Chloroplast polypeptides synthesized by leaf segments and isolated chloroplasts during senescence in barley.

Starting from senescent barley (Hordeum vulgare L. cv Hassan) leaf segments receiving light and hormone treatments affecting senescence, the plastid polypeptides synthesized by isolated chloroplasts and by leaf segments were analyzed by radiolabelling followed SDS-PAGE and fluorography. Among 20 to 30 polypeptides detected, a few were specifically synthesized (by chloroplasts and/or leaf segments) after each senescence treatment. Apparently, the polypeptides labelled in assays with isolated chloroplasts are truly synthesized in vivo, because most of them were also labelled in assays with leaf segments. The comparison of polypeptide profiles, for every senescence treatment, after labelling with isolated chloroplasts or leaf segments, suggests that most plastid polypeptides synthesized during senescence are coded in plastid DNA.     

Drought induces many forms of cysteine proteases not observed during natural senescence

Drought-induced senescence and natural senescence was characterised in the cowpea leaf, with a focus on cysteine proteases. Soluble protein content and ribulose 1,5-bisphosphate carboxylase (Rubisco) content declined as senescence progressed. Endopeptidase activity with Rubisco as a physiological substrate exhibited significant increase at acidic (pH 4.8) than at neutral (pH 7.0) during drought induced senescence and declined during recovery. Natural senescence was associated with a several-fold increase in the endopeptidase activity at both the pHs. Cysteine proteases were analyzed using western blot with polyclonal antibodies raised against papain. Several polypeptides of molecular weights 57, 52, and 43 kDA were recognized by the antibodies, the levels of which showed an increase under water deficit conditions, followed by a decrease during recovery. Three polypeptides of molecular weights 69, 60, and 48 kDa appeared only during the water stress conditions, whereas, during natural senescence, only a single 48 kDa polypeptide with maximum intensity at 9 days after flowering was observed. The results suggests the possibility of distinguishing drought-induced and natural senescence.     

Energy charge and emergence of the coleoptile and radicle at varying oxygen levels in Echinochloa crus-galli

The role of protein synthesis in senescence and in the inhibition of senescence by light and kinetin was studied in barley ( Hordeum vulgare L. cv. Hassan) leaves with different inhibitors of protein synthesis. A comparison of the actions of D- and L-chloramphenicol was made to compensate for the effects of D-chloramphenicol not mediated by inhibition of protein synthesis. The involvement of phytochrome was also studied. The results suggest that: 1) cytoplasmic protein synthesis is required for senescence in the light and in the dark; 2) chloroplasts, in the dark, synthesize protein which accelerates senescence; 3) kinetin inhibits the synthesis by chloroplasts of senescence-accelerating protein; 4) light changes the type of protein synthesized by chloroplasts from those accelerating to those retarding senescence; and 5) lightretar-dation of senescence is mediated by phytochrome and, probably, by photophos-phorylation.     

Phytochrome Control of Chlorophyll Content in Mature Attached Leaves of Petunia axillaris

Petunia axillaris plants were grown under white light (photoperiod10 h). Pulses of end-of-day far-red light, but not red lightat the end of the day slowed down the accumulation of chlorophyllin expanding, greening leaves and reduced chlorophyll contentin mature leaves. The chlorophyll a / b ratio was unaffected.Low phytochrome photoequilibria reduced chlorophyll contentin mature leaves without affecting leaf area or d. wt. Thiseffect occurred even before senescence was triggered in thecontrol plants having high phytochrome photoequilibria     

The impact of light intensity on shade-induced leaf senescence

Plants often have to cope with altered light conditions, which in leaves induce various physiological responses ranging from photosynthetic acclimation to leaf senescence. However, our knowledge of the regulatory pathways by which shade and darkness induce leaf senescence remains incomplete. To determine to what extent reduced light intensities regulate the induction of leaf senescence, we performed a functional comparison between Arabidopsis leaves subjected to a range of shading treatments. Individually covered leaves, which remained attached to the plant, were compared with respect to chlorophyll, protein, histology, expression of senescence-associated genes, capacity for photosynthesis and respiration, and light compensation point (LCP). Mild shading induced photosynthetic acclimation and resource partitioning, which, together with a decreased respiration, lowered the LCP. Leaf senescence was induced only under strong shade, coinciding with a negative carbon balance and independent of the red/far-red ratio. Interestingly, while senescence was significantly delayed at very low light compared with darkness, phytochrome A mutant plants showed enhanced chlorophyll degradation under all shading treatments except complete darkness. Taken together, our results suggest that the induction of leaf senescence during shading depends on the efficiency of carbon fixation, which in turn appears to be modulated via light receptors such as phytochrome A.     

Chloroplast Protein Synthesis During Barley Leaf Growth and Senescence: Effect of Leaf Excision

Chloroplast protein synthesis was measured during the expansion,maturity and senescence of the oldest leaf of barley, Hordeumvulgare L., var. Hassan. A maximum rate of protein synthesisoccurred near the end of the expansion stage 9 d after sowing.Protein synthesis increased again at the beginning of senescenceand reached a new maximum at day 14 after sowing. Detachmentand incubation of leaves in the dark stimulated chioroplastprotein synthesis by fully expanded or by senescent leaves butnot by expanding leaves. If the detached leaves were kept inthe light, chloroplast protein synthesis was stimulated in fullyexpanded but not in senescent leaves. Short treatments (18 h)of leaf segments with growth substances in either light or indarkness, significantly changed the rate of protein synthesisshown by chloroplasts. The relationship between chloroplastprotein synthesis and leaf senescence is discussed. Key words: Hormones, light, maturity     

Phytochrome‐mediated regulation of plant respiration and photorespiration

The expression of genes encoding various enzymes participating in photosynthetic and respiratory metabolism is regulated by light via the phytochrome system. While many photosynthetic, photorespiratory and some respiratory enzymes, such as the rotenone‐insensitive NADH and NADPH dehydrogenases and the alternative oxidase, are stimulated by light, succinate dehydrogenase, subunits of the pyruvate dehydrogenase complex, cytochrome oxidase and fumarase are inhibited via the phytochrome mechanism. The effect of light, therefore, imposes limitations on the tricarboxylic acid cycle and on the mitochondrial electron transport coupled to ATP synthesis, while the non‐coupled pathways become activated. Phytochrome‐mediated regulation of gene expression also creates characteristic distribution patterns of photosynthetic, photorespiratory and respiratory enzymes across the leaf generating different populations of mitochondria, either enriched by glycine decarboxylase (in the upper part) or by succinate dehydrogenase (in the bottom part of the leaf).     

Photomorphogenically Defined Light and Resistance of Poa pratensis to Drechslera sorokiniana

Photomorphogenic light definitions were derived by mathematical determination of the estimated phytochrome photoequilibrium for each light treatment spectrum. A wide range of photomorphogenic light treatments represented by spectra with estimated phytochrome photoequilibria of 0.45, 0.54, 0.60, 0.67, and 0.71 was utilized to determine the influence of photomorphogenically defined light on resistance of Poa pratensis L. to pathogenesis by Drechslera sorokiniana. Accurate resolution of D. sorokiniana leaf spot development required evaluation of separate leaf ages due to the sequential appearance, development, and senescence of P. pratensis leaves. Disease development (all light treatments) was greatest on leaf 4 (oldest, postmature) followed by leaf 1 (youngest, premature). Low levels of disease occurred on leaves 2 and 3 (mature). Photomorphogenic light defined by estimated phytochrome photoequilibria greater than 0.60 (natural light 0.60) was most disease promotive on leaf 1. Conversely, photomorphogenic light defined by estimated phytochrome photoequilibria of less than 0.60 was most disease promotive on leaf 4. These responses indicate that inherent resistance or susceptibility expressed by P. pratensis to pathogenesis by D. sorokiniana is regulated in part by leaf age (developmental senescent stage) and by photomorphogenically defined light quality. A hypothesis is presented and discussed which integrates and speculates on these observations with respect to the literature.     

Vacuole Cysteine Proteases and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Degradation during Monocarpic Senescence in Cowpea Leaves

Characterisation of proteases degrading ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO, EC: 4.1.1.39) was studied in the cowpea leaf during monocarpic senescence 3 and 9 d after flowering (DAF), representing early and mid pod fill. The stage at 3 DAF coincided with decrease in the metabolic parameters characterising senescence, i.e., contents of total soluble proteins, RuBPCO, and leaf nitrogen. At 9 DAF, there was a decline in total soluble proteins and an appearance of a 48 kDa cysteine protease. Characterisation of the proteases was done using specific inhibitors. Subcellular localisation at 3 DAF was studied by following the degradation of RuBPCO large subunit (LSU) in the vacuole lysates using immunoblot analyses. Cysteine proteases played a predominant role in the degradation of RuBPCO LSU at the crude extract level. At 9 DAF, expression of cysteine protease isoforms was monitored using polyclonal antibodies against papain and two polypeptides of molecular masses 48 and 35 kDa were observed in the vacuole lysates. We confirmed thus the predominance of cysteine proteases in the vacuoles during different stages of pod development in cowpea leaf.     

Effects of growth regulators and light on chloroplasts NAD(P)H dehydrogenase activities of senescent barley leaves

The activities NADH and NADPH dehydrogenases were measured with ferricyanide as electron-acceptor (NADH-FeCN-ox and NADPH-FeCN-ox, respectively) in mitochondria-free chloroplasts of barley leaf segments after receiving various treatments affecting senescence. NADPH-FeCN-ox declined during senescence in the dark, in a way similar to chlorophyll and Hill reaction, and increased when leaf segments were incubated at light. These results suggest that NADPH-FeCN-ox is related to some photosynthetic electron transporter activity (probably ferredoxin-NADP⁺ oxidoreductase). In contrast, NADH-FeCN-ox is notably stable during senescence in the dark and at light. This activity increased during incubation with kinetin or methyl-jasmonate (Me-JA) but decreased when leaf segments were treated with abscisic acid (ABA). The effects of the inhibitors of protein synthesis cycloheximide and chloramphenicol suggest that the changes of NAD(P)H dehydrogenase activities may depend on protein synthesis in chloroplasts. In senescent leaf, chloroplast NADH dehydrogenase might be a way to dissipate NADH produced in the degradation of excess carbon which is released from the degradation of amino acids.Abbreviations ABA abscisic acid - DCPIP 2,6-dichlorophenol-indo-phenol - DOC deoxycholate - Me-JA methyl jasmonate - NADH-FeCN-ox NADH ferricyanide oxidoreductase - NADPH-FeCN-ox NADPH ferricyanide oxidoreductase     

Secretory protein biosynthesis in snail hemocytes: in vitro modulation by larval schistosome excretory-secretory products

Circulating hemocytes of the snail, Biomphalaria glabrata, synthesize and secrete a variety of polypeptides when maintained in vitro in serum-free medium containing [35S] methionine. SDS-PAGE/fluorographic analysis of supernatants from resistant snail (10-R2-OK strain) hemocyte cultures revealed the presence of numerous labeled polypeptides ranging in Mr from 220 to 14 kDa. Most of these same proteins were also produced by hemocytes of a susceptible B. glabrata strain (M-line), but the overall rate of secretory protein synthesis was reduced from that of resistant snail cells. In addition, excretory-secretory (ES) products contained in supernatants from Schistosoma mansoni miracidial transformation and 1-day primary sporocyst cultures stimulated increases in the synthesis of various polypeptides. Particularly striking was a 3-fold increase in the synthesis of a 66-kDa secretory polypeptide by hemocytes of both snail strains, and a concomitant increase in M-line hemocytes and decrease in 10-R2-OK cells of a 63-kDa polypeptide. Overall, however, the level of ES product-induced secretory protein synthesis was greater in 10-R2-OK snail hemocytes than in those of the M-line strain. Exposure of a nonhemocytic B. glabrata cell line to parasite culture supernatants had no stimulatory/inhibitory effect on labeled protein ouput, suggesting that the observed hemocyte response may be snail cell-type specific. Finally, the larval ES components responsible for modulating hemocyte protein metabolism are mainly concentrated in a heat-stable fraction composed of molecules of greater than 30 kDa. However, the loss of the ability of heated parasite products to stimulate synthesis of certain hemocyte proteins and the presence of minor stimulating activity in a low molecular weight fraction (less than 10 kDa) implies the possible existence of multiple larval components affecting formation of specific hemocyte secretory polypeptides. It is concluded that snail hemocytes are capable of in vitro synthesis and secretion of a variety of methionine-containing polypeptides, and that ES products of early larval schistosomes can modulate (i.e., stimulate or inhibit) this metabolic process. A differential response of susceptible vs. resistant hemocytes to larval products suggests that the degree to which these cells can be metabolically activated may determine their cytotoxic effectiveness.     

Analysis of phytochrome kinetics in light-grown Avena sativa L. seedlings

The phytochrome content, the rate of phytochrome accumulation after a light/dark transition and the rate of phytochrome destruction after a 1.5 d reaccumulation period in darkness were measured in light grown Avena sativa L. seedlings. The results using spectrophotometrical methods (Norflurazon treated seedlings) and the radio-immunoassay (RIA) (green seedlings) were almost identical. The rate of phytochrome synthesis was analysed by measuring the activity of poly(A⁺)-RNA coding for the phytochrome apoprotein. It was demonstrated that the rate of phytochrome synthesis is different in light and in dark. These results were confirmed by measuring the incorporation of radioactive label in vivo. Five minutes red (and 5 min far-red) light strongly reduces the rate of phytochrome synthesis. Even after prolonged dark periods only 50% of the initial rate of phytochrome synthesis is recovered for light and dark grown seedlings which received one red light pulse.     

Oxygen concentration, nitrogenase activity and heterocyst frequency in the leaf cavities of Azolla filiculoides Lam

Following exposure to UV light the synthesis of six polypeptides (112, 100, 89, 76, 71 and 65 kDa) was found to be enhanced or induced in the alga Chlamydomonas reinhardtii. Treatment with 4-nitroquinoline-N-oxide (NQO) resulted in the enhanced/induced synthesis of six polypeptides with molecular masses similar to those enhanced following exposure to UV light. Heat shock resulted in the enhanced synthesis of five polypeptides (89, 76, 71, 60 and 22 kDa), three of which (89, 76 and 71 kDa) had apparently identical mobilities to polypeptides whose synthesis was enhanced following UV treatment.     

Regulation of senescence in bean leaf discs by light and chemical growth regulators

The senescence of excised discs of primary leaves of Phaseolus vulgaris, L., var. Red Kidney was followed by measuring the net breakdown of protein and chlorophyll. The chemical growth regulators indoleacetic acid, 2,4-dichlorophenoxy-acetic acid, gibberellic acid, kinetin, and 6-benzylaminopurine were relatively ineffective in retarding senescence in this tissue. White light, on the other hand, was very effective in senescence retardation. The response to light did not have the characteristics of a low energy (phytochrome) response and was blocked by concentrations of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea which inhibited photosynthesis in the leaf discs. The light-induced retardation of senescence was concluded to be dependent on photosynthesis.     

Production and characterization of three polyclonal antibodies raised against cyst wall proteins of a hypotrichous ciliate.

Three polyclonal antibodies raised against Paraurostyla sp. cyst wall polypeptides of molecular weight 110,000 (p110), 66,000 (p66) and 52,000 (p52) have been obtained. The specificity of the antisera was tested by immunoblotting. Anti-p110 antibody detected five bands of 300, 170, 135, 110 and 40 kDa, respectively. Antiserum obtained against p66 recognized only this protein. Anti-p52 antiserum showed reaction for two different bands of 52 and 44 kDa, respectively. The precise localization of these proteins in the cyst wall was assessed by light microscope immunocytochemistry. Anti-p110 antiserum produced a strong positive reaction in both the ectocyst and endocyst. Both anti-p66 and anti-p52 antibodies recognized the ectocyst.     

Production and Characterization of Three Polyclonal Antibodies Raised Against Cyst Wall Proteins of a Hypotrichous Ciliate

ABSTRACT Three polyclonal antibodies raised against Paraurostyla sp. cyst wall polypeptides of molecular weight 110,000 (p110), 66,000 (p66) and 52,000 (p52) have been obtained. The specificity of the antisera was tested by immunoblotting. Anti-p110 antibody detected five bands of 300, 170, 135, 110 and 40 kDa, respectively. Antiserum obtained against p66 recognized only this protein. Anti-p52 antiserum showed reaction for two different bands of 52 and 44 kDa, respectively. The precise localization of these proteins in the cyst wall was assessed by light microscope immunocytochemistry. Anti-p110 antiserum produced a strong positive reaction in both the ectocyst and endocyst. Both anti-p66 and anti-p52 antibodies recognized the ectocyst.     

Senescence in wheat leaves: is a cysteine endopeptidase involved in the degradation of the large subunit of Rubisco?

In wheat (Triticum aestivum L.), leaf senescence can be initiated by different factors. Depending on the plant system (intact plants or detached leaves) or the environmental conditions (light, nutrient availability), the symptoms of senescence differ. The aim of this work was to elucidate the catabolism of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC. 4.1.1.39) under various senescence-inducing conditions. Leaf senescence was initiated in intact plants by darkness or by N-deprivation and in leaf segments by exposure to light or darkness. Depending on the treatment, a 50 kDa fragment of Rubisco was observed. The formation of this fragment was enhanced by leaf detachment and low light. In segments exposed to high light and in intact plants induced to senesce by N-deprivation, the fragment was essentially absent. Since an antibody against the N-terminus of a large subunit of Rubisco (LSU) did not cross-react with the fragment, it appears likely that a smaller fragment was removed from the N-terminus of LSU. Inhibitor studies suggest that a cysteine endopeptidase was involved in the formation of the 50 kDa fragment. Non-denaturing-PAGE followed by SDS-PAGE revealed that the fragment was produced while LSU was integrated in the holoenzyme complex, and that it remained there after being produced. It remains open how the putative endopeptidase reaches the stromal protein Rubisco. The results indicate that depending on the senescence-inducing conditions, different proteolytic enzymes may be involved. The involvement of vacuolar proteases must be considered as occurring during LSU degradation, which takes place in darkness, low light or under carbon limitation.     

Changes in polypeptide expression following Trypanosoma cruzi differentiation from trypomastigotes to amastigotes.

Changes in the dynamic of expression of polypeptides following the differentiation from infective trypomastigotes to multiplicative amastigote forms of Trypanosoma cruzi were mapped by two-dimensional gel electrophoresis and quantitatively analyzed by laser densitometry. Following the differentiation from trypomastigotes to amastigotes the expression of the polypeptides 212, 183, 176, 149, 50-55, 43, 39, 34 and 28 kDa is turned off in multiplicative amastigotes, whereas the expression of the polypeptides 80, 66 (p.Is. 6.75-7.50), 42 and 38 kDa is turned on. After complete differentiation from trypomastigotes to amastigotes the expression of the polypeptides 43, 42, 33, 32, 29 and 23 kDa is up-regulated in amastigotes, whereas the expression of the acidic polypeptides 66 (p.Is. 6.27-6.64), 45-48 and 41-43 kDa is down-regulated.     

Electrophoretic mobility of gamma-glutamyltransferase in rat liver subcellular fractions.Evidence for structure difference from the kidney enzyme.

Adult rat liver gamma-glutamyltransferase (GGT) has been poorly characterized because of its very low concentration in the tissue. In contrast with the kidney, the liver enzyme is inducible by some xenobiotics, and its relationship to hepatic ontogeny and carcinogenesis seems to be important. Liver GGT polypeptides were identified by immunoblot analysis in subcellular fractions (rough endoplasmic reticulum, smooth endoplasmic reticulum, Golgi membranes and plasma membranes). Rat liver GGT appeared as a series of polypeptides corresponding to different maturation steps. Polypeptides related to the heavy subunit of GGT were detected in rough endoplasmic reticulum at 49, 53 and 55 kDa, and in Golgi membranes at 55, 60 and 66 kDa. Two polypeptides related to the light subunit of GGT were also observed in Golgi membranes. In plasma membranes GGT was composed of 100 kDa, 66 kDa and 31 kDa polypeptides. The 66 kDa component could correspond to the heavy subunit of the rat liver enzyme, and if so has a molecular mass higher than that of the purified rat kidney form of GGT (papain-treated). These data suggest different peptide backbones for the heavy subunits of liver GGT and kidney GGT.