Pulse-labeling Studies on Protein Synthesis in Developing Pea Seeds and Evidence of a Precursor Form of Legumin Small Subunit (original) (raw)
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The Journal of Cell Biology, 1982
Cotyledons of developing pea seeds (Pisurn sativum L.) were labeled with radioactive amino acids and glucosamine, and extracts were prepared and separated into fractions rich in endoplasmic reticulum (ER) or protein bodies . The time-course of synthesis of the polypeptides of legumin and vicilin and the site of their assembly into protein oligomers were studied using immunoaffinity gels and sucrose density gradients. When cotyledons were pulselabeled (1-2 h), newly synthesized legumin was present in polypeptides with Mr 60,000-65,000, and newly synthesized vicilin was present as a series of polypeptides with Mr 75,000, 70,000, 50,000, and 49,000. These radioactive polypeptides were found primarily in the ER (Chrispeels et al ., 1982, /. Cell BioL, 93 :5-14) . During a subsequent chase period, newly synthesized reserve proteins were initially present in the protein bodies in the above-named polypeptides . Between 1 and 20 h later, radioactive legumin subunits (M r 40,000 and 19,000) and smaller vicilin polypeptides (Mr 34,000, 30,000, 25,000, 18,000, 14,000, 13,000, and 12,000) appeared in the protein bodies . The appearance of these labeled polypeptides in the protein bodies was not the result of a slow transport from the ER (or cytoplasm) .
Control of storage protein accumulation during legume seed development
Journal of Plant Physiology, 2001
The regulation of partitioning of carbohydrate skeletons into different storage products of developing seeds is still not understood. We explored two ways to gain more insight in the process. First we analyzed mechanisms that control storage protein accumulation in Vicia faba seeds of contrasting protein content. As expected, the seeds of the high protein genotypes (HP) contained more protein and total nitrogen as compared to the low protein genotypes (LP) whereas starch and total amounts of carbon were not altered. There was no major difference in the proportion of amino acids delivered from seed coats into the endospermal cavity of either HP or LP genotypes. However, HP genotype cotyledons contained twofold higher levels of free amino acids at the later developmental stages when their higher protein content was expressed. After four hours of incubation, in vitro uptake rates of 14 C glutamine by HP cotyledons were significantly higher for the protein rich cotyledons indicating a possible higher capacity to take up amino acids. In both genotypes 14 C-glutamine was rapidly converted into glutamate and then partly decarboxylated to γ-amino butyric acid. However, in the HP cotyledons the ratio of 14 C-glutamine to 14 C-glutamate was higher as compared to the LP cotyledons reflecting the observed higher glutamine uptake rate. In a second approach we studied Vicia narbonensis seeds which expressed ADP glucose pyrophosphorylase in antisense orientation. These seeds contained less starch and more sucrose and water but also more protein. In addition, blocking the starch synthesis pathway caused pleiotropic effects on water content and induced temporal changes in seed development. The resulting longer seed fill duration period could partially explain the observed elevated protein content in the AGP-antisense seeds. Key words: assimilate partitioning-storage protein-amino acid metabolism-nutrient transportlegume seed development Abbreviations: AAP amino acid permeases.-AGP ADP-glucose pyrophosphorylase.-GABA γ-aminobutyric acid.-HP high protein.-LP low protein.-PEPC phosphoenolpyruvate carboxylase * This paper is more focussed than the oral presentation at the meeting since a more general review has been published recently (Wobus and Weber 1999).
Protein Deposition and Mobilization in Seeds
2006
Cereal and Leguminous plant’s seeds take a large place of human food consumption. The germinating seed depends on its reserve material till the photosynthetic system develops. Storage proteins deposited in the seed during the maturation and have a purpose to provide free amino acids to the growing seedling. The present knowledge on protein storage during the seed development and enzymes involved in mobilization of the storage proteins has been summarized.
Biochemie und Physiologie der Pflanzen, 1991
The legJ subfamily of genes in garden pea, Pisum sativum L., encodes "minor" legumin seed storage protein polypeptides. Data on the differential expression of the 3 genes (legJ,K,L) within this subfamily is reported. The expression of one gene (legJ) is specifically upregulated during the desiccation phase of cotyledon development, when other storage protein genes are downregulated. The complete sequence of a second gene in the subfamily, legK, shows that the failure to observe any expression ofthis gene is due to the mutation of its initiator ATG (methionine) codon to a GTG (valine) codon. The third gene in the subfamily, legL, shows maximal expression during the cotyledon expansion phase of seed development, Le. like other storage protein genes. Evidence for the use of alternative polyadenylation addition signal sequences in these genes is also presented.
Patterns of protein synthesis in dormant and growing vegetative buds of pea
Planta, 1988
Lateral buds on intact pea plants (Pisum sativum L. cv. Alaska) remain dormant until they are stimulated to develop by decapitating the terminal bud. Using two-dimensional gel electrophoresis, we have examined the protein content of terminal and lateral buds from intact plants and from plants at various times after decapitation. Silverstaining and in-vivo-labeling demonstrated very different sets of proteins. The level of expression of 18 stained and 25 labeled proteins was altered when growth was stimulated; this represents 3.4% and 9.1% of the total proteins detected by each method, respectively. Within 24 h of being stimulated, lateral buds doubled in length and their protein content was qualitatively nearly the same as that of terminal buds. Six hours after decapitation, before the onset of detectable growth, the overall pattern of protein synthesis in lateral buds was more like that of growing lateral buds or of terminal buds than that of dormant lateral buds. Direct application of N6-furfurylaminopurine (kinetin) to buds on intact plants stimulated their growth and resulted in the same pattern of protein synthesis as did decapitation. Inhibition of bud growth by addition of indole-3-acetic acid to the stumps of decapitated plants resulted in the synthesis of dormancy-related proteins. Lateral buds at all stages of development incorporated labeled amino acids at similar rates, indicating that metabolic activity is not a component of dormancy in these buds.
The changes of protein patterns during one week of germination of some legume seeds and roots
This study was carried out in order to evaluate some Egyptian legume seeds (Vicia faba, Cicer arietinum and Lupinus termes) as raw and germinated foods, as sources of plant proteins. The work was extended to study the changes of the seed and root proteins during 7 days of germination. The results are sum-marised as follows: (1) The proteins of ungerminated seeds were resolved on SDS-PAGE into a number of bands and their molecular weights were determined: chick-peas, 19 (8-78 kDa); faba beans, 20 (1 l-96 kDa) and lupins 19 (14-86 kDa). (2) The changes in the seed and root proteins during 7 days of germination of the V. faba, C. arietinum and L. termes showed the following: l
Journal of Experimental Botany, 1997
A fraction enriched in endoplasmic reticulum and Golgi membranes from developing cotyledons of Pisum sativum L. has proved to be a convenient source for the isolation of prolegumin, the precursor of the major 11S storage globulin of pea seeds. Two pro-proteins were isolated with molecular masses of 60 kDa and 75 kDa, respectively. A monoclonal antibody, designated 2B1, against prolegumin was raised using the in vitro immunization technique. This antibody recognizes the 60 kDa precursor polypeptide, but only the 20 kDa /7-subunit of mature legumin. Prolegumin, like the ^-subunit of the mature legumin, is a hydrophobic protein. After import into the protein storage vacuole, and after formation of the protein bodies trimeric 9S prolegumin assembles into 12S hexamers without prior processing of the precursor. Since prolegumin in vitro does not oligomerize into more than 9S trimers these results suggest that a protein-mediated assembly of 9S prolegumin trimers into 12S prolegumin hexamers probably occurs in the lumen of the protein storage vacuole. Prolegumin, but not mature legumin, binds very tightly to membranes. This property points to a possible way of identifying a putative prolegumin receptor.
cDNA and protein sequence of a major pea seed albumin (PA 2 : Mr?26 000)
Plant Molecular Biology, 1987
Pea albumin 2 (PA2 :Mr -26000) is a major component of the albumin fraction derived from aqueous salt extracts of pea seed. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and chromatography on DEAE-Sephacel resolve PA2 into two closely related components (PA2a and PA2b). A cDNA clone coding for one of these components has been sequenced and the deduced amino acid sequence compared with partial, chemically-determined sequences for cyanogen bromide peptides from both PA2 components. Complete amino acid sequences were obtained for the C-terminal peptides. The PA2 molecule of 230 amino acids contains four imperfect repeat sequences each of approximately 57 amino acids in length.
Proteins of cotyledons of mature horse chestnut seeds
Russian Journal of Plant Physiology, 2006
This is the first characterization of proteins from axial organs of recalcitrant horse chestnut seeds during deep dormancy, dormancy release, and germination. We demonstrated that, during the entire period of cold stratification, axial organs were enriched in easily soluble albumin-like proteins and almost devoid of globulins. About 80% of the total protein was found in the cytosol. Approximately one third of cytosolic proteins were heat-stable polypeptides, which were major components of total proteins. Heat-stable proteins comprised three groups of polypeptides with mol wts of 52-54, 24-25, and 6-12 kD with a predominance of low-molecular-weight proteins. The polypeptide patterns of heat-stable and thermolabile proteins differed strikingly. Heat-stable proteins accumulated in axes during the late seed maturation, comprising more than 30% of the total protein in axes of mature seeds. The polypeptide patterns of the total protein of axial organs and its particular fractions did not change in the course of seed dormancy and release. At early germination, the content of heat-stable proteins in axes decreased and their polypeptide pattern changed both in the cytosol and cell structures. We believe that at least some heat-stable proteins can function as storage proteins in the axes. Localization of storage proteins in the cells of axial organs and the role of heat-stable proteins in recalcitrant seeds are discussed.