Subchloroplast localization of polypeptides synthesized by chloroplasts during senescence

To study the localization of polypeptides synthesized by isolated senescent chloroplasts we have fractionated the chloroplasts into stroma, envelope and thylakoid components. The validity of the fractionation procedure was tested by assaying both chlorophyll and enzyme markers, as well as the polypeptide composition of each fraction. Plastids in the transition of etioplast to chloroplast, senescent chloroplasts and kinetin-treated chloroplasts produced acceptable fractions, although their polypeptide compositions varied considerably during the ontogeny, particularly those of the envelope. Most of the polypeptides synthesized by isolated senescent chloroplasts were incorporated into the thylakoids except for a 58 kDa polypeptide localized in the stroma and some minor polypeptides present in both stroma and envelope. Although most of the polypeptides synthesized by isolated chloroplasts from kinetin-treated leaves were incorporated into the thylakoid membrane, several polypeptides were found in the stroma (90, 80, 65 and 54 kDa) and in the envelope (100, 75, 48 and 28–30 kDa). The results indicate that early in senescence, the polypeptides of the envelope change but, that probably, most of the new polypeptides are synthesized in the cytoplasm.     

Protein synthesis by chloroplasts during the senescence of barley leaves

Chloroplasts were isolated from senescent leaf segments of barley ( Hordeum vulgare L. var. Mozoncillo) and assayed for protein synthesis. Protein synthesis activity of the chloroplasts greatly increased after 10–20 h of incubation of leaf segments in the dark in spite of an intense degradation of chloroplast rRNA. The rise in the activity of protein synthesis was more pronounced when kinetin was present in the incubation medium. However, as deduced from SDS-polyacrylamide gel electrophoresis of the products, different proteins were synthesized under the two conditions of incubation of the leaf segments. The activity of protein synthesis of the chloroplasts decreased during the first hours of incubation of the leaf segments in the light.
Cutting and incubation in the dark of the leaf segments enhanced the synthesis of a few proteins also formed by chloroplasts in attached senescing leaves. Hormone and senescence treatments changed the type and the rate of the protein synthesized by chloroplasts, which suggests that hormones may control senescence through a modulation of the protein synthesized by the chloroplasts.     

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.     

Changes in chloroplast peroxidase activities in relation to chlorophyll loss in barley leaf segments

Total peroxidase activity increased during senescence of excised barley ( Hordeum vulgare L. cv. Kashimamugi) leaves. Kinetin treatment furter increased total peroxidase activity but repressed chlorophyll degradation in excised barley leaves. When isoperoxidases were extracted from barley leaf segments. 4 cationic and 4 anionic isozymes were found in polyacrylamide gel electrophorests during leaf senescence. The chloroplasts contained only two cationic isoperoxidase activities. One (designated C4) was repressed by kinetin. and the other (C3) was increased by kinetin. Glucosamine, which also repressed the degradation of chlorophyll, completely repressed C4 activity but did not affect C3 activity. The induction with senescence, and the repression with kinetin and glucosamine, suggest chat chloroplast isoperoxidase C4 may function as a chlorophyll-degrading enzyme during barley leaf senescence.     

Dismantling of Arabidopsis thaliana mesophyll cell chloroplasts during natural leaf senescence

One of the earliest events in the process of leaf senescence is dismantling of chloroplasts. Mesophyll cell chloroplasts from rosette leaves were studied in Arabidopsis thaliana undergoing natural senescence. The number of chloroplasts decreased by only 17% in fully yellow leaves, and chloroplasts were found to undergo progressive photosynthetic and ultrastructural changes as senescence proceeded. In ultrastructural studies, an intact tonoplast could not be visualized, thus, a 35S-GFP::δ-TIP line with a GFP-labeled tonoplast was used to demonstrate that chloroplasts remain outside of the tonoplast even at late stages of senescence. Chloroplast DNA was measured by real-time PCR at four different chloroplast loci, and a fourfold decrease in chloroplast DNA per chloroplast was noted in yellow senescent leaves when compared to green leaves from plants of the same age. Although chloroplast DNA did decrease, the chloroplast/nuclear gene copy ratio was still 31:1 in yellow leaves. Interestingly, mRNA levels for the four loci differed: psbA and ndhB mRNAs remained abundant late into senescence, while rpoC1 and rbcL mRNAs decreased in parallel to chloroplast DNA. Together, these data demonstrate that, during senescence, chloroplasts remain outside of the vacuole as distinct organelles while the thylakoid membranes are dismantled internally. As thylakoids were dismantled, Rubisco large subunit, Lhcb1, and chloroplast DNA levels declined, but variable levels of mRNA persisted.     

Transport of methotrexate in Chinese hamster ovary cells: a mutant defective in methotrexate uptake and cell binding

A regulatory role for cytoplasmically derived proteins in chloroplast translation in organello was examined by analyzing protein synthesis in plastids isolated from cells of Euglena gracilis which had been treated with cycloheximide (CHI). Incorporation of [35S]methionine by chloroplasts from CHI-inhibited Euglena was reduced approximately 40 and 90% by exposure of the cells to the antibiotic for 2 and 4 h, respectively. The chloroplast translation products were then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. The synthesis of polypeptides in the soluble compartment of the plastid was substantially diminished by as little as 15 min of CHI pretreatment. No qualitative alterations of the polypeptide pattern were detected. Qualitative changes were seen in the thylakoid fraction, however. Comparison of the stainable polypeptides and fluorographs of thylakoid membranes from CHI-treated cells with those of controls showed several instances in which the more slowly migrating member of a doublet accumulated with a concomitant depletion of a more rapidly migrating component. A pair of polypeptides at 28 and 30 kDa, which we believe are the Euglena homologs of the photogene product and its precursor, respectively, are representative of this phenomenon. Additionally, thylakoids from cells pretreated with CHI sometimes synthesized novel polypeptides larger than 65 kDa. Finally, when intact chloroplasts from CHI-inhibited Euglena were incubated with a postchloroplast supernatant from normal cells, there was a partial reversion of the anomalies seen in the fluorographs. These data are interpreted to indicate the cytoplasmic origin of one or more proteins whose function is to process chloroplast translation products.     

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.     

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     

Phytochrome and hormone control of polypeptides synthesized by chloroplasts of senescent barley leaves

To identify the polypeptides involved in the mechanism of leaf senescence, light-driven protein synthesis was assayed with chloroplasts isolated from barley leaf segments incubated during 20 h under different light and hormone treatments affecting senescence. The radioactive products were analyzed by SDS-PAGE and fluorography. The synthesis of some polypeptides was stimulated by ABA (66, 44, 30, 22, 20, kDa) and ethylene (66, 50, 48, 44, kDa) which accelerate senescence. Kinetin and red light (in an effect mediated by phytochrome), which retard senescence, inhibited the synthesis of some polypeptides (50, 48, 37, kDa) and stimulated the synthesis of others (54, 32, kDa). Probably phytochrome and hormones control senescence by affecting the synthesis of specific polypeptides.     

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     

Control by phytochrome of the synthesis of protein related to senescence in chloroplasts of barley (Hordeum vulgare)

Protein synthesis has been measured in chloroplast isolated from detached leaves of barley ( Hordeum vulgare L. cv. Hassan). The effects of hormone and light treatments of the leaves on chloroplast protein synthesis have been compared with effects on other senescence symptoms. Interruption of the dark with red light retards senescence and increases chloroplast protein synthesis. The effect of red light was reversed by far-red light. Red light did not act additively with kinetin, and it competed with ethylene and abscisic acid, accelerators of senescence, which decreased protein synthesis. In contrast to the interruption of the dark with red light, continuous light decreased chloroplast protein synthesis. It may be concluded effects on chloroplast protein synthesis. The retardation of senescence by continuous light is not necessarily related to P_u Instead, energy provided by photosynthesis may be an important factor.     

Optimization of protein synthesis in isolated higher plant chloroplasts. Identification of paused translation intermediates

Protein synthesis in isolated, intact pea chloroplasts was optimized and compared to translation within chloroplasts in vivo. Many polypeptides labeled with [35S]methionine in isolated intact chloroplasts did not comigrate with polypeptides which were labeled within chloroplasts in vivo. Antibodies to the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (EC 4.1.1.39) immunoprecipitated [35S]-labeled large subunit plus several lower-molecular-mass translation products of isolated chloroplasts. The lower-molecular-mass soluble translation products synthesized in pulse-labeled chloroplasts were converted into full-length large-subunit polypeptides during a subsequent chase period. This result suggests that many of the polypeptides observed in pulse-labeled chloroplasts are incomplete translation products which are the result of ribosome pausing at discrete points along chloroplast mRNAs. The pulse-chase technique was used to follow synthesis of the 34.5-kDa precursor of the psb A gene product and its processing to the mature 32-kDa polypeptide in isolated chloroplasts. Chloroplast translation profiles obtained using the pulse-chase assay were very similar to translation profiles obtained in vivo thus extending the utility of protein synthesis in isolated chloroplasts.     

Differential effects of abscisic acid and methyl jasmonate on endoproteinases in senescing barley leaves

Endoproteinase activity was analyzed in chloroplasts isolated from barley leaf segments incubated in the dark with various hormonal senescence effectors. As a control, the endoproteinase activity of the supernatant fraction obtained during chloroplast preparation was also analyzed. Measured against azocaseine as substrate, the endoproteinase activity in chloroplasts increased 18 fold during the induction of senescence. This rise in activity was inhibited by kinetin (the activity increased only 10 fold) and very strongly stimulated by abscisic acid (ABA) (117 fold) and methyl jasmonate (Me-JA) (57 fold). Although less so, the endoproteinase activity of the supernatant fraction, mainly vacuolar and with acid pH optimum, was affected in the same way by all three effectors. Among the five endoproteinases (EC) found in chloroplasts, EC2 and EC4 were induced after incubation in water. ABA increased the levels of EC2 and EC4 (5 fold), and induced the development of EC3 and EC5, while Me-JA totally inhibited EC2 and EC4, and induced the development of EC1. At least one of the endoproteinases, EC2, is synthesized in chloroplasts. Among the six endoproteinases found in the supernatant fraction (E), E1, E2, E3 and E5, which are very probably extrachloroplastic endoproteinases, are stimulated by ABA to varying degrees. However, Me-JA stimulates E1 to a greater extent and totally inhibits E3. The differential effects of ABA and Me-JA on chloroplast and supernatant fraction endoproteinases suggest different action mechanisms for both senescence promotors.Abbreviations ABA abscisic acid - DTT dithiothreitol - E supernatant fraction endoproteinase - EC chloroplast endoproteinase - Me-JA methyl jasmonate - PNP p-nitrophenol - SDS-PAGE polyacrylamide gel electrophoresis containing sodium dodecyl sulphate - TCA trichloroacetic acid     

In vitro synthesis and assembly of photosystem II proteins of spinach chloroplasts

The synthesis and assembly of photosystem II (PS II) proteins of spinach chloroplasts were investigated in three different in vitro systems, i.e., protein synthesis in isolated chloroplasts (in organello translation), read-out translation of thylakoid-bound ribosomes, and transport of translation products from spinach leaf polyadenylated RNA into isolated chloroplasts. Polyacrylamide gel electrophoresis of labeled thylakoid polypeptides in the presence of sodium dodecyl sulfate revealed that the first two systems were capable of synthesizing the reaction center proteins of PS II (47 and 43 kDa), the herbicide-binding protein, and cytochrome b559. The reaction center proteins synthesized in organello were shown to bind chlorophyll and to assemble properly into the PS II core complex. One of the reaction center proteins translated by the thylakoid-bound ribosomes (47 kDa) was also found to be integrated in situ into the complex but was lacking bound chlorophyll. Incorporation of radioactivity into the three extrinsic proteins of the oxygen-evolution system (33, 24, and 18 kDa) was detected only when intact chloroplasts were incubated with the translation products from polyadenylated RNA, showing that these proteins are coded for by nuclear DNA. The occurrence of a precursor polypeptide 6 kDa larger than the 33-kDa protein was immunochemically detected in the translation products.     

Protein synthesis in chloroplasts. VIII. Differential synthesis of chloroplast proteins during spinach leaf development

Excised primary leaves of spinach (Spinacia oleracea) incorporate [35S]-methionine into a number of chloroplast polypeptides. The ratio of incorporation of isotope into the large subunit of ribulose bisphosphate carboxylase relative to a thylakoid polypeptide (peak D) decreases during leaf development in whole leaves; this changing pattern of incorporation is also observed in isolated chloroplasts where these two polypeptides are the major products of protein synthesis. Chloroplast RNA prepared from developing leaves was translated in a reticulocyte lysate extract to yield full-length carboxylase large subunit and peak D polypeptides. The fidelity of translation of these two polypeptides was checked by partial protease digestion. Changes in the synthesis of the large subunit of the carboxylase and peak D in developing leaves are reflected in changes in the amount of translatable mRNA for these two polypeptides.     

THE INFLUENCE OF KINETIN AND ANTIMETABOLITES ON THE SYNTHESIS OF FATTY ACIDS IN LEAVES

Green leaf tissues contain relatively higher proportions of unsaturated fatty acids, especially α-linolenic acid, than do etiolated or senescent tissues. There appear to be developmental changes in the fatty acid composition of leaves during maturation and senescence. The normal rate of development of spinach (Spinacia oleracea L.) and bean (Phaseolus vulgaris L.) leaf tissues was altered by the application of kinetin and antimetabolites. Spinach was used for the kinetin studies and bean for the antimetabolite studies. Supposedly the kinetin retarded senescence and the antimetabolites retarded normal development. Special emphasis was placed on the incorporation of acetate into palmitate, the most abundant saturated fatty acid, and into linolenate, the most abundant unsaturated fatty acid. Kinetin does not enhance linolenate synthesis, but kinetin-treated tissues contain proportionately more linolenate. In contrast, tissues treated with antimetabolites contain proportionately less linolenate. Actinomycin-D and puromycin seem to have a greater effect on the synthesis of linolenate than on the synthesis of palmitate. Chloramphenicol does not have this same differential effect. The possible influence of antimetabolites on the synthesis of unsaturated fatty acids is discussed.     

Senescence in oat leaves: Changes in translatable mRNAs

Changes in translatable mRNA populations during the senescence of oat (Avena sativa L. cv. Victory) leaves were examined by analyzing the in vitro translation products of isolated RNA. Total RNA was isolated from oat leaves of 7-day-old seedlings, and also after these leaves were aged for different lengths of time under various conditions. Polypeptides from in vitro translations were separated by two-dimensional gel electrophoresis to estimate any changes in translatable mRNA populations associated with senescence. Corresponding leaf samples were monitored for loss of chlorophyll as a measure of the extent of senescence. The aging of excised leaves in the light for 4 days resulted in the disappearance or substantial quantitative decrease of a number of mRNA species, while only five new translatable mRNA species were produced. Three of these mRNAs were unique to aging of leaves under light. Two of these mRNA species were also produced during the early stages of senescence in attached leaves of seedlings grown under light. The translatable mRNA populations of leaves aged for 4 days either on intact seedlings or detached and kept in the light in the presence of kinetin were very similar. Aging of excised leaves in the dark on water for 24 h resulted in very extensive changes in translatable mRNA populations. Over thirty polypeptides disappeared or were substantially reduced in quantity, while about an equal number appeared de novo or were substantially increased in quantity. Aging of these leaves for an additional 24 or 48 h resulted in only a few additional changes in translatable mRNAs. The presence of kinetin during aging of excised leaves in the dark inhibited few of the numerous changes in mRNAs that occured during the first 24 h, but did inhibit most of the changes that occured after 48 or 72 h of aging in the dark. When leaves were first aged in the dark and then returned to light, most of the initial changes in translatable mRNAs expression were reversed. Such changes in mRNAs thus appear to be light-regulated and not necessarily associated with senescence.     

Internal Water Status of Kinetin-treated, Salt-stressed Plants

Stomatal resistances and turgor pressures were measured during a 12-day period on leaves of bean (Phaseolus vulgaris L. cv. Contender) which were treated with kinetin, were salinized or were treated with kinetin and salinized. Stomatal resistances were highest in salt-stressed plants, and progressively lower in salt-stressed and kinetin-treated plants, control plants, and kinetin-treated plants. Turgor pressures were highest in salt-stressed plants, and progressively lower in control plants, kinetin-treated plants, and salt-stressed and kinetin-treated plants. Stomata appeared to be kept open more widely under kinetin treatment than under control conditions, even when turgor pressures were lower in kinetin-treated plants than in control plants.     

Translation in chloroplasts

The discovery that chloroplasts have semi-autonomous genetic systems has led to many insights into the biogenesis of these organelles and their evolution from free-living photosynthetic bacteria. Recent developments of our understanding of the molecular mechanisms of translation in chloroplasts suggest selective pressures that have maintained the 100-200 genes of the ancestral endosymbiont in chloroplast genomes. The ability to introduce modified genes into chloroplast genomes by homologous recombination and the recent development of an in vitro chloroplast translation system have been exploited for analyses of the cis-acting requirements for chloroplast translation. Trans-acting translational factors have been identified by genetic and biochemical approaches. Several studies have suggested that chloroplast mRNAs are translated in association with membranes.