In Vitro Stability and Inactivation of Peptide Hydrolases Extracted from Phaseolus vulgaris L

Endopeptidase activity against azocasein had a higher temperature optimum (50°C) in leaf extracts than in cotyledon extracts (37°C). The temperature optima for aminopeptidase (46°C) and for carboxypeptidase (53°C) were similar in leaf and cotyledon extracts. The endopeptidase activity showed an excellent stability in crude extracts from leaves even at 37°C, while the endopeptidase in cotyledon extracts was less stable. Carboxypeptidase was very stable in both leaf and cotyledon extracts. Aminopeptidase was the least stable of the enzymes investigated and its inactivation rate depended on the source of the extract. A moderate stability was observed in extracts of leaves or of ungerminated seeds, but this enzyme was rapidly inactivated in cotyledon extracts at pH 5.4. At pH 7.5 aminopeptidase remained active longer than at pH 5.4. From experiments with mixed extracts it could be concluded that in cotyledons an aminopeptidase inactivating factor was formed during germination. This factor was heat sensitive, excluded by Sephadex G-25, precipitated by 75% ammonium sulfate and inhibited by tosyl-L-lysine chloromethyl ketone. These data suggest that the factor is a protein and considering the similar properties it appears possible that it is the endopeptidase formed during germinatio

Effect of changed source/sink relations on proteolytic activities and on nitrogen mobilization in field-grown wheat (Triticum aestivum L.)

Nitrogen mobilization and the pattern of proteolytic enzymes were investigated in leaves and glumes of field-grown winter wheat (Triticum aestivum L.) during maturation. Source/sink relations were changed by removal of the ear, the flag leaf or the lower leaves shortly after anthesis. Removal of the ear was most effective, resulting in delayed senescence of the flag leaf with the chlorophyll, aminopeptidase and carboxypeptidase activities remaining high in contrast to the control, whereas neutral endopeptidase activity increased more slowly. No major changes were observed in the second leaf from the top in plants with either ears or flag leaves removed. Nitrogen mobilization and proteolytic activities in glumes and the remaining leaves were influenced only slightly by leaf removal. In earless plants, nitrogen was transported from the second leaf into the leaf sheath and stem, but in the flag leaf the total reduced nitrogen remained high and free amino groups increased. The increase in endopeptidase activity was influenced by the source/sink relations. However, the accumulation of amino groups and the increasing endopeptidase activity in the flag leaf of earless plants suggest that the nitrogen sink capacity did not greatly control protein degradation; it remains to be seen whether phytohormones, accumulated amino acids or other factors delayed the increase in endopeptidase activit

Changes in nitrogen contents and in proteolytic activities in different parts of field-grown wheat ears (Triticum aestivum L.) during maturation

Aminopeptidase, carboxypeptidase and neutral endopeptidase activities were analyzed in glumes and in kernels of field-grown wheat (Triticum aestivum L.) during ear development. Kernels harvested on two dates were subdivided into outer pericarp, cross cells, endosperm and embryo. In developing parts with a net nitrogen influx (young glumes, embryo, endosperm) the aminopeptidase activity is high, but in nitrogen-mobilizing tissues (senescing glumes, Outer pericarp) this activity decreases. Carboxypeptidase is most active in fully expanded tissues. Neutral endopeptidase shows the highest activity in the nitrogen mobilizing parts and extremely low activity in the embryo and the endosper

Senescence and protein degradation in leaf segments of young winter wheat: influence of leaf age

Leaf senescence in intact wheat plants can be strongly influenced by altered source/sink relations. Interactions with other plant parts are no longer possible in detached leaves and therefore differences in their senescence behaviour reflect the physiological status of the leaf before cutting. The net degradation of chlorophylls and of selected enzyme proteins (detected by SDS-PAGE and immunoblotting) was delayed in detached young leaves as compared to senescing or mature leaves excised from the same field-grown wheat plants. The physiological leaf age was therefore decisive for the velocity of artificial senescence. Net degradation rates of the enzymes investigated varied in detached leaves. The protein quantities of plastidial glutamine synthetase, phosphoribulokinase and phosphoglycolate phosphatase decreased more rapidly than those of ferredoxin-dependent glutamate synthase and nitrite reductase. Differences were also detected between two enzymes involved in the same metabolic pathway (photorespiratory carbon cycle) but located in different subcellular compartments: the plastidial enzyme phosphoglycolate phosphatase was lost more rapidly than glycolate oxidase (peroxisomal enzyme

Effects of an extended drought period on physiological properties of grassland species in the field

A very high percentage (around 70%) of the agronomic area in Switzerland is covered by grasslands at various altitudes where environmental conditions, management, community structure and productivity vary widely. As heat waves and drought are predicted to increase in future climate, survival of plant species in grasslands is a major issue of concern in Central Europe. The effect of summer drought on representative grasslands in Switzerland was studied through drought experiments (using rain-out shelters avoiding natural precipitation) to understand the response of predominant species to changed climatic conditions. The physiological performance (gas exchange, leaf water potential) of selected species was investigated at three locations in Switzerland. The pre-dawn leaf water potential of all species was lower (more negative) under the dryer conditions at the three sites. Net photosynthesis and stomatal conductance of forb and legume species did not show major changes under drought, while grass species showed large decreases at the lowland site. These differences between forb-legume and grass species were not observed at the pre-alpine and alpine site. The apparent drought tolerance of the forb-legume species seems to be due—at least partially—to increased water use efficiency under drought condition

Nitrogen metabolism and remobilization during senescence

Senescence is a highly organized and well‐regulated process. As much as 75% of total cellular nitrogen may be located in mesophyll chloroplasts of C3‐plants. Proteolysis of chloroplast proteins begins in an early phase of senescence and the liberated amino acids can be exported to growing parts of the plant (e.g. maturing fruits). Rubisco and other stromal enzymes can be degraded in isolated chloroplasts, implying the involvement of plastidial peptide hydrolases. Whether or not ATP is required and if stromal proteins are modified (e.g. by reactive oxygen species) prior to their degradation are questions still under debate. Several proteins, in particular cysteine proteases, have been demonstrated to be specifically expressed during senescence. Their contribution to the general degradation of chloroplast proteins is unclear. The accumulation in intact cells of peptide fragments and inhibitor studies suggest that multiple degradation pathways may exist for stromal proteins and that vacuolar endopeptidases might also be involved under certain conditions. The breakdown of chlorophyll‐binding proteins associated with the thylakoid membrane is less well investigated. The degradation of these proteins requires the simultaneous catabolism of chlorophylls. The breakdown of chlorophylls has been elucidated during the last decade. Interestingly, nitrogen present in chlorophyll is not exported from senescencing leaves, but remains within the cells in the form of linear tetrapyrrolic catabolites that accumulate in the vacuole. The degradation pathways for chlorophylls and chloroplast proteins are partially interconnecte

Influence of the activation status and of ATP on phosphoribulokinase degradation

The light-regulated choroplast enzyme phosphoribulokinase (EC 2.7.1. 19) exists in two forms. In darkness this enzyme is present in an oxidized form, which is inactive. It is activated in the light by a thioredoxin-mediated reduction. In extracts from young wheat leaves (Triticum aestivum L.) phosphoribulokinase as well as some other thioredoxin-modulated enzymes can be activated by the artificial reductant dithiothreitol (DTT). The influence of the activation status and of the substrate ATP on phosphoribulokinase stability was investigated in the presence of endogenous endopeptidases from senescing wheat leaves. Similar experiments were performed with purified phosphoribulokinase from spinach in the presence of exogenous, purified endopeptidases (chymotrypsin and trypsin). Phosphoribulokinase stability was analysed by immunoblotting and activity measurements. Both systems led to similar conclusions. DTT (reductant) and ATP (substrate) stabilized phosphoribulokinase in wheat leaf extracts as well as partially purified phosphoribulokinase from spinach. The combination of both effectors was far more protective than either effector alone. DTT had hardly any effect on the degradation of thioredoxin-independent chloroplast enzymes such as glutamate synthase and glutamine synthetase. These results suggest that the activation status and substrate concentrations are not only important for the activity of phosphoribulokinase, but are also relevant for the susceptibility of this enzyme to proteolysi

Selective Transport of Zinc, Manganese, Nickel, Cobalt and Cadmium in the Root System and Transfer to the Leaves in Young Wheat Plants

• Background and Aims The uptake, translocation and redistribution of the heavy metals zinc, manganese, nickel, cobalt and cadmium are relevant for plant nutrition as well as for the quality of harvested plant products. The long-distance transport of these heavy metals within the root system and the release to the shoot in young wheat (Triticum aestivum ‘Arina') plants were investigated. • Methods After the application of 65Zn, 54Mn, 63Ni, 57Co and 109Cd for 24 h to one seminal root (the other seminal roots being excised) of 54-h-old wheat seedlings, the labelled plants were incubated for several days in hydroponic culture on a medium without radionuclides. • Key Results The content of 65Zn decreased quickly in the labelled part of the root. After the transfer of 65Zn from the roots to the shoot, a further redistribution in the phloem from older to younger leaves was observed. In contrast to 65Zn, 109Cd was released more slowly from the roots to the leaves and was subsequently redistributed in the phloem to the youngest leaves only at trace levels. The content of 63Ni decreased quickly in the labelled part of the root, moving to the newly formed parts of the root system and also accumulating transiently in the expanding leaves. The 54Mn content decreased quickly in the labelled part of the root and increased simultaneously in leaf 1. A strong retention in the labelled part of the root was observed after supplying 57Co. • Conclusions The dynamics of redistribution of 65Zn, 54Mn, 63Ni, 57Co and 109Cd differed considerably. The rapid redistribution of 63Ni from older to younger leaves throughout the experiment indicated a high mobility in the phloem, while 54Mn was mobile only in the xylem and 57Co was retained in the labelled root without being loaded into the xyle

Light-independent degradation of stromal proteins in intact chloroplasts isolated from Pisum sativum L. leaves: requirement for divalent cations

Intact chloroplasts were isolated from mature pea (Pisum sativum L.) leaves in order to study the degradation of several stromal proteins in organello. Changes in the abundances of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), phosphoribulokinase (EC 2.7.1.19), glutamine synthetase (EC 6.3.1.2) and ferredoxin-dependent glutamine:α-ketoglutarate aminotransferase (glutamate synthase; EC 1.4.7.1) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Coomassie-staining of the gels or immunoblotting using specific antibodies for the different enzymes. Degradation of several stromal proteins was strongly stimulated when intact chloroplasts were incubated in the light in the presence of dithiothreitol. Since free radicals may artificially accumulate in the chloroplast under such conditions and interfere with the stability of stromal proteins, the general relevance of these processes remains questionable. In the absence of light, proteolysis proceeded slowly in isolated chloroplasts and was not stimulated by dithiothreitol. Inhibition by ethylenediaminetetraacetic acid (EDTA), 1,10-phenanthroline or excess zinc ions as well as the requirement for divalent cations suggested that a zinc-containing metalloprotease participated in this process. Furthermore, light-independent degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase and phosphoribulokinase was enhanced in chloroplasts isolated from leaves in which senescence was accelerated by nitrogen starvation. Our results indicate that light-independent stromal protein degradation in intact chloroplasts may be analogous to proteolysis that occurs in intact leaves during senescence

Requirements for the light-stimulated degradation of stromal proteins in isolated pea (Pisum sativum L.) chloroplasts

Chloroplasts from 17-d-old pea leaves (Pisum sativum L.) were isolated to elucidate the requirements for the light-induced degradation of stromal proteins. The influence of electron transport through the thylakoids and the influence of ATP on protein degradation were investigated. When chloroplasts were incubated in the light (45 μmol m−2s−1), glutamine synthetase, the large subunit of ribulose-1,5-bisphosphate carboxylase and glutamate synthase were degraded, whereas phosphoribulokinase, ferredoxin-NADP+ reductase and the 33 kDa protein of photosystem II remained more stable. Major protein degradation was not observed over 240 mm in darkness. The electron transport inhibitor dichlorophenyldimethylurea reduced protein degradation in the light over several hours, whereas dibromothymoquinone was less effective. Inhibiting the production of ATP with tentoxin or by destroying the δpH with the ionophores valinomycin and nigericin had no effect or even a stimulating influence on protein degradation when chloroplasts were exposed to light. Furthermore, adding ATP to chloroplasts incubated in the dark had no effect on proteolysis. From these results it is concluded that the transport of electrons through the thylakoids or photooxidative processes associated with it (especially in presence of DTT), rather than the availability of ATP caused the acceleration of stromal protein degradation by light in isolated pea chloroplast