A study on senescence in tobacco leaf disks I. Inhibition by benzylaminopurine of decrease in protein level

The drop in protein level as an index of senescence in tobaccoleaf disks was examined in the dark in the presence of BA and/orinhibitors of protein synthesis. Cycloheximide at 10^–6–10^–5M and 10^–5 g/ml actinomycin-D accelerated the senescenceand interfered with the anti-senescence action of 10^–6M BA. Chloramphenicol (10^–8– 10^–4 M) and puromycin(10^–8–10^–4M) did not modify the senescenceor the action of BA. Cycloheximide was more effective at earlierstages of the dark culture of the disks. The rate of ¹⁴C-leucineincorporation into the protein fraction was increased by BAand decreased with senescence, and this drop was removed byBA. The incorporation was also suppressed by cycloheximide equallyin the presence and absence of BA. The drop in labeled proteinof the disks during the chasing period was retarded by not onlyBA but cycloheximide also. The conclusion reached, based onthese and relevant findings, was that BA affects the anti-senescenceaction by promoting synthesis and at the same time inhibitingdegradation of protein. (Received December 2, 1974; )     

Chloroplasts regulate leaf senescence: delayed senescence in transgenic ndhF-defective tobacco

Mitochondrial involvement has not been identified in the programmed cell death (PCD) of leaf senescence which suggests that processes such as those involving reactive oxygen species (ROS) are controlled by chloroplasts. We report that transgenic tobacco (DeltandhF), with the plastid ndhF gene knocked-out, shows low levels of the plastid Ndh complex, homologous to mitochondrial complex I, and more than a 30-day-delay in leaf senescence with respect to wt. The comparison of activities and protein levels and analyses of genetic and phenotypic traits of wtxDeltandhF crosses indicate that regulatory roles of mitochondria in animal PCD are assumed by chloroplasts in leaf senescence. The Ndh complex would increase the reduction level of electron transporters and the generation of ROS. Chloroplastic control of leaf senescence provides a nonclassical model of PCD and reveals an unexpected role of the plastid ndh genes that are present in most higher plants.     

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.     

Effects of senescence on chloroplasts of the tobacco leaf

Chloroplast fine structure, oxygen evolution and plastid composition studies were made on ageing leaves over a 30-day period. Plastids in leaves which were approaching maximum levels of chlorophyll generated large numbers of alveolate suborganelles, named plastosomes, which were released into the cytoplasm. There was some evidence that these were of lipid composition. The relationship between net oxygen evolution by leaf discs and chlorophyll decline in leaf tissue varied with age of leaf or its position on the stalk. In no case studied was there a continuous parallel between the two processes. The rate of oxygen used nearly doubled for some leaves during the 30-day period. The volume of plastid pellet obtained from homogenized leaf samples rapidly declined immediately after maximum chlorophyll levels were reached. Plastid chlorophyll and protein also declined but not at the same rate. Absorption spectra of methanol extracts showed no qualitative change in pigments during the ageing process.     

Studies on senescing tobacco leaf disks with special reference to peroxidase

Summary Senescing tobacco leaf disks were treated with hydroxyproline (hypro). Chlorophyll breakdown and -amino-nitrogen increase were partially inhibited, but the decrease in soluble protein was stimulated. The normal increase in absolute acid-phosphatase activity was reversed, but the increase in peroxidase activity occurred although after a lag phase. Kinetin inhibition of senescence was reversed by hypro and kinetin actually reinforced the effects of hypro. Proline only partially overcame the effects of hypro plus kinetin. In the presence of kinetin, proline considerably stimulated the increase in peroxidase activity.Discussed are the way in which hypro may be acting, and the possibility of peroxidase being involved in proline hydroxylation.     

Cytokinin biochemistry in relation to leaf senescence. VIII. Translocation, metabolism and biosynthesis of cytokinins in relation to sequential leaf senescence of tobacco

Cytokinin bases (zeatin and dihydrozeatin) and ribosides (zeatin riboside and dihydrozeatin riboside) were identified as major cytokinins in tobacco xylem sap by radioimmunoassay. When ³H-labelled zeatin riboside or dihydrozeatin riboside were supplied to tobacco plants via the xylem, leaves of differing maturity did not differ appreciably in level of radioactivity or in metabolism of the cytokinin. The major metabolites of zeatin riboside in leaves were adenine, adenosine and adenine nucleotides, whereas that of dihydrozeatin riboside was dihydrozeatin 7-glucoside. Incorporation of [¹⁴C]adenine into zeatin was evident in upper green leaves. indicating that young leaves have the capacity to synthesize cytokinins in situ. In contrast, fully expanded green leaves and senescing tobacco leaves exhibited little or no incorporation of [¹⁴C]adenine into cytokinins. This difference in cytokinin biosynthetic capacity may contribute to the differing cytokinin levels in leaves of different matirity, and may participate in control of sequential leaf senescence in tobacco.     

Cytokinin biochemistry in relation to leaf senescence. VI. Effect of nitrogenous nutrients on cytokinin levels and senescence of tobacco leaves

The effect of nitrogenous nutrients on endogeneous cytokinins and senescence of tobacco leaves was investigated. Ammonium nitrate was the most effective in retarding senescence and its activity was attributed principally to NH₄⁺ ions. Repeated applications or a continuous supply of ammonium nitrate was required for maximal retardation of tobacco leaf senescence. Ammonium nitrate solution supplied via the petioles reduced the senescence retarding effect of dihydrozeatin applied directly to the laminae of detached tobacco leaves. Ammonium nitrate also elevated the endogenous levels of cytokinins (especially zeatin and dihydrozeatin) particularly in growing tobacco leaves excised from near the apex of the plant. Ammonium nitrate induced retardation of leaf senescence may be mediated at least partly by its effect on foliar cytokinin content.     

Cytokinin biochemistry in relation to leaf senescence. VII. Endogenous cytokinin levels and exogenous applications of cytokinins in relation to sequential leaf senescence of tobacco

The cytokinin complex in tobacco leaves of various maturities was characterized by radioimmunoassay and mass spectrometry. Zeatin was the major base, whereas zeatin riboside was identified as the main riboside. in leaves of all maturities studied. Relative to upper younger leaves, the basal yellow leaves had reduced levels of both cytokinin bases and ribosides. Exogenous applications of dihydrozeatin and zeatin to detached tobacco leaves in amounts sufficient to delay senescence, elevated cytokinin base and riboside levels 2–5 fold. Presenescent and senescent leaves of intact plants showed quantitatively similar changes in cytokinin content. which therefore appear to be of significance in control of senescence. When supplied exogenously, the principal cytokinin bases found to occur in tobacco leaves (zeatin and dihydrozeatin) were markedly more effective than auxins and gibberellic acid in retarding senescence. Localised application of cytokinins to leaf blades of detopped plants was much less effective than application to intact plants. The cytokinin induced senescence retardation in tobacco leaves was independent of effects on directed metabolite transport. Evidence that endogenous levels of active cytokinins in intact tobacco leaves are involved in control of sequential leaf senescence is discussed.     

Photorespiration: stimulation of glycine decarboxylation by oxygen in tobacco leaf disks and corn leaf segments

Glycine decarboxylation, shown by us as an intermediate stepin CO₂ evolution during photorespiration, was enhanced by oxygentensions of up to 100%, whereas CO₂ evolution from glucose didnot increase when oxygen tensions were raised above 20%. Thus,we concluded that enhancement of photorespiration by oxygenis not only due to stimulation of glycolate oxidation, but alsoto stimulation of glycine decarboxylation. ¹ This work was reported at the Annual Meeting (1971) of theJapanese Society of Plant Physiologists in Okayama. ² The Okayama Tobacco Experiment Station, Japan Monopoly Corporation,Tamashima, Kurashiki, Okayama. ³ The Hatano Tobacco Experiment Station, Japan Monopoly Corporation,Naganuki, Hatano, Kanagawa. (Received October 19, 1971; )     

Ethylene as a regulator of senescence in tobacco leaf discs

The regulatory role of ethylene in leaf senescence was studied with excised tobacco leaf discs which were allowed to senesce in darkness. Exogenous ethylene, applied during the first 24 hours of senescence, enhanced chlorophyll loss without accelerating the climacteric-like pattern of rise in both ethylene and CO₂, which occurred in the advanced stage of leaf senescence. Rates of both ethylene and CO₂ evolution increased in the ethylene-treated leaf discs, especially during the first 3 days of senescence. The rhizobitoxine analog, aminoethoxy vinyl glycine, markedly inhibited ethylene production and reduced respiration and chlorophyll loss. Pretreatment of leaf discs with Ag⁺ or enrichment of the atmosphere with 5 to 10% CO₂ reduced chlorophyll loss, reduced rate of respiration, and delayed the climacteric-like rise in both ethylene and respiration. Ag⁺ was much more effective than CO₂ in retarding leaf senescence. Despite their senescence-retarding effect, Ag⁺ and CO₂, which are known to block ethylene action, stimulated ethylene production by the leaf discs during the first 3 days of the senescing period; Ag⁺ was more effective than CO₂. The results suggest that although ethylene production decreases prior to the climacteric-like rise during the later stages of senescence, endogenous ethylene plays a considerable role throughout the senescence process, presumably by interacting with other hormones participating in leaf senescence.     

Nucleotide pools in leaf and root tissue of tobacco plants: Influence of leaf senescence

The content of the different ribonucleotides, nucleoside diphosphate sugars, NAD⁺ and NADP⁺ was determined in the leaves and roots of single plants of Nicotiana tabacum L. cv. Samsun using a new procedure. The determination makes use of extraction with HClO₄ followed by extract purification and separation by a combination of anion-exchange and reversed phase high performance liquid chromatography. The largest pools were the adenine nucleotides and the uracil nucleotides with nucleoside diphosphate sugars as the main fraction. Leaf senescence was accompanied by a strong reduction of the nucleotide content of the leaves, but the adenine nucleotide-derived energy charge remained at a high level and even increased at the later stages of senescence. This indicates a balanced metabolism also in the older leaves and excludes the possibility that leaf senescence is triggered by the energy charge.     

Chloroplast and cytoplasmic enzymes 1, 2, 3. Subunit structure of pea leaf aldolases.

The pea leaf chloroplastic and cytoplasmic forms of aldolase are very similar in structure. The subunit molecular weights determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate are approximately 37.000. Both aldolases appear to terminate in the same sequence, SerAlaTyrCOOH, and the amino-terminal sequence H₂NGlySerTyrAla was obtained for each. The previously reported differences in kinetic properties and in isoelectric points of the two pea leaf enzymes probably are the consequence of minor differences in amino acid composition or conformation.     

Chloroplast substitution overcomes leaf chlorosis in a Moricandia arvensis-based cytoplasmic male sterile Brassica juncea

 A male sterile Brassica juncea line based on Moricandia arvensis cytoplasm was developed previously by backcrossing the somatic hybrid M. arvensis+B. juncea, and the gene for restoring fertility was introgressed. The CMS line is very severely chlorotic because of the presence of alien chloroplasts and flowering is delayed by 30–40 days, making it unsuitable for the exploitation of heterosis. We have resorted to another cycle of protoplast fusion between green fertile B. juncea and chlorotic male sterile B. juncea, and developed green male-sterile plants. Molecular analysis revealed that in green male-sterile plants chloroplasts of M. arvensis origin were substituted by those from B. juncea, giving rise to intergeneric cytoplasmic hybrids with mitochondria of M. arvensis origin. With the development of dark-green male-sterile plants, the CMS fertility restoration system is suitable for the production of hybrid mustard. Received: 23 February 1998 / Accepted: 12 May 1998     

Reactive oxygen species formation and cell death in catalase-deficient tobacco leaf disks exposed to cadmium

The physiological responses of tobacco (Nicotiana tabacum L.) to oxidative stress induced by cadmium were examined with respect to reactive oxygen species (ROS) formation, antioxidant enzymes activities, and cell death appearance in wild-type SR1 and catalase-deficient CAT1AS plants. Leaf disks treated with 100 or 500 µM CdCl₂ increased Evans blue staining and leakage of electrolytes in SR1 or CAT1AS plants, more pronouncedly in the transgenic cultivar, but without evidence of lipid peroxidation in any of the cultivars compared to controls. Cadmium significantly reduced the NADPH oxidase-dependent O ₂ ^? formation in a dose dependent manner in SR1 very strongly at 500 µM (to 5% of the activity in the nontreated SR1 leaf disks). In CAT1AS, the NADPH oxidase activity was constitutively reduced at 50% with respect to that of SR1, but the magnitude of the decay was less prominent in this cultivar, reaching an average of 64% of the C at 21 h, for both Cd concentrations. Hydrogen peroxide formation was only slightly increased in SR1 or CAT1AS leaf disks at 21 h of exposure compared to the respective controls. Cd increased superoxide dismutase activity more than six times at 21 h in CAT1AS, but not in SR1 and reduced catalase activity by 59% at 21 h of treatment only in SR1 plants. Despite that catalase expression was constitutively lower in CATAS1 compared to SR1 nontreated leaf disks, 500 µM CdCl₂ almost doubled it only in CAT1AS at 21 h. The mechanisms underlying Cd-induced cell death were possibly not related exclusively to ROS formation or detoxification in tobacco SR1 or CAT1AS plants.