Protein Mobilization and Proteolytic Enzyme Activities (original) (raw)
Related papers
Zeitschrift für Naturforschung C, 2006
The protein mobilization from attached and detached seeds of Vicia faba L. cv. Eresen 87 (Fabaceae) was investigated. While the total soluble protein content decreased, the free amino acid content increased during the 7 days germination period. Among the three proteolytic enzymes, only endopeptidase activity was found to be affected by the removal of the embryonic axis. Leucine aminopeptidase activity was high at the beginning, then it decreased; carboxypeptidase activity reached the highest value at day 5. In order to examine the effects of plant growth regulators on detached cotyledons incubated with plant growth regulators [10 Ð4 m benzyladenine (BA), gibberellic acid (GA 3 ), indole acetic acid (IAA) and 10 Ð5 m abscisic acid (ABA)], only benzyladenine was found promotive on protein mobilization. Our results suggest that the removal of the embryonic axis in seeds of Vicia faba L. cv. Eresen 87 decreases protein mobilization and endopeptidase activity.
Plant Molecular Biology, 2000
Families of papain- and legumain-like cysteine proteinases (CPR) were found in Vicia seeds. cDNAs and antibodies were used to follow organ specificity and the developmental course of CPR-specific mRNAs and polypeptides. Four papain-like cysteine proteinases (CPR1, CPR2, proteinase A and CPR4) from vetch seeds (Vicia sativa L.) were analysed. CPR2 and its mRNA were already found in dry embryonic axes. CPR1 was only detected there during early germination. Both CPR1 and CPR2 strongly increased later during germination. In cotyledons, both CPR1 and CPR2 were only observed one to two days later than in the axis. Proteinase A was not found in axes. In cotyledons it could only be detected several days after seeds had germinated. CPR4 mRNA and polypeptide were already present in embryonic axes and cotyledons during seed maturation and decreased in both organs during germination. Purified CPR1, CPR2 and proteinase A exhibited partially different patterns of globulin degradation products in vitro. Although the cDNA-deduced amino acid sequence of the precursor of proteinase A has an N-terminal signal peptide, the enzyme was not found in vacuoles whereas the other papain-like CPRs showed vacuolar localization. Four different legumain-like cysteine proteinases (VsPB2, proteinase B, VnPB1 and VnPB2) of Vicia species were analysed. Proteinase B and VnPB1 mRNAs were detected in cotyledons and seedling organs after seeds had germinated. Proteinase B degraded globulins isolated from mature vetch seeds in vitro. VsPB2 and proteinase B are localized to protein bodies of maturing seeds and seedlings, respectively, of V. sativa. Like VsPB2 from V. sativa, also VnPB2 of V. narbonensis corresponds to vacuolar processing enzymes (βVPE). Based on these results different functions in molecular maturation and mobilization of storage proteins could be attributed to the various members of the CPR families.
Brazilian Archives of …, 1999
In this work, a woody species [A. peregrina (L.) Speg.] was studied in order to observe the effect of ABA and GA 3 at the biochemical level during the process of seed germination. Embryos incubated in sucrose solution containing ABA and/or GA 3 were analyzed through SDS-PAGE to observe the mobilization pattern of storage proteins during the beginning of germination. Cotyledons isolated from seeds incubated in aqueous solutions containing ABA and/or GA 3 , were also analyzed through SDS-PAGE and by PAGE/Activity Gels (polyacrylamide gels copolymerized with substrate for enzymes) to observe the mobilization pattern of storage proteins and protease activity after the beginning of the germination. Results of these experiments show that ABA blocks protein mobilization by inhibiting protease activity in cotyledons. This inhibition is not sufficient to prevent germination showing that the effect of ABA on germination is not dependent on protease activity. The blockage of storage protein mobilization was also observed in embryos, but no protease activity inhibition was clearly detected. ABA was able to induce the synthesis of proteins in cotyledons but not in embryos. A polypeptide with an approximate molecular weight of 17 kD, was degraded within 6 hours in control embryos, but this degradation was blocked by ABA and GA 3 . Using the same concentrations of ABA and GA 3 on embryos and cotyledons, the effect of ABA was counteracted by GA 3 in embryos, but not in cotyledons. Although the effects of ABA and GA 3 were not so different from those shown in the literature, the behavior of 17 kD-polypeptide contradicts these reports suggesting that specific studies should be performed.
American Journal of Plant Sciences, 2018
Proteolysis of seed storage proteins (SSP) during germination provides a steady supply of amino acids to the embryo development into seedling. This process is coordinated by different peptidases that act sequentially and overlaid mode. These enzymes are an ancient group evolved separately in a wide structural and functional diversity and have many applications in medicine, pharmacy and industry. However, the knowledge about seed peptidases during germination was obtained from studies almost restricted to the cultivated species. This restriction implies caution about generalizations made from these studies, as well limits the biological knowledge about plant kingdom and technological use from plant peptidases. In this work, a scan of the proteolytic activity was held in germinating seeds of a leguminous subtropical woody tree. Eleven proteolytic activities were detected in protein extracts from embryonic axis and cotyledons. The presence and intensity of these activities varied over time and between these tissues. There was indication that aspartyl-endopeptidases (phytepsins) and cysteine-carboxypeptidases (plant cathepsins) were involved in A. colubrina SSP hydrolysis. These peptidases differ to that commonly involved in germination of the cultivated leguminous. In addition, one of detected phytepsins showed stability on pH scale, which is important for industrial uses. There was also detected a metallo-carboxypeptidase activity, which has been not described in plants. These peptidases must be isolated to confirm or not these indications. However, these data indicate the biological and technological importance of extending the studies about plant peptidases on a diverse genetic basis.
Regulation of reserve protein metabolism in the cotyledons of mung bean seedlings
Proceedings of the National Academy of Sciences, 1976
Seedling growth in mung beans ( Phaseolus aureus , Roxb.) is accompanied by the metabolism of the reserve proteins, and the appearance in the cotyledons of a proteolytic enzyme with endopeptidase activity. Enzyme activity increases 25-fold during the first 5 days of growth. Cotyledon extracts prepared from seeds imbibed for 24 hr with water do not react with rabbit endopeptidase antiserum, which suggests that the enzyme is not present in the seeds as a zymogen. Labeling experiments show that the enzyme is synthesized in the course of seedling growth. The endopeptidase is localized in the protein bodies, and the specific activity of the enzyme in these organelles increases 30-fold. Ultrastructural studies show that the rough endoplasmic reticulum proliferates and may give rise to vesicles which fuse with the protein bodies prior to reserve protein digestion. These vesicles could be the primary lysosomes which transport the enzyme from its site of synthesis to its site of action.
Planta, 2011
In the present manuscript, we report on the proteolytic enzymes acting in the Araucaria bidwillii megagametophyte throughout seed germination. At seed maturity the megagametophyte contains a bulk of reserves for the growing embryo, thus representing the major storage tissue of the bunya pine seed. Soon after seed germination the megagametophyte undergoes storage protein mobilization, degenerating as a no longer needed tissue by the late germinative stages. By using in-solution and in-gel assays, and mass spectrometric analyses we detected exopeptidases and proteinases differently active in this tissue at selected germinative stages, and obtained preliminary data on the nature of an endopeptidase active at the late stages. Early germination stages were characterized by aminopeptidase and aspartic, metallo and cysteine proteinase activities; carboxypeptidases and serine proteinases became highly active by the late stages. Partial sequencing of a protein responsible for late stage serine peptidase activity sensitive to the caspase-6 inhibitor, showed a set of amino acid sequences with various degrees of identity with various plant subtilisin-like serine proteinases. The participation of the early stage proteases in the storage protein mobilization and the involvement of the late stage proteases in the megagametophyte cell death are proposed and discussed.
European Journal of Biochemistry, 1995
Proteinase B, an asparagine-specific endopeptidase, has been purified from germinating vetch (Vicia sativa L.) seeds. The final preparation consists of two enzymically active proteins with molecular masses of approximately 39 kDa and 37 kDa. Synthetic substrates were used to confirm cleavage specificity of the proteinase B preparation. As expected, the enzyme cleaves the substrates at the C-terminal side of Asn residues. The octapaptide ETRNGVEE was digested most efficiently. When Gly was replaced by Ile or Glu, cleavage took place with lower efficiency. Polyclonal antibodies displayed both proteins in cotyledon extracts of germinated vetch seeds. In addition, a strong cross-reacting protein band was found in cotyledon extracts of developing seeds, indicating the presence of a very similar enzyme during seed development.
Journal of Plant Physiology, 2008
Cynara cardunculus L. seeds were germinated in vitro under environmentally controlled conditions. Seeds showed a 60% germination rate, and three growth stages were established based on the seedling mean relative growth rate (RGR). Root, stem and cotyledons were compared in these stages with respect to the emergence of total proteases and cardosin activity and its allocation in the seedling. In growth stage I (1st-5th post-germinative days), seedlings grew very slowly. Total proteases and cardosins were already active at the onset of seedlings in the stem. Total soluble protein remained constant in cardoon seedlings during stage I, and the content of all free amino acids (aa) but proline (Pro) was equally allocated on the 1st post-germinative day. In growth stage II (5th-10th post-germinative days), seedlings grew intensively and exhibited fully developed cotyledons. A pronounced increase in the content of all free aa up to the middle of growth stage II in both stems and roots was observed. In addition, the allocation of the total proteolytic activity and cardosins followed a gradient from the root to the seedling shoot. However, the whole seedling soluble protein remained constant up to the 7th day in and tended to peak on the 10th post-germinative day, being allocated mainly to the seedling stem. In growth stage III (10th-15th post-germinative days), cardoon seedlings exhibited the lowest mean RGR and the highest R/S growth ratio. An intensive degradation of total soluble protein present in the whole seedling except for cotyledons (ca. 5-fold) was observed. Nevertheless, in growth stage III, both the gradients exhibited by total proteases and cardosins activities between the root and the seedling shoot were ARTICLE IN PRESS www.elsevier.de/jplph 0176-1617/$ -see front matter
Enzyme expression in soybean cotyledon, callus, and cell suspension culture
Canadian Journal of Botany-revue Canadienne De Botanique, 1979
MATTHEWS. B. F., iind J . M. WIDHOLM. 1979. Enzymeexpression in soybean cotyledon, callus, and cell suspension culture. Can. J . Bot. 57: 299-304. Feedback control of enzymes in the pathway for the biosynthesis of aspartate-klmily amino acids was examined using Glycine i?rrr.r callus, suspension cultures. and seedlings to determine if the controls are different in the three systems. Aspartate kinase activity (EC 2.7.2.4)derived from soybean cell suspension cultures is 5% inhibited by I.0mM L-threonine and 5Wh inhibited by I .OmM L-lysine. In contrast, aspartate kinase activities fr-om 3-day-old seedling cotyledons and from callus cultures grown from cell suspension cultures are inhibited approximately 7% by