Senescence program in rice (Oryza sautiva L.) leaves: Analysis of the blade of the second leaf at the tissue and cellular levels

Summary Previously, we showed that all greening mesophyll cells in the coleoptiles of rice (Oryza sauva L. cv. Nippon-bare) follow the identical program of senescence, which features the early degradation of chloroplast DNA (cpDNA) and subsequent nuclear condensation and disorganization. Following the coleoptile study, we analyzed the senescence-associated changes in the blade of the second leaf of rice at the tissue and cellular levels. Under the experimental conditions, the second leaf started to elongate rapidly 2 days after sowing and emerged on day 3. The blade of the second leaf completed its growth on day 4, although the sheath continued to grow until day 7. The amount of soluble protein and chlorophyll (Chl) per blade reached a maximum on day 7, and then declined. When blades were divided into three parts (the tip, mid-region, and base), levels of both soluble protein and Chl in the tip segment peaked earlier and decreased at a faster rate than in the other parts, demonstrating a longitudinal gradient of senescence from the tip to the base of the blade. In cross sections through the center of the tip and base segments, all the mesophyll cells senesced synchronously. They passed through the following steps in order: (i) degradation of cpDNA, (ii) decrease in the size of the chloroplast with degeneration of the chloroplast inner membranes, and (iii) condensation and disorganization of the nuclei. Although some differences were shown between the coleoptile and the second leaf in the timing and rate of each event, the order of those senescence-related events was conserved, suggesting an identical program of senescence exists in rice leaves.Abbreviations Chl chlorophyll - cpDNA chloroplast DNA - cpnucleoid chloroplast nucleoid - DAPI 4,6-diamidino-2-phenylindole - DiOC₇ 3,3-dihexyloxacarbocyanine iodide - VB vascular bundle - VIMPCS video-intensified microscope photon-counting system     

Three-dimensional analysis of the senescence program in rice (Oryza sativa L.) coleoptiles

The cytological sequence of senescence-related changes in coleoptiles of rice (Oryza sativa L. cv. Nippon-bare) was studied using fluorescence and electron microscopy. The coleoptiles reach full size 3 d after sowing, then rapidly senesce and wither completely by day 7. The interveinal region in cross-sections taken 1 mm from the tip of the coleoptile was selected for this analysis. Fluorescence microscopy using samples embedded in Technovit 7100 resin, electron microscopy and immunoelectron microscopy using DNA-specific antibodies were used to elucidate the sequence of senescence-related events. These occur in the following order: (i) degradation of the chloroplast DNA (cpDNA); (ii) condensation of the nucleus in conjunction with a decrease in the size of the dense-chromatin region, shrinkage of the chloroplast, degradation of ribulose-1, 5-bisphosphate carboxylase/oxygenase, dilation of the thylakoid membranes, increase in size and number of osmiophilic globules, condensation of the cytoplasm; (iii) disorganization of the nucleus, degeneration of the tonoplast; (iv) complete loss of the cytoplasmic components, distortion of the cell wall, invasion of microorganisms into the intercellular spaces and ultimately into the cell itself. The mitochondria maintain their ultrastructural integrity and a constant level of mitochondrial DNA throughout senescence. In young mesophyll cells, invagination of the tonoplast into the vacuole frequently occurs. This occasionally includes cytoplasmic material, which is digested in the vacuole as senescence proceeds. Immunoelectron microscopy suggests that cpDNA degradation involves rough digestion first, rather than rapid, direct decomposition of the DNA into nucleotides. The fragmented cpDNA is then dispersed throughout the chloroplast and cytoplasm. Received: 9 April 1998 / Accepted: 11 June 1998     

Preferential digestion of chloroplast nuclei (nucleoids) during senescence of the coleoptile ofOryza sativa

Summary The coleoptile ofOryza sativa develops, grows and ages within 4 days that follow imbibition. It is, thus, a very useful system for experimental analysis of the life cycle of organelles, for example, the development, growth and aging of plastids in higher plants. We examined the behavior and levels of DNA and chlorophyll in the plastid by epifluorescence microscopy after staining with 4-6-diamidino-2-phenylindole (DAPI), and by fluorimetry with a video-intensified-photon counting system (VIMPCS). The whitish yellow coleoptile appeared soon after imbibition and, between the first 24 and 60 h that followed imbibition, it grew markedly in a longitudinal direction, with concomitant elongation of the cells, and an increase in the volume of plastids and in the amount of DNA in the plastids. The chlorophyll content per plastid began to increase when the coleoptile turned green, 48 h after imbibition, and reached a plateau value when the coleoptile was 3.5 mm in length, 72 h after imbibition. More than 12 h later, the chlorophyll disappeared just before the breakdown of chloroplasts was initiated. Proplastids in young coleoptiles, contained a plastid nucleus which was located in the central area of the plastids and each nucleus consisted of approximately 6 copies of plastid DNA (ptDNA). The number of copies of ptDNA per plastid increased gradually, with a concomitant increase in the volume of the plastids after imbibition, and reached approximately 130 times the value in the young proplastids, 60 h after imbibition, when the plastid developed into a chloroplast. However, each plastid nucleus did not scatter throughout the entire interior region of each chloroplast. The disappearance of each plastid nucleus occurred more than 12 h before the degeneration of the chloroplasts. The number of plastids per cell increased from 10 to 15 in young coleoptiles within 12 h after imbibition. Yet the number remained constant throughout subsequent growth and aging of the coleoptile. Thus the preferential reduction in the amount of chloroplast DNA was not due to the division of the plastid but could, perhaps, be associated directly with the aging of the cells of the coleoptile which precedes senescence of the coleoptiles.     

Changes in the Number and Composition of Chloroplasts during Senescence of Mesophyll Cells of Attached and Detached Primary Leaves of Wheat (Triticum aestivum L.)

Changes in the number and composition of chloroplasts of mesophyll cells were followed during senescence of the primary leaf of wheat (Triticum aestivum L.). Senescence was due to the natural pattern of leaf ontogeny or was either induced by leaf detachment and incubation in darkness, or incubation of attached leaves in the dark. In each case discrete sections (1 centimeter) of the leaf, representing mesophyll cells of the basal, middle, and tip regions, were examined. For all treatments, senescence was characterized by a loss of chlorophyll and the protein ribulose 1,5-bisphosphate carboxylase (RuBPCase). Chloroplast number per mesophyll cell remained essentially constant during senescence. It was not until more than 80% of the plastid chlorophyll and RuBPCase was degraded that some reduction (22%) in chloroplast number per mesophyll cell was recorded and this was invariably in the mesophyll cells of the leaf tip. We conclude that these data are consistent with the idea that degradation occurs within the chloroplast and that all chloroplasts in a mesophyll cell senesce with a high degree of synchrony rather than each chloroplast senescing sequentially.     

The coleoptile chloroplast: Distinct distribution of xanthophyll cycle pigments,and enrichment in Photosystem II

Recent studies have shown that coleoptile chloroplasts operate the xanthophyll cycle, and that their zeaxanthin concentration co-varies with their sensitivity to blue light. The present study characterized the distribution of photosynthetic pigments in thylakoid pigment–protein complexes from dark-adapted and light-treated coleoptile and mesophyll chloroplasts, the low temperature fluorescence emission spectra, and the rates of PS I and PS II electron transport in both types of chloroplasts from 5-day-old corn seedlings. Pigments were extracted from isolated PS I holocomplex, LHC IIb trimeric and LHC II monomeric complexes and analyzed by HPLC. Chlorophyll distribution in coleoptile thylakoids showed 31% of the total collected Chl in PS I and 65% in the light harvesting complexes of PS II. In mesophyll thylakoids, the values were 44% and 54%, respectively. Mesophyll and coleoptile PS I holocomplexes differed in their Chl t a/Chl t b ratios (8.1 and 6.1, respectively) and -carotene content. In contrast, mesophyll and coleoptile LHC IIb trimers and LHC II monomers had similar Chl t a/Chl t b ratios and -carotene content. The three analyzed pigment–protein complexes from dark-adapted coleoptile chloroplasts contained zeaxanthin, whereas there was no detectable zeaxanthin in the complexes from dark-adapted mesophyll chloroplasts. In both chloroplast types, zeaxanthin and antheraxanthin increased markedly in the three pigment–protein complexes upon illumination, while violaxanthin decreased. In mesophyll thylakoids, zeaxanthin distribution as a percentage of the xanthophyll cycle pool was: LHC II monomers > LHC IIb trimers > PS I holocomplex, and in coleoptile thylakoids, it was: LHC IIb trimers > LHC II monomers = PS I holocomplex. Low temperature (77 K) fluorescence emission spectra showed that the 686 nm emission of coleoptile chloroplasts was approximately 50% larger than that of mesophyll chloroplasts when normalized at 734 nm. The pigment and fluorescence analysis data suggest that there is relatively more PS II per PS I and more LHC I per CC I in coleoptile chloroplasts than in mesophyll chloroplasts. Measurements of t in vitro uncoupled photosynthetic electron transport showed approximately 60% higher rates of electron flow through PS II in coleoptile chloroplasts than in mesophyll chloroplasts. Electron transport rates through PS I were similar in both chloroplast types. Thus, when compared to mesophyll chloroplasts, coleoptile chloroplasts have a distinct PS I pigment composition, a distinct chlorophyll distribution between PS I and PS II, a distinct zeaxanthin percentage distribution among thylakoid pigment–protein complexes, a higher PS II-related fluorescence emission, and higher PS II electron transport capacity. These characteristics may be associated with a sensory transducing role of coleoptile chloroplasts.     

Ultrastructure of Mesophyll Cells and Pigment Content in Senescing Leaves of Maize and Barley

Leaf senescence is a genetically regulated stage in the plant life cycle leading to death. Ultrastructural analysis of a particular region of the leaf and even of a particular mesophyll cell can give a clear picture of the time development of the process. In this study we found relations between changes in mesophyll cell ultrastructure and pigment concentration in every region of the leaf during leaf senescence in maize and barley. Our observations demonstrated that each mesophyll cell undergoes a similar senescence sequence of events: a) chromatin condensation, b) degradation of thylakoid membranes and an increase in the number of plastoglobules, c) damage to internal mitochondrial membrane and chloroplast destruction. Degradation of chloroplast structure is not fully correlated with changes in photosynthetic pigment content; chlorophyll and carotenoid content remained at a rather high level in the final stage of chloroplast destruction. We also compared the dynamics of leaf senescence between maize and barley. We showed that changes to the mesophyll cells do not occur at the same time in different parts of the leaf. The senescence damage begins at the base and moves to the top of the leaf. The dynamics of mesophyll cell senescence is different in leaves of both analyzed plant species; in the initial stages, the process was faster in barley whereas in the later stages the process occurred more quickly in maize. At the final stage, the oldest barley mesophyll cells were more damaged than maize cells of the same age.     

Effects of γ-irradiation on elongation and indole-3-acetic acid level of maize (Zea mays) coleoptiles

The effects of γ-irradiation on elongation and the level of indole-3-acetic acid (IAA) of maize (Zea mays) coleoptiles were investigated. When 3-day-old seedlings of maize were exposed to γ-radiation lower than 1 kGy, a temporal retardation of coleoptile elongation was induced. This retardation was at least partly ascribed to a temporal decrease in the amount of free IAA in coleoptile tips on the basis of the following facts: (1) the reactivity to IAA of the elongating coleoptile cells was not altered by irradiation; (2) endogenous IAA level in the tip of irradiated coleoptiles was at first unchanged, but then declined before returning to nearly the same level as that of the non-irradiated control; and (3) the amount of IAA that diffused from coleoptile tip sections showed a similar pattern to that of endogenous IAA. The rate of conversion between free and conjugated IAA was not significantly affected by irradiation. These results suggest that a temporal inhibition of maize coleoptile elongation induced by γ-irradiation can be ascribed to the reduction of endogenous IAA level in the coleoptile tip, and this may originate from the modulation in the rate of IAA biosynthesis or catabolism.     

Senescence in the nongreening region of the rice (Oryza sativa) coleoptile

Summary The coleoptile of rice (Oryza sativa L. cv. Nippon-bare) emerges from the imbibed seed on day 2 after sowing and ceases its growth on day 3. In cross section, the cells near the outer epidermis turn into green between days 2 and 3, while those near the inner epidermis remain colorless. In this study, the complete process of the development in the nongreening cells in the coleoptile was examined by fluorescence and electron microscopy. Embryonic morphology on day 0 was rapidly converted into the differentiated greening or nongreening cells between days 1 and 2. Senescence in the inner, nongreening region first appeared on day 4 in the third or fourth cell layer from the inner epidermis and then spread towards both the inner and the outer epidermis, and the inner cells collapsed completely before the outer cells senesced. Cells adjacent to the inner epidermis, which senesced slowly, followed a sequence of events during development: (1) degradation of plastid DNA; (2) dispersal of nuclear chromatin, differentiation of plastids into amyloplasts, degradation of mitochondrial DNA; (3) degradation of the starch in amyloplasts; (4) disorganization of plastids; (5) condensation of the nucleus, shrinkage of mitochondria; (6) complete loss of cellular components, distortion of cell walls. In the interior cells, the early events including degeneration of plastid DNA and mitochondrial DNA occurred in parallel with those in the cells adjacent to the inner epidermis, yet rapid collapse of all the cellular components proceeded between days 3 and 5, and nuclear condensation could not be detected.Abbreviations cpDNA chloroplast DNA - DAPI 4,6-diamidino-2-phenylindole - DiOC₇ 3,3-dihexyloxacarbocyanine - IE inner epidermis - mtDNA mitochondrial DNA - mt-nucleoid mitochondrial nucleoid - OE outer epidermis - ptDNA plastid DNA - pt-nucleoid plastid nucleoid     

Chloroplast Development in Rye Coleoptiles

In the parenchyma cells of 1-d-old dark-grown rye coleoptiles (Secale cereale) proplastids occurred which sometimes contained starch grains. During coleoptile growth in darkness starch-filled amyloplasts are formed from the preexisting proplastids. No prolamellar bodies were observed in the stroma of the plastids of the etiolated coleoptile. After irradiation of 3-d-old etiolated coleoptiles with continuous white light three different types of plastids occurred. In the epidermal cells proplastids were observed. The parenchyma cells below the stomata of the outer epidermis (above the two vascular bundles) contained mature, spindle-shaped chloroplasts with a well-developed thylakoid system. In the parenchyma cells that surround the vascular bundles amyloplasts with some thylakoid membranes (chloroamyloplasts) occurred. The mesophyll cells of the primary leaves of dark-grown seedlings contained etioplasts with large prolamellar bodies. In the primary leaves of irradiated plants chloroplasts similar to those of the parenchyma cells of the coleoptile were observed. Our results show that the rye coleoptile, which grows underground as a heterotrophic organ, is capable of developing mature chloroplasts upon reaching the light above the soil surface. The significance of this expression of photosynthetic capacity for the carbon economy of the developing seedling is discussed.     

Coleoptile Senescence in Rice (Oryza sativa L.)

We investigated the cellular events associated with cell deathin the coleoptile of rice plants (Oryza sativa L.). Seeds germinatedunder submergence produced coleoptiles that were more elongatedthan those grown under aerobic conditions. Transfer of seedlingsto aerobic conditions was associated with coleoptile opening(i.e. splitting) due to death of specific cells in the sideof the organ. Another type of cell death occurred in the formationof lysigenous aerenchyma. Senescence of the coleoptile was alsonoted, during which discolouration of the chlorophyll and tissuebrowning were apparent. DNA fragmentation was observed by deoxynucleotidyltransferase-mediateddUTP nick end labelling (TUNEL) assay, and further confirmedby the appearance of oligonucleosomal DNA ladders in senescentcoleoptile cells. Two nucleases (Nuc-a and Nuc-b) were detectedby in-gel-assay from proteins isolated from coleoptiles. Nuc-a,commonly observed in three cell death phases required eitherCa²⁺or Mg²⁺, whereas Nuc-b which appeared during senescencerequired both Ca²⁺and Mg²⁺. Both nucleases were strongly inhibitedby Zn²⁺. Copyright 2000 Annals of Botany Company Aerenchyma, rice, cell death, coleoptile, fragmentation, nuclease, Oryza sativa, senescence, split, submergence, TUNEL     

Leaf anatomy and chloroplast ultrastructure of Mn-deficient orange plants

Leaf samples of Mn-deficient and Mn-sufficient (control) ‘Navelate’ orange plants grown in a greenhouse were taken to investigate the effects of Mn deficiency in leaf structure and chloroplast ultrastructure. Total leaf chlorophyll concentration was significantly lower in Mn-deficient plants than in control ones. Entire lamina thickness was not altered due to Mn deficiency. However, Mn deficiency resulted in disorganization of mesophyll cells, mainly of palisade parenchyma cells. The number of mesophyll chloroplasts per cellular area and their length were both affected negatively. The membranous system of chloroplasts was also disorganized. The percentages of starch grains and plastoglobuli per chloroplast of Mn-deficient leaves were significantly greater than those of control leaves.     

The hypocotyl chloroplast plays a role in phototropic bending of Arabidopsis seedlings: developmental and genetic evidence

Chloroplasts of guard cells and coleoptiles have been implicated in the sensory transduction of blue light. The present study was aimed at establishing whether the chloroplast of the hypocotyl from Arabidopsis, another blue light-responding organ, has similar characteristics to that of sensory-transducing guard cell and coleoptile chloroplasts. Results showed that the phototropic curvature and arch length induced by blue light in Arabidopsis seedlings matched the distribution of mature chloroplasts in the bending hypocotyl. The bending arch consistently included the region of the hypocotyl containing mature chloroplasts, and never extended beyond that region. Manipulation of the extent of greening of dark-grown hypocotyls by varying red light pretreatments elicited blue light-stimulated curvatures and arch lengths that depended on the duration of the red light pretreatment and on the distribution of mature chloroplasts in the hypocotyl. Albino psd2 mutants of Arabidopsis, which lack mature chloroplasts, are devoid of phototropic sensitivity under conditions in which wild-type seedlings show large curvatures. The star mutant of Arabidopsis has a delayed greening and a delayed phototropic response as compared with wild type. Measurements of photosynthetic oxygen evolution and carbon fixation, dark respiration, and light-dependent zeaxanthin formation in the hypocotyl showed features similar to those of guard cells and coleoptiles, and distinctly different from those of mesophyll tissue. These results indicate that the hypocotyl chloroplast has characteristics similar to those associated with guard cell and coleoptile chloroplasts, and that phototropic bending of Arabidopsis hypocotyls appears to require mature chloroplasts.     

HISTOLOGICAL STUDIES ON THE COLEOPTILE. I. TISSUE AND CELL TYPES IN THE COLEOPTILE TIP

The uppermost 1-4 mm of 25-mm coleoptiles of oats and wheat have been studied at the optical microscope level, using newer histological methods and sections 1-4 μ thick. The outer epidermal wall, which shows very fine wrinkling, is continuous with the thinner wall of the inner epidermis through the pore. The cells of both epidermal layers have acidophilic cytoplasm with long transvacuolar strands. Both inner and outer epidermis have stomata, those of the outer epidermis having kidney-shaped guard cells like those of dicotyledons. The guard-cell walls are lignified in their inner layers only and are thinly cuticularized. In the vascular bundles the sieve tubes terminate apically about 250 μ below the end of the xylem; the xylem in turn terminates about 400 μ below the extreme apex. A number of clearly undifferentiated cells, with highly basophilic cytoplasm and many mitochondria, separate the xylem elements from the inner epidermis. Towards the outer epidermis there are a few sieve elements, each of which is associated with a special cell having an elongated nucleus supported on fine cytoplasmic strands. The parenchyma of both the tip and the shaft of the coleoptile are generally interpenetrated by air-spaces, but where they are adjacent to the inner epidermis there is heavy interposition of readily stained intercellular material, especially in Triticum. Plastids are widely distributed throughout the tissue, but their greening in light takes place preferentially towards the phloem side of the vascular bundles. The observations are discussed in reference to earlier literature and with regard to the function of the coleoptile as a protecting and guiding organ for the shoot system of the seedling.     

Time course of phytochrome-mediated changes in growth gradients on coleoptiles of Triticum aestivum L.

The length of parenchyma cells along the axis of dark-grown coleoptiles of Triticum aestivum L. and the pattern of competence for red-light-(R-) induced stimulation or inhibition of cell elongation in the course of coleoptile development were determined by microscopic measurements in a file of 240 cells from the tip to the base. On the basis of these measurements distinct zones (responding in different ways to R) were selected for studying the early time course of phytochrome-mediated growth-rate changes in intact coleoptiles by use of a sensitive transducer system. Between 2 d and 4 d after sowing dark-grown coleoptiles showed a graded incline in cell growth activity from the apex to the base (growth gradient). Whereas cell elongation in the coleoptile base ceased 4 d after sowing, cell elongation speeded up in the tip and middle region at that time. Those cells that grew slowly in darkness (tip and middle region between 2d and 3 d after sowing) were stimulated in growth by R-pulse irradiation (1 min R, 660 nm, 1000 J · m^–2). In contrast, the growth of fast-growing cells (base between 2 d and 4 d after sowing, tip and middle region between 4 d and 5 d after sowing) was inhibited by R. However, the starting time for R-induced growth changes was different for different coleoptile zones. The respective data point to the storage of a phytochrome-mediated signal in the cells of the middle region, until these cells become competent to respond to it; alternatively, Pfr, the far-red-light-absorbing form of phytochrome, may be stored in a stable form. Continuous recordings on the effect of R, far-red (FR) and R/FR on the zonal growth responses were made on intact coleoptiles, selected 3 d after sowing. During a 5-h investigation period the R-induced changes in growth rate could be divided into two phases: (i) A transient growth inhibition which started approx. 15 min after R. This response was qualitatively the same in all coleoptile zones investigated (tip, middle region, base). (ii) Zonal-specific growth responses which became measurable approx. 2.5 h after R, i.e. growth promotion in the tip, growth inhibition in the base and an adaptation of growth rate to the dark control level in the middle region. The R-induced growth rate changes were reversible by FR for both phases. Additional growth experiments on excised coleoptile segments under R and auxin application indicated that the zonal-specific growth promotion or inhibition may be not mediated by an influence of R on the auxin level.Abbreviations FR far-red light - Pfr far-red-light-absorbing form of phytochrome - R red light The technical assistance of Mrs. B. Liebe is gratefully acknowledged.     

Endonuclease activities in the coleoptile and the first leaf of developing etiolated wheat seedlings

DNase activity in coleoptiles and the first leaf apices of winter wheat (Triticum aestivum L., cv. Mironovskaya 808) etiolated seedlings was found to increase significantly during seedling growth, peaking on the eighth day of plant development. The maximum of DNase activity was coincident with apoptotic internucleosomal DNA fragmentation in these organs. Wheat endonucleases are capable of hydrolyzing both singleand double-stranded DNA of various origins. The leaf and coleoptiles were found to exhibit nuclease activities that hydrolyzed the lambda phage DNA with N⁶-methyladenine and 5-methylcytosine more actively compared to the hydrolysis of similar unmethylated DNAs. Thus, the endonucleases of wheat seedlings are sensitive to the methylation status of their substrate DNAs. The leaves and coleoptiles exhibited both Ca²⁺/Mg²⁺- and Zn²⁺-dependent nuclease activities that underwent differential changes during development and senescence of seedling organs. EDTA at a concentration of 50 mM fully inhibited the total DNase activity. Electrophoretic heterogeneity was observed for DNase activities operating simultaneously in the coleoptile and the first leaf at different stages of seedling development. Proteins exhibiting DNase activity (16–80 kD mol wt) were revealed in the first leaf and the coleoptile; these proteins were mostly nucleases with the pH optimum around 7.0. Some endonucleases (mol wts of 36, 39, and 28 kD) were present in both organs of the seedling. Some other DNases (mol wts of 16, 56, and about 80 kD) were found in the coleoptile; these DNases hydrolyzed DNA in the nucleus at terminal stages of apoptosis. Different suites of DNase activities were revealed in the nucleus and the cytoplasm, the nuclear DNase activities being more diverse than the cytoplasmic ones. Thus, the cellular (organspecific) and subcellular heterogeneity in composition and activities of DNases has been revealed in wheat plants. These DNases undergo specific changes during seedling development, serving at various stages of programmed cell death in seedling tissues.     

Gradients of polyamines and their biosynthetic enzymes in coleoptiles and roots of corn

The distribution of diamines, polyamines, and their biosynthetic enzymes arginine decarboxylase and ornithine decarboxylase in roots and coleoptiles of corn (Zea mays var Golden Cross Bantam) seedlings have been determined. Putrescine content, expressed on either a fresh weight or protein basis, increases from the tip to the base in both roots and coleoptiles. In roots, this gradient is paralleled by an activity gradient of arginine and ornithine decarboxylases. Spermidine is distributed equally along the length of coleoptiles; in roots, this is true only on a protein basis. Free spermine is detectable only in the root tip, but a bound form is present throughout the root and coleoptile. The results are compared with gradients in protein and DNA content and discussed in relation to the possible cellular roles of polyamines.     

Cytokinin promotes catalase and ascorbate peroxidase activities and preserves the chloroplast integrity during dark-senescence

Increased oxidative stress displayed during dark-senescence of wheat leaves (Triticum aestivum L.) is caused not only by the increased levels of radicals but also by a loss of antioxidant capacity. Mature leaves were incubated in 6-benzylaminopurine (BAP 10⁻⁴ M) or water (control) during 6 d in the dark. The senescence-delaying effect of BAP was associated with the retention of the chloroplast structure, 60% of the initial content of chlorophyll (Chl) and 77% of the initial content of protein. BAP reduced the degradation of the light-harvesting chlorophyll a/b binding protein (LHCP-2), and the large (LSU) and small subunits (SSU) of Rubisco. Our results indicated that the presence of the NADPH:protochlorophyllide oxidoreductase (POR, EC.1.6.99.1) was not promoted by the cytokinin, leading to the conclusion that BAP maintains the level of Chl, preventing its degradation, rather than inducing Chl biosynthesis. The internal structure of chloroplasts was maintained in BAP-treated leaves for up to 6 d, with well-organized grana thylakoids and small plastoglobuli; in contrast, chloroplasts of control leaves deteriorated rapidly from day 4 with disorganized internal membranes, and more and larger plastoglobuli. BAP increased the activities of catalase (CAT, EC 1.11.1.6) and ascorbate peroxidase (APX, EC 1.11.1.11) and reduced the level of H₂O₂ in the delayed-senescence tissue. The present research indicates that BAP reduces levels of reactive oxygen species (ROS), and enhances the activity of antioxidant enzymes (CAT, APX). Our results suggest that BAP protects the cell membranes and the photosynthetic machinery from oxidative damage during delay of senescence in the dark.     

Can Lateral Redistribution of Auxin Account for Phototropism of Maize Coleoptiles?

Elongation growth of intact, red-light grown maize (Zea mays L.) coleoptiles was studied by applying a small spot of an indole acetic acid (IAA)-lanolin mixture to the coleoptile tip. We report that: (a) endogenous auxin is limiting for growth, (b) an approximately linear relation holds between auxin concentration and growth rate over a range which spans those rates occurring in phototropism, and (c) an auxin gradient established at the coleoptile tip is well sustained during its basipetal transport. We argue that the growth differential underlying coleoptile phototropism (first-positive curvature) can be explained by redistribution of auxin at the coleoptile tip.     

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 C(3)-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 interconnected.     

Degradation of chloroplast DNA in second leaves of rice (Oryza sativa) before leaf yellowing

Summary The second leaf ofOryza sativa develops, grows and ages within the 10 days that follow imbibition under our controlled continuous-light conditions. Proplastids in the leaf cells develop, mature to become chloroplasts and then age and disintegrate. In an examination of this life process, we studied first the behavior and the number of copies of plastid DNA and levels of chlorophyll by epifluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI), and by fluorimetry with a video-intensified microscope photon-counting system (VIMPCS). The results indicated that the number of copies of the plastid DNA per plastid increased and reached to plateau value of approximately 100 at the time when the elongation of the mesophyll cells and the enlargement of chloroplasts ceased 96 h after imbibition. However, 24 h later, the number of copies of plastid DNA per chloroplast began to decrease and fell rapidly to approximately 30 copies within 168 h after imbibition. Our examination of the number of chloroplasts per mesophyll cell indicated that no division of chloroplasts occurred more than 72 h after imbibition. The results suggest that the decrease in number of copies of plastid DNA per chloroplast was not due to an increase in the number of chloroplasts, but that this decrease was caused by degradation by unidentified enzymes. Since visible senescence of leaves, which was characterized by development of a yellowish color, began 168 h after imbibition, the degradation of plastid DNA seemed to occur 48 h before the visible leaf senescence. When we tested the nucleolytic activities in the second leaves after imbibition by digestion of plasmids in vitro and DNA-SDS polyacrylamide gel electrophoresis, five Ca²⁺–, four Zn²⁺–, and four Mn²⁺–dependent nucleases were detected in the leaf blades, and one of the Ca²⁺–, two of the Zn²⁺–, and two of the Mn²⁺–dependent nucleases were also identified in a purified preparation of intact chloroplasts. When the activity of the Zn²⁺–dependent nucleases (51 kDa and 13 kDa) increased markedly, degradation of the plastid DNA occurred. These results suggest that the destruction of chloroplast DNA, which occurs approximately 48 h before leaf yellowing, could be due to the activation of some metallo-nucleases and, furthermore, this enzymatic degradation propels the leaf towards senescence.