Control of storage-protein synthesis during seed development in pea (Pisum sativum L.)
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Abstract:
The tissue-specific syntheses of seed storage proteins in the cotyledons of developing pea (Pisum sativum L.) seeds have been demonstrated by estimates of their qualitative and quantitative accumulation by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and rocket immunoelectrophoresis respectively. Vicilin-fraction proteins initially accumulated faster than legumin, but whereas legumin was accumulated throughout development, different components of the vicilin fraction had their predominant periods of synthesis at different stages of development. The translation products in vitro of polysomes isolated from cotyledons at different stages of development reflected the synthesis in vivo of storage-protein polypeptides at corresponding times. The levels of storage-protein mRNA species during development were estimated by ‘Northern’ hybridization using cloned complementary-DNA probes. This technique showed that the levels of legumin and vicilin (47000-Mr precursors) mRNA species increased and decreased in agreement with estimated rates of synthesis of the respective polypeptides. The relative amounts of these messages, estimated by kinetic hybridization were also consistent. Legumin mRNA was present in leaf poly(A)+ RNA at less than one-thousandth of the level in cotyledon poly(A)+ (polyadenylated) RNA, demonstrating tissue-specific expression. Evidence is presented that storage-protein mRNA species are relatively long-lived, and it is suggested that storage-protein synthesis is regulated primarily at the transcriptional level.Keywords:
Legumin
Vicilin
Polysome
Legumin
Vicilin
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The two storage-protein fractions of pea seeds, legumin and vicilin, have each been resolved electrophoretically on a cellulose acetate gel matrix into multiple molecular species distinguished by electrophoretic mobility and by quantitative or qualitative differences in subunit composition or both. Electrophoretograms of these apparent holoproteins from a range of lines and cultivars were found to be genotype-specific, generally showing three strong bands, together with up to three additional minor bands, assignable to a vicilin series of components. A further three or four bands could be assigned to a legumin series, although the slowest of these showed apparent admixture of certain polypeptides typical of the vicilin fractions. Each putative holoprotein band in both the legumin and vicilin series behaved additively in the electrophoretograms of F1 offspring from reciprocal crosses between lines showing distinct patterns. For comparison with the proteins distinguished electrophoretically on cellulose acetate gels, storage proteins from lyophilized protein bodies were fractionated on the basis of differential solubility at various ionic strengths and pH values. The single legumin and three vicilin fractions obtained by this method showed sedimentation velocities typical of the respective holoproteins. No overlap in the polypeptide composition of legumin with that of vicilin fractions was observed. The components represented in the four fractions accounted for all the major polypeptides in total storage-protein extracts and in the bands eluted from cellulose acetate gels. The distinctive polypeptide pattern and electrophoretic mobility of vicilin fraction 4 identified this protein as a contaminant of slow legumin bands in cellulose acetate gels, and as an additional vicilin species not recognized directly from the electrophoretic analysis.
Vicilin
Legumin
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Legumin
Vicilin
Immunogold labelling
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Legumin
Vicilin
Cotyledon
Transcription
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Gel electrophoresis has been used to investigate genetically controlled variation in storage-protein constituents forming five series of bands (LA-LE) derived from legumin fractions, and three series of bands (VA-VC) from vicilin fractions, of pea seeds. In each variant system, the phenotypes of the storage-protein polypeptides from F1 seeds were additive with respect to the band patterns of the parental lines, and identical in reciprocal crosses. Neither dominance nor formation of new interaction products was observed. Variation in the three systems involving vicilin polypeptides and two of those involving legumin components was found to be based on allelic alternatives at single loci designated Vicilin A (Vca), Vicilin B (Vcb), Vicilin C (Vcc), Legumin A (Lga) and Legumin C (Lgc). For each of these variant systems, the gene products involved and the basis of the phenotypic variation have been discussed. Variants of the VC band complex, in which mobility of two bands both composed of 12 and 14 kdalton polypeptides is altered, appear likely to correspond to vicilin variants described previously. Type lines are specified for each of the variant phenotypes analysed, and for the genes designated.
Vicilin
Legumin
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Suspensor
Vicilin
Legumin
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Vicilin
Legumin
Epicotyl
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The tissue-specific syntheses of seed storage proteins in the cotyledons of developing pea (Pisum sativum L.) seeds have been demonstrated by estimates of their qualitative and quantitative accumulation by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and rocket immunoelectrophoresis respectively. Vicilin-fraction proteins initially accumulated faster than legumin, but whereas legumin was accumulated throughout development, different components of the vicilin fraction had their predominant periods of synthesis at different stages of development. The translation products in vitro of polysomes isolated from cotyledons at different stages of development reflected the synthesis in vivo of storage-protein polypeptides at corresponding times. The levels of storage-protein mRNA species during development were estimated by ‘Northern’ hybridization using cloned complementary-DNA probes. This technique showed that the levels of legumin and vicilin (47000-Mr precursors) mRNA species increased and decreased in agreement with estimated rates of synthesis of the respective polypeptides. The relative amounts of these messages, estimated by kinetic hybridization were also consistent. Legumin mRNA was present in leaf poly(A)+ RNA at less than one-thousandth of the level in cotyledon poly(A)+ (polyadenylated) RNA, demonstrating tissue-specific expression. Evidence is presented that storage-protein mRNA species are relatively long-lived, and it is suggested that storage-protein synthesis is regulated primarily at the transcriptional level.
Legumin
Vicilin
Polysome
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An 8S storage globulin from buckwheat seed, which resembles the structure and features common to the vicilin-like family of seed storage proteins, was analyzed for this paper. It was found that expression of the 8S globulin gene precedes that of the 13S globulin (the main buckwheat storage protein) and starts from an early stage of buckwheat seed development (9−11 days after flowering), continuing to accumulate throughout seed development to contribute ∼7% of total seed proteins. This protein fraction might be more interesting for biotechnological application than the 13S buckwheat legumin consisting of 23−25 kDa subunits reported to be the major buckwheat allergen. A partial cDNA was also isolated, showing high homology with cDNAs coding for vicilin-like storage proteins from various plant species, and its expression profile throughout seed development as well as in different buckwheat tissues was analyzed. Keywords: Buckwheat; Fagopyrum esculentum; storage proteins; vicilin-like; biosynthesis; expression
Vicilin
Legumin
Fagopyrum
Glutelin
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