corollas > androecium, gynoecium. The relative abundance of LSU in sepals (74%) compared to photosynthetically competent organs [leaves (100%) and cotyledons (81%)] suggests that sepals may be photosynthetically competent in supporting development of the other floral organs.

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Ellen T. Paparozzi, Joshua R. Widhalm, and M. Elizabeth Conley

Common swedish ivy plants when exposed to nitrogen (N) stress display typical nitrogen deficiency symptoms such as reddening of stems and petioles and yellowing of leaves. When N levels are restored, leaves of swedish ivy plants will re-green without leaf loss. An experiment was conducted to determine how proteins change when leaves were re-greened after N deficiency. Cuttings of Plectranthus australis were rooted under mist and allowed to yellow. Plants were then potted up and fertilized with one of two treatments: complete nutrients with N at 150 ppm or complete nutrients with 0.8 ppm N. The experimental design was a randomized complete-block design with six blocks. Each block had the two N treatments and six plants per treatment. After 3–4 weeks, all plants in the 150-ppm N treatment had re-greened and leaf samples for protein analysis were taken. Plants in four of the six blocks were then switched to the other treatment. After leaves had re-greened once again, leaf samples were taken and the experiment was terminated. Two-dimensional polyacrylamide gel electrophoresis was used to compare the treatments. No obvious differences in protein absence or presence were noted. However, Rubisco appeared to be differentially expressed between the two treatments. 2-D gel analysis with subsequent Western blots showed that for most of the leaf samples, the large subunit of Rubisco (56kD) was quantitatively about 1.3 times more concentrated in the N-deficient plants and possibly modified. The small subunit (12kD) was not reliably detectable. Additional protein results for repeated leaf re-greening and the role Rubsico may play in leaf re-greening will be discussed.

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Stephen P. Lee, Paul M. Chen, Tony H.H. Chen, Diane M. Varga, and Eugene A. Mielke

A proportion of `d'Anjou' pear fruit (Pyrus communis L.) developed a disorder, “black speck” or “skin speckling”, after prolonged controlled atmosphere (CA) storage (1% O2, - 0.5 C). A comparative study of biochemical components revealed that there was no significant difference in succinic, citric, fumaric, and pyruvic acids between the speckled' and normal skin tissues. The content of malic acid in the affected tissue was almost three times lower than that in the normal tissue. The specific activity of NADP-malic enzyme (EC 1.1.1.40) in the affected tissue was also lower, but the total activities were similar. The affected tissue contained higher percentages of dry matter and soluble proteins than the normal tissue. Two-dimensional gel electrophoresis of proteins showed that two groups of novel polypeptides appeared only in the affected skin tissue. This study indicated that a certain proportion of `d'Anjou' pear fruit might have been exposed to unfavorable preharvest environmental stresses, and, therefore, could no longer tolerate the subsequent semi-anaerobic and chilling stresses during prolonged CA storage.

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Gregory A. Lang and Joshua Tao

Plant dormancy research has long been stifled by the lack of appropriate biochemical markers to characterize the changing physiological status of dormant vegetative or reproductive buds. Two sets of experiments were conducted in an attempt to identify changes in soluble protein profiles during endodormancy of peach and blueberry reproductive apices. Bud samples from the peach cultivars `La Festival' (low chilling requirement) and `La White' (moderate chilling requirement) were taken every 15 days in the orchard during December and January, extracted for soluble proteins, and analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Outshoots were forced at 25C in a growth chamber to determine the intensity of endodormancy. A further experiment utilized potted `Bluechip' and `Meader' (troth high chilling requirement) blueberry plants given varying periods of cold (4.5C) chamber treatment, followed by forcing at 25C in a growth chamber. Bud samples were taken following cold treatment for extraction and SDS-PAGE. The relationship of the resulting protein profiles to chilling unit accumulation and intensity of endodormancy will be discussed.

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Ryutaro Tao, Hisayo Yamane, Akira Sugiura, Hideki Murayama, Hidenori Sassa, and Hitoshi Mori

This report identifies S-RNases of sweet cherry (Prunus avium L.) and presents information about cDNA sequences encoding the S-RNases, which leads to the development of a molecular typing system for S-alleles in this fruit tree species. Stylar proteins of sweet cherry were surveyed by two dimensional polyaclylamide gel electrophoresis (2D-PAGE) to identify S-proteins associated with gametophytic self-incompatibility. Glycoprotein spots linked to S-alleles were found in a group of proteins which had Mr and pI similar to those of other rosaceous S-RNases. These glycoproteins were present at highest concentration in the upper segment of the mature style and shared immunological characteristics and N-terminal sequences with those of S-RNases of other plant species. cDNAs encoding these glycoproteins were cloned based on the N-terminal sequences. Genomic DNA and RNA blot analyses and deduced amino acid sequences indicated that the cDNAs encode S-RNases; thus the S-proteins identified by 2D-PAGE are S-RNases. Although S1 to S6-alleles of sweet cherry cultivars could be distinguished from each other with the genomic DNA blot analysis, a much simpler method of PCR-based typing system was developed for the six S-alleles based on the DNA sequence data obtained from the cDNAs encoding S-RNases.

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Hisayo Yamane, Ryutaro Tao, Akira Sugiura, Nathanael R. Hauck, and Amy F. Iezzoni

This report demonstrates the presence of S-ribonucleases (S-RNases), which are associated with gametophytic self-incompatibility (SI) in Prunus L., in styles of self-incompatible and self-compatible (SC) selections of tetraploid sour cherry (Prunus cerasus L.). Based on self-pollen tube growth in the styles of 13 sour cherry selections, seven selections were SC, while six selections were SI. In the SI selections, the swelling of pollen tube tips, which is typical of SI pollen tube growth in gametophytic SI, was observed. Stylar extracts of these selections were evaluated by two-dimensional polyacrylamide gel electrophoresis. Glycoproteins which had molecular weights and isoelectric points similar to those of S-RNases in other Prunus sp. were detected in all selections tested. These proteins had immunological characteristics and N-terminal amino acid sequences consistent with the S-RNases in other Prunus sp. Two cDNAs encoding glycoproteins from `Erdi Botermo' were cloned. One of them had the same nucleotide sequence as that of S4-RNase of sweet cherry (Prunus avium L.), while the amino acid sequence from the other cDNA encoded a novel S-RNase (named Sa-RNase in this study). This novel RNase contained two active sites of T2/S type RNases and five regions conserved among other Prunus S-RNases. Genomic DNA blot analysis using cDNAs encoding S-RNases of sweet cherry as probes indicated that three or four S-RNase alleles are present in the genome of each selection regardless of SI. All of the selections tested seemed to have at least one S-allele that is also found in sweet cherry. Genetic control of SI/SC in tetraploid sour cherry is discussed based on the results obtained from restriction fragment length polymorphism analysis.