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     

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.     

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.     

The Effect of Light and Hormones on Leaf Senescence

With wheat leaves as material, the changes of superoxide dismutase (SOD), lipid peroxi-dation and membrane permeability during leaf senescence in light or dark, and treated withphytohormones (KT or ABA) have been studied. The changes of chlorophyll content, lipidperoxidation and fine structure of spinach chloroplasts senescing in light or dark have alsobeen studied. When leaves senesce in light, the activity of SOD increased at first then decreased. The increase of SOD activity was able to result from the synthesis of new protein. Lightwas found to delay the leaf senescence obviously but also accelerate leaf senescence by causinglipid peroxidation when prolonged the illumination time. The delay or acceleration of leafsenescence by exogenous hormones were observed, it may be due to the control of lipid peroxi-dation by adjusting the activity of SOD. O2-participated the chlorophyll decomposition andlipid peroxidation during chloroplasts senesce in light. A favourable role of light in mainta-lng the fine structure of isolated chloroplasts was clear.     

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     

A Vertical Gradient of the Chloroplast Abundance among Leaves of Chenopodium album

The abundances of chloroplasts in leaves on the main stems ofChenopodium album at different height levels were investigatedin relation to the photosynthetic capacity and light environmentof the leaves. (1) The number of chloroplasts per mesophyllcell decreased with descending position of leaves, except foryoung developing leaves at the top of plants that had smallerchloroplast numbers per cell than matured leaves beneath them.Contents of chlorophyll and ribulose-1,5-bisphosphate carboxylase/oxygenaseper leaf area that were highest in the topmost young leavesand decreased with decreasing height level indicate that thereis a vertical gradient of chloroplast abundance per leaf areadecreasing from the top of the leaf canopy with depth. (2) Light-saturatingrate of photosynthetic oxygen evolution per leaf area of maturedleaves decreased more steeply with decreasing leaf positionthan the chloroplast number per cell. Gradients of chlorophylland the enzyme protein contents were also steeper than thatof the chloroplast number. Loss of photosynthesis in lower leavesis, therefore, ascribed partly to loss of whole chloroplastsand partly to reduced photosynthetic capacities of the remainingchloroplasts. (3) The chloroplast number per cell in newly expandedsecond leaves was comparable to those in leaves that have developedat later stages of the plant growth but decreased graduallyduring leaf senescence both in the dark and light. The formationof the vertical gradient of chloroplast abundance is, therefore,ascribed to loss of whole chloroplasts during senescence ofleaves. (4) Irradiance a leaf receives decreased sharply fromthe top of the canopy with depth. The physiological or ecophysiologicalsignificance of the vertical distribution of chloroplasts amongleaves was discussed taking light environments of leaves intoconsideration. (Received July 31, 1995; Accepted October 20, 1995)     

Phytochrome-mediated accumulation of chloroplast DNA in pea leaves

Pea seedlings grown for 5 days in the dark were treated with red light for 5 min and grown for 2 more days in the dark. Effects of the red light on chloroplast DNA levels in the pea leaves were examined using probe DNA of the chloroplast-coded large subunit and nuclear-coded small subunit of ribulosebisphosphate carboxylase/oxygenase. The gene dosage of the large subunit, but not of the small subunit, was increased by red light. The increase was inhibited by subsequent far-red light treatment. These results indicate that accumulation of chloroplast DNA in the cell is mediated by phytochrome. Probably the replication of chloroplast DNA is mediated by phytochrome.     

Phytochrome mediated induction of nitrate reductase activity in etiolated maize leaves

The effects of red and far-red light on the enhancement of in vitro nitrate reductase activity and on nitrate accumulation in etiolated excised maize leaves were examined. Illumination for 5 min with red light followed by a 4-h dark period caused a marked increase in nitrate reductase activity, whereas a 5-min illumination with far-red light had no effect on the enzyme activity. The effect of red light was completely reversed by a subsequent illumination with the same period of far-red light. Continuous far-red light also enhanced nitrate reductase activity. Both photoreversibility by red and far-red light and the operation of high intensity reaction under continuous far-red light indicated that the induction of nitrate reductase was mediated by phytochrome. Though nitrate accumulation was slightly enhanced by red and continuous far-red light treatments by 17% and 26% respectively, this is unlikely to account for the entire increase of nitrate reductase activity. The far-red light treatments given in water, to leaves preincubated in nitrate, enhanced nitrate reductase activity considerably over the dark control. The presence of a lag phase and inhibition of increase in enzyme activity under continuous far-red light-by tungstate and inhibitors of RNA synthesis and protein synthesis-rules out the possibility of activation of nitrate reductase and suggests de novo synthesis of the enzyme affected by phytochrome.     

Control of Some Enzymes of Nitrogen Metabolism during Senescence of Detached Barley (Hordeum vulgare L.) Leaves

The effects of chloramphenicol, cycloheximide and kinetin onthe changes in activity of glutamate dehydrogenase (GDH), glutamatepyruvate transaminase (GPT), glutamate oxaloacetate transaminase(GOT) and nitrite reductase were studied during the senescenceof detached barley leaves in the light and dark. The four enzymesseemed to be synthesized at least during the first hours ofsenescence. The rate of synthesis of GDH was clearly higherthan that of its degradation, thus continuously increasing duringsenescence. Chloramphenicol and kinetin delayed the enzyme degradationprocesses of senescence in the dark. However, chloramphenicolaccelerated senescence in the light. Kinetin had no significanteffect on the enzyme activities in the light. Cycloheximidetreatments produced lower enzyme levels than their respectivecontrols in both the light and dark, but the enzyme levels werehigher in cycloheximide treated leaves in the light than inthe controls in water in the dark. The results are discussedwith reference to the requirement for protein synthesis in thedifferent processes of senescence. (Received August 17, 1981; Accepted February 22, 1982)     

Phytochrome-mediated assembly of polyribosomes in etiolated bean leaves. Evidence for post-transciptional regulation of development.

Light operating through phytochrome controls the proportion of total ribosomes present as polyribosomes in etiolated leaves of Phaseolus vulgaris. Similar responses but with slightly different time courses are elicited by brief red light treatment and by continuous far-red or white light. The increase in polyribosome proportions after red light treatment reaches a maximum within 2 h, after which the proportion steadily declines. Light treatment appears to lead to increased proportions of polyribosomes in higher size classes. This is most evident with continuous white light, but probably also occurs with red and far-red light. The increase in polyribosomes is due principally to cytoplasmic ribosomes although proportionately greater effects are observed in chloroplast ribosomes. Although cordycepin inhibits RNA synthesis by 85-90% within 3 h there is no depression of the light-mediated increase in polyribosome proportions when leaves are preincubated in the inhibitor for 3 h. The data therefore indicate that phytochrome is capable of controlling protein synthesis, and thus development, at a post-transciptional level.     

Effect of light on the chloroplast division cycle and DNA synthesis in cultured leaf discs of spinach

The effects of light on both the division cycle of chloroplasts and the synthesis of chloroplast DNA were investigated in cultured discs taken from the distal end of 2-centimeter spinach (Spinacia oleracea) leaves. Comparisons were made of discs cultured for a maximum of 4 days in a shaking liquid medium under continuous white light, darkness, and of discs cultured for 1 day in light following 3 days in darkness. In continuous white light the shortest generation time of chloroplasts observed in this study was 19.4 hours and the duration of spherical, ovoid, and dumbbell-shaped stages in the division cycle were 13.4, 2.8, and 3.1 hours, respectively. In darkness the generation times of chloroplasts extended to 51.5 hours. Under these conditions the duration of spherical, ovoid, and dumbbell-shaped stages were 22.8, 8.4, and 20.2 hours, respectively, suggesting that in darkness the separation of dumbbell-shaped chloroplasts may be the rate limiting step. When discs cultured in the dark were transferred to light, most dumbbell-shaped chloroplasts separated into daughter chloroplasts in less than an hour. Measurements of chloroplast DNA established that the cellular level of chloroplast DNA increased 10-fold over the 4 days of culture in continuous white light. Comparisons of the plastids of dark and light grown discs showed that the synthesis of chloroplast DNA was enhanced by light. Observations of DAPI stained dividing chloroplasts indicate that DNA partitioning can take place during the final stage of chloroplast division and that it does not precede plastid division.     

The Effect of Benzimidazole and Red Light on the Senescence of Detached Leaves of Oryza sativa cv. Ratna

Excised rice leaves (Oryza sativa L. cv. Ratna) werefloated on a 10^–3M solution of benzirnidazole under dark or continuous red light. Compared to the water control a degradation of chlorophyll, protein, RNA, DNA and a decrease in the activity of alkaline inorganic pyrophosphatase was delayed at the same time as an increase of α-amino nitrogen and the activity of acid inorganic pyrophosphatase occurred, Benzimidazole was more effective under red light than in the dark in retarding senescence. The possible role of inorganic pyrophosphatases is discussed with respect to biosynthesis during leaf senescence.     

Photoperiod and light quality effects on rate of dark respiration and on photosynthetic capacity in developing seedlings of Chenopodium rubrum

Development and acclimation of energy transduction were studied in seedlings of Chenopodium rubrum L. ecotype selection 184 (50° 10' N; 105° 35' W) in response to photomorphogenic and photoperiodic treatments. Dark respiration and photosynthetic capacity [nmol O₂ (pair of cotyledons)⁻¹ h⁻¹] were measured with an oxygen electrode. Changes in chloroplast ultrastructure were analyzed concomitantly. After germination, seedlings were grown at constant temperature either in darkness or in continuous light (white, red, far-red and blue) or were subjected to diurnal cycles of light/dark or changes in light quality. Dark respiration was low in far-red light treated seedlings. In red light treated seedlings dark respiration was high and the mean value did not depend on fluence rate or photoperiod. Blue light stimulated transitorily and modulated dark respiration in photoperiodic cycles. Photosynthetic capacity was reduced by far-red light and increased by red light. In response to blue light photosynthetic capacity increased, with indications of a requirement for continuous energy input. Phytochrome and a separate blue light receptor seemed to be involved. In continuous red light a clear cut circadian rhythm of dark respiration was observed. Blue light had a specific effect on chloroplast structure.     

Light induction of the Euglena chloroplast protein synthesis elongation factors: relative effectiveness of different wavelength ranges

The abilities of different wavelength ranges of light to promote the increase in the activities of the Euglena chloroplast protein synthesis elongation factors (EFs) during chloroplast biogenesis have been determined. Blue light was far more effective than either green light or red light in increasing the level of chloroplast EF-G, a nuclear encoded gene product. This observation suggests that the induction of EF-Gchl is under the control of the blue photoreceptor that has been identified in Euglena. Blue light was also the most effective wavelength range in facilitating the increase in EF-Ts, a nuclear gene product, and EF-Tu, a chloroplast gene product. However, red light and surprisingly green light were also effective. These results are not consistent with either of the known blue or blue/red photoreceptor systems in Euglena being the sole component involved in the light induction of these two factors and suggest that a green photoresponse may also be important in the development of the chloroplast. The specific activity of the Euglena mitochondrial protein biosynthetic translocase (EF-Gmt) decreased in cells exposed to light. Blue light caused an immediate decline in EF-Gmt activity; whereas, there was a temporal delay in the decrease in EF-Gmt activity when cells were exposed to either red or green light.     

Senescence in Detached Oat Leaves I. Changes in Free Amino Acid Levels

Changes in the levels of free amino acids have been measuredduring light and dark senescence of oat leaf segments. Leaveswere aged either on water, 5 ppm kinetin or 30 ppm abscisicacid. The patterns with which levels of individual amino acidschange differ a great deal in leaves senescing either in darkor light, signifying that different mechanisms may regulateoat leaf senescence in light and dark. Levels of serine andmost of the other amino acids that increase substantially duringdark senescence of oat leaves change parallel to mitochondrialrespiration. Kinetin depresses the rise in amino acids justas it does with respiration in the dark. The synthesis of serineproteases does not seem to be limited by the availability ofendogenous serine. The levels of glutamine increase dramaticallyin leaves kept in light (ca. 2,200% of initial value within7 days) but only a little in the dark, which may reflect a possiblerole of photorespiration during the senescence of oat leavesin the light. Abscisic acid enhances the release of amino acidsmore strongly in the light than dark. The senescence promotingeffect of abscisic acid in the light seems to bring about changesin amino acid levels similar to those that appear in leavessenescing on water in the dark. ¹ Present address: C.F. Kettering Research Laboratory, 150 EastSouth College Street, Yellow Springs, Ohio 45387, U.S.A. (Received June 24, 1981; Accepted October 30, 1981)     

Multiple actions of abscisic acid in senescence of oat leaves

The modifications induced by abscisic acid (ABA) on the senescence of oat leaves in darkness have been studied and are compared with its well-known effects in light. Contrary to the action in light, ABA preserves chlorophyll (Chl) in the dark almost as well as kinetin. Chlorophylla is decolorized more extensively thanb, and the content ofb is maintained by ABA almost at its initial level for 4 days. ABA also prevents proteolysis in darkness just as completely as chlorophyll loss, the relationship of both breakdown processes to ABA concentration being strictly log-linear over the range from 1 to 100 M. In line with this action, ABA inhibits formation of the neutral protease in the dark but not in the light. The data suggest that ABA and kinetin operate to preserve chlorophyll and protein by different mechanisms, since their actions are neither independent nor synergistic but actually interfere with one another. In this connection, protein values given by the Lowry and Bradford methods have been compared. In parallel with the effect on senescence, ABA slowly opens the stomata in the dark. This effect increases with time, and by day 3 the stomata in ABA are as open as in leaves on water in light. Thus all these effects of ABA in darkness are strikingly opposite to those commonly observed on leaves in natural lighting. In addition, ABA powerfully inhibits the formation of ethylene in the dark by the detached oat leaves, and this inhibition also tends to increase with time. Finally, a slight antagonism to ABA's action on senescence is exerted byp-coumaric acid in the light but not in the dark.     

Quantitative studies on ferredoxin in greening bean leaves

Two methods of measuring small amounts of the iron-sulphur protein ferredoxin are described. One involves measurements of the signal at g=1.96 produced by reduced ferredoxin in an e.p.r. (electron-paramagnetic-resonance) spectrometer; the other depends on the rate of ferredoxin-dependent electron transport in a chloroplast bioassay measured in an O(2) electrode. These methods of measurement were used to examine the development of ferredoxin during the greening of etiolated bean leaves. Ferredoxin is present in low concentrations in the leaves and cotyledons of 14-day-old etiolated beans (Phaseolus vulgaris L. var. Canadian Wonder), and develops in a linear manner with time when the leaves are illuminated. This synthesis appears to be independent of chlorophyll synthesis during the early stages of greening. However, the chlorophyll/ferredoxin ratio reaches a final value of approx. 360 irrespective of the light intensity, indicating the existence of a control mechanism operative in deciding the stoicheiometry of these components in the mature chloroplast. The ferredoxin synthesis appears to be light-dependent, and red light is the most effective in its promotion. The effect of red illumination is not reversed by far-red light, indicating the absence of a phytochrome control of ferredoxin synthesis. From experiments using specific inhibitors of chloroplast protein synthesis, it is concluded that ferredoxin is synthesized on cytoplasmic ribosomes.     

The Effect of Age on the Phytochrome-Mediated Chlorophyll Formation in Dark-grown Bean Leaves

The effect of red and far-red treatment on chlorophyll synthesis in dark-grown bean leaves was studied at various ages. Although the effect was pronounced in the old leaves, no effect was observed in the young ones (4 days old). In the 5-day old leaves a measurable effect of red light pretreatment can be observed, whereas the far-red reversal effect was not observed. — The length of the dark period between the red pretreatment and the continuous illumination is also age dependent. Leaves older than 6 days show a maximum at about six hours, while in the young leaves the red light effect increases with the time of dark incubation up to the 24 hours tested. — The reversal effect of far-red light on protochlorophyllide regeneration was also examined. The far-red light has no reversal effect on leaves younger than 6 days old, while on the old leaves it has such an effect.