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.     

Identification of the Ndh (NAD(P)H-plastoquinone-oxidoreductase) complex in etioplast membranes of barley: changes during photomorphogenesis of chloroplasts

In the last few years the presence in thylakoid membranes of chloroplasts of a NAD(P)H-plastoquinone oxidoreductase complex (Ndh complex) homologous to mitochondrial complex I has been well established. Herein, we report the identification of the Ndh complex in barley etioplast membranes. Two plastid DNA-encoded polypeptides of the Ndh complex (NDH-A and NDH-F) were relatively more abundant in etioplast membranes than in thylakoids from greening chloroplasts. Conversion of etioplast into chloroplast, after light exposure of barley seedlings grown in the dark, was accompanied by a decrease in the NADH dehydrogenase activity associated to plastid membranes. Using native-PAGE and immunolabelling techniques we have determined that a NADH specific dehydrogenase activity associated with plastid membranes, which was more active in etioplasts than in greening chloroplasts, contained the NDH-A and NDH-F polypeptides. These results complemented by those obtained through blue-native-PAGE indicated that NDH-A and NDH-F polypeptides are part of a 580 kDa NADH dependent dehydrogenase complex present in etioplast membranes. This finding proves that accumulation of the Ndh complex is independent of light. The decrease in the relative levels and specific activity of this complex during the transition from etioplast to chloroplasts was accompanied by a parallel decrease in the specific activity of peroxidase associated to plastid membranes. Based on the mentioned observations it is proposed that an electron transport chain from NADH to H2O2 could be active in barley etioplasts.     

Location,expression and orientation of the putative chlororespiratory enzymes,Ndh and IMMUTANS,in higher-plant plastids

The chloroplasts of many plants contain not only the photosynthetic electron transport chain, but also two enzymes, Ndh and IMMUTANS, which might participate in a chloroplast respiratory chain. IMMUTANS encodes a protein with strong similarities to the mitochondrial alternative oxidase and hence is likely to be a plastoquinol oxidase. The Ndh complex is a homologue of complex I of mitochondria and eubacteria and is considered to be a plastoquinone reductase. As yet these components have not been purified to homogeneity and their expression and orientation within the thylakoid remain ill-defined. Here we show that the IMMUTANS protein, like the Ndh complex, is a minor component of the thylakoid membrane and is localised to the stromal lamellae. Protease digestion of intact and broken thylakoids indicates that both Ndh and IMMUTANS are orientated towards the stromal phase of the membrane in Spinacia oleracea L. Such an orientation is consistent with a role for the Ndh complex in the energisation of the plastid membrane. In expression studies we show that IMMUTANS and the Ndh complex are present throughout the development of both Pisum sativum L. cv Progress No. 9 and Arabidopsis thaliana (L.) Heynh. leaves, from early expansion to early senescence. Interestingly, both the Ndh complex and the IMMUTANS protein accumulate within etiolated leaf tissue, lacking the photosystem II complex, consistent with roles outside photosynthetic electron transport.Abbreviations PQ plastoquinone - PSI, PSII photosystem I, II     

The impact of photosynthesis on initiation of leaf senescence

Senescence is the last stage of leaf development preceding the death of the organ, and it is important for nutrient remobilization and for feeding sink tissues. There are many reports on leaf senescence, but the mechanisms initiating leaf senescence are still poorly understood. Leaf senescence is affected by many environmental factors and seems to vary in different species and even varieties of plants, which makes it difficult to generalize the mechanism. Here, we give an overview on studies reporting about alterations in the composition of the photosynthetic electron transport chain in chloroplasts during senescence. We hypothesize that alternative electron flow and related generation of the proton motive force required for ATP synthesis become increasingly important during progression of senescence. We address the generation of reactive oxygen species (ROS) in chloroplasts in the initiation of senescence, retrograde signaling from the chloroplast to the nucleus and ROS‐dependent signaling associated with leaf senescence. Finally, a few ideas for increasing crop yields by increasing the chloroplast lifespan are presented.     

Chlororespiration and poising of cyclic electron transport. Plastoquinone as electron transporter between thylakoid NADH dehydrogenase and peroxidase

Polypeptides encoded by plastid ndh genes form a complex (Ndh) which could reduce plastoquinone with NADH. Through a terminal oxidase, reduced plastoquinone would be oxidized in chlororespiration. However, isolated Ndh complex has low activity with plastoquinone and no terminal oxidase has been found in chloroplasts, thus the function of Ndh complex is unknown. Alternatively, thylakoid hydroquinone peroxidase could oxidize reduced plastoquinone with H(2)O(2). By immunoaffinity chromatography, we have purified the plastid Ndh complex of barley (Hordeum vulgare L.) to investigate the electron donor and acceptor specificity. A detergent-containing system was reconstructed with thylakoid Ndh complex and peroxidase which oxidized NADH with H(2)O(2) in a plastoquinone-dependent process. This system and the increases of thylakoid Ndh complex and peroxidase activities under photooxidative stress suggest that the chlororespiratory process consists of the sequence of reactions catalyzed by Ndh complex, peroxidase (acting on reduced plastoquinone), superoxide dismutase, and the non-enzymic one-electron transfer from reduced iron-sulfur protein (FeSP) to O(2). When FeSP is a component of cytochrome b(6).f complex or of the same Ndh complex, O(2) may be reduced with NADH, without requirement of light. Chlororespiration consumes reactive species of oxygen and, eventually, may decrease their production by lowering O(2) concentration in chloroplasts. The common plastoquinone pool with photosynthetic electron transport suggests that chlororespiratory reactions may poise reduced and oxidized forms of the intermediates of cyclic electron transport under highly fluctuating light intensities.     

Aspects of programmed cell death during leaf senescence of mono- and dicotyledonous plants

Summary Leaf senescence is a highly regulated stage in the plant life cycle, leading to cell death, recently examined as a type of the programmed cell death (PCD). One of the basic features of PCD is the condensation of nuclear chromatin which is caused by endonucleolytic degradation of nuclear DNA (nDNA). In our investigations, we applied the technique of the single-cell electrophoresis system (“comet assay”) in order to determine the type of nDNA fragmentation during leaf senescence. The comet assay, a sensitive method revealing nonrandom internucleosomal damage that is specific for PCD, is especially useful for the detection of nDNA degradation in isolated viable cells. Simultaneously, we analyzed the mesophyll cell ultrastructure and the photosynthetic-pigment concentration in the leaves of two species,Ornithogalum virens andNicotiana tabacum, representing mono- and dicotyledonous plants which differ in the pattern of leaf differentiation. These investigations demonstrated that, in both species, the comet assay revealed nDNA degradation in yellow-leaf protoplasts containing chloroplasts that showed already changed ultrastructure (swelled or completely degraded thylakoids) and cell nuclei with a significant condensation of chromatin. There was no nDNA degradation in green-leaf protoplasts containing differentiated chloroplasts with numerous grana stacks and nuclei with dispersed chromatin. The analysis of intermediate developmental stage showed that the degradation of nDNA precedes condensation of nuclear chromatin. Thus the comet assay is a very useful and sensitive method for early detection of PCD. Moreover, results of our studies indicate that leaf senescence involves PCD.     

Identification of a functional respiratory complex in chloroplasts through analysis of tobacco mutants containing disrupted plastid ndh genes.

The plastid genomes of several plants contain homologues, termed ndh genes, of genes encoding subunits of the NADH:ubiquinone oxidoreductase or complex I of mitochondria and eubacteria. The functional significance of the Ndh proteins in higher plants is uncertain. We show here that tobacco chloroplasts contain a protein complex of 550 kDa consisting of at least three of the ndh gene products: NdhI, NdhJ and NdhK. We have constructed mutant tobacco plants with disrupted ndhC, ndhK and ndhJ plastid genes, indicating that the Ndh complex is dispensible for plant growth under optimal growth conditions. Chlorophyll fluorescence analysis shows that in vivo the Ndh complex catalyses the post-illumination reduction of the plastoquinone pool and in the light optimizes the induction of photosynthesis under conditions of water stress. We conclude that the Ndh complex catalyses the reduction of the plastoquinone pool using stromal reductant and so acts as a respiratory complex. Overall, our data are compatible with the participation of the Ndh complex in cyclic electron flow around the photosystem I complex in the light and possibly in a chloroplast respiratory chain in the dark.     

Changes in the protein and activity levels of the plastid NADH–plastoquinone–oxidoreductase complex during fruit development

Plastids contain an NADH dehydrogenase complex (Ndh complex) homologous to the mitochondrial complex I (EC 1.6.5.3). In this work, we have analysed the changes in the Ndh complex during ripening of pepper (Capsicum annum L., cv. Maor) and tomato (Lycopersicon esculentum Mill., cv. Marglobe) fruits. The Ndh complex was mainly present in the outer pericarp of tomato fruits, whereas it was evenly distributed in the pericarp of pepper. In both kinds of fruit we observed a decrease in the total amount of Ndh complex from the green to the red stage of development. This decrease corresponds to parallel decreases in the content and activity of the complex in plastids during the transition from chloroplasts to chromoplasts. Levels of plastidial quinol peroxidase activity were also higher during the first stages of tomato fruit development than during the latter stages of ripening. However, when referred to total plastid protein, the amount and activity of the Ndh complex in chloroplasts isolated from green fruits was higher than in chloroplasts isolated from leaves. These results strongly suggest that function of the Ndh complex, probably related to a plastidial electron transport chain, can be important during the first stages of fruit development.     

Studies of the Ndh complex and photosystem II from mesophyll and bundle sheath chloroplasts of the C4-type plant Zea mays

In C(4) plants, granal mesophyll (MS) chloroplasts contain higher photosystem (PS) II and lower PS I activity than agranal bundle sheath (BS) chloroplasts. The maize NAD(P)H dehydrogenase or NAD(P)H-plastoquinone oxidoreductase (also named Ndh complex) from MS and BS chloroplasts, contains at least 11 subunits (NdhA-K) and is homologous to NADH dehydrogenase or Complex I from mitochondria and bacteria. The amount of Ndh complex is higher in BS compared with MS chloroplasts. However, there is little information about the interdependence of the PS II and Ndh complex in chlororespiration and linear and cyclic electron transport in C(4) plants. To characterize the expression of the PS II and Ndh complex in maize plastids, we used cytochrome b559 (cyt b559) antibodies and Ndh immunoglobulins (IgG) to analyze the Ndh complex and PS II in both MS and BS chloroplasts from maize leaves by Western blotting and immunolabeling. In Western blot experiments, it was found that the amount of cyt b559 (a marker for PS II) is 7-8 times higher in MS than BS chloroplasts. Conversely, the NdhH, -J, -K and -E content is 2.5-3 times higher in BS than MS chloroplasts. Similar results were obtained in immunolabeling experiments using Ndh IgGs and cyt b559 antibodies in MS and BS chloroplasts. These data suggest that in BS chloroplasts, ATP could be produced mainly by cyclic electron transport around PS I and Ndh complexes. Conversely, the linear electron transport in BS chloroplasts via PS II could have a lower production of ATP. These results also suggest that the contribution of the Ndh complex in the production of ATP in MS chloroplasts is minimal and that instead, this complex could have a chlororespiratory role.     

Inactivation of a plastid evolutionary conserved gene affects PSII electron transport, life span and fitness of tobacco plants

Chloroplasts contain a plastoquinone-NADH-oxidoreductase (Ndh) complex involved in protection against stress and the maintenance of cyclic electron flow. Inactivation of the Ndh complex delays the development of leaf senescence symptoms. Chlorophyll a fluorescence measurements, blue native gel electrophoresis, immunodetection and other techniques were employed to study tobacco (Nicotiana tabacum) Ndh-defective mutants (DeltandhF). The DeltandhF mutants compared with wild-type plants presented: (i) higher photosystem II : photosystem I (PSII : PSI) ratios; (ii) similar or higher levels of ascorbate, carotenoids, thylakoid peroxidase and superoxide dismutase, yield (Phi(PSII)) and maximal photochemical efficiency of PSII levels (F(v)/F(m)) than wild-type plant leaves of the same age; (iii) lower values of nonphotochemical quenching yield (Phi(NPQ)), but not at very high light intensities or during induced leaf senescence; (iv) a similar decrease of antioxidants during senescence; (v) no significant differences in the total foliar area and apical growth rate; and (vi) a production of viable seeds significantly higher than wild-type plants. These results suggest that the Ndh complex is involved in one of the redundant mechanisms that play a safety role in photosynthesis under stress, which has been conserved during evolution, but that its deletion increases fitness when plants are grown under favourable controlled conditions.     

Changes in the structure of DNA molecules and the amount of DNA per plastid during chloroplast development in maize

We examined the DNA from chloroplasts obtained from different tissues of juvenile maize seedlings (from eight to 16 days old) and adult plants (50-58 days old). During plastid development, we found a striking progression from complex multigenomic DNA molecules to simple subgenomic molecules. The decrease in molecular size and complexity of the DNA paralleled a progressive decrease in DNA content per plastid. Most surprising, we were unable to detect DNA of any size in most chloroplasts from mature leaves, long before the onset of leaf senescence. Thus, the DNA content per plastid is not constant but varies during development from hundreds of genome copies in the proplastid to undetectable levels in the mature chloroplast. This loss of DNA from isolated, mature chloroplasts was monitored by three independent methods: staining intact chloroplasts with 4',6-diamidino-2-phenylindole (DAPI); staining at the single-molecule level with ethidium bromide after exhaustive deproteinization of lysed chloroplasts; and blot-hybridization after standard DNA isolation procedures. We propose a mechanism for the production of multigenomic chloroplast chromosomes that begins at paired DNA replication origins on linear molecules to generate a head-to-tail linear concatemer, followed by recombination-dependent replication.     

New subunits NDH-M, -N, and -O, encoded by nuclear genes, are essential for plastid Ndh complex functioning in higher plants

In higher plants, the Ndh complex reduces plastoquinones and is involved in cyclic electron flow around photosystem I, supplying extra-ATP for photosynthesis, particularly under environmental stress conditions. Based on plastid genome sequences, the Ndh complex would contain 11 subunits (NDH-A to -K), but homologies with bacterial complex indicate the probable existence of additional subunits. To identify missing subunits, tobacco (Nicotiana tabacum) NDH-H was His tagged at its N terminus using plastid transformation. A functional Ndh subcomplex was purified by Ni(2+) affinity chromatography and its subunit composition analyzed by mass spectrometry. Five plastid encoded subunits (NDH-A, -H, -I, -J, and -K) were identified as well as three new subunits (NDH-M, -N, and -O) homologous to cyanobacterial and higher plant proteins. Arabidopsis thaliana mutants missing one of these new subunits lack a functional Ndh complex, and NDH-M and NDH-N are not detected in a tobacco transformant lacking the Ndh complex. We discuss the involvement of these three nuclear-encoded subunits in the functional integrity of the plastidial complex.     

Isolation and structural characterization of the Ndh complex from mesophyll and bundle sheath chloroplasts of Zea mays

Complex I (NADH: ubiquinone oxidoreductase) is the first complex in the respiratory electron transport chain. Homologs of this complex exist in bacteria, mitochondria and chloroplasts. The minimal complex I from mitochondria and bacteria contains 14 different subunits grouped into three modules: membrane, connecting, and soluble subcomplexes. The complex I homolog (NADH dehydrogenase or Ndh complex) from chloroplasts from higher plants contains genes for two out of three modules: the membrane and connecting subcomplexes. However, there is not much information about the existence of the soluble subcomplex (which is the electron input device in bacterial complex I) in the composition of the Ndh complex. Furthermore, there are contrasting reports regarding the subunit composition of the Ndh complex and its molecular mass. By using blue native (BN)/PAGE and Tricine/PAGE or colorless-native (CN)/PAGE, BN/PAGE and Tricine/PAGE, combined with mass spectrometry, we attempted to obtain more information about the plastidal Ndh complex from maize (Zea mays). Using antibodies, we detected the expression of a new ndh gene (ndhE) in mesophyll (MS) and bundle sheath (BS) chloroplasts and in ethioplasts (ET). We determined the molecular mass of the Ndh complex (550 kDa) and observed that it splits into a 300 kDa membrane subcomplex (containing NdhE) and a 250 kDa subcomplex (containing NdhH, -J and -K). The Ndh complex forms dimers at 1000-1100 kDa in both MS and BS chloroplasts. Native/PAGE of the MS and BS chloroplasts allowed us to determine that the Ndh complex contains at least 14 different subunits. The native gel electrophoresis, western blotting and mass spectrometry allowed us to identify five of the Ndh subunits. We also provide a method that allows the purification of large amounts of Ndh complex for further structural, as well as functional studies.     

Cyclic electron flow around photosystem I in C(3) plants. In vivo control by the redox state of chloroplasts and involvement of the NADH-dehydrogenase complex

Cyclic electron flow around photosystem (PS) I has been widely described in vitro in chloroplasts or thylakoids isolated from C(3) plant leaves, but its occurrence in vivo is still a matter of debate. Photoacoustic spectroscopy and kinetic spectrophotometry were used to analyze cyclic PS I activity in tobacco (Nicotiana tabacum cv Petit Havana) leaf discs illuminated with far-red light. Only a very weak activity was measured in air with both techniques. When leaf discs were placed in anaerobiosis, a high and rapid cyclic PS I activity was measured. The maximal energy storage in far-red light increased to 30% to 50%, and the half-time of the P(700) re-reduction in the dark decreased to around 400 ms; these values are comparable with those measured in cyanobacteria and C(4) plant leaves in aerobiosis. The stimulatory effect of anaerobiosis was mimicked by infiltrating leaves with inhibitors of mitochondrial respiration or of the chlororespiratory oxidase, therefore, showing that changes in the redox state of intersystem electron carriers tightly control the rate of PS I-driven cyclic electron flow in vivo. Measurements of energy storage at different modulation frequencies of far-red light showed that anaerobiosis-induced cyclic PS I activity in leaves of a tobacco mutant deficient in the plastid Ndh complex was kinetically different from that of the wild type, the cycle being slower in the former leaves. We conclude that the Ndh complex is required for rapid electron cycling around PS I.     

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.     

Delayed wheat flag leaf senescence due to removal of spikelets is associated with increased activities of leaf antioxidant enzymes,reduced glutathione/oxidized glutathione ratio and oxidative damage to mitochondrial proteins

Removal of reproductive ‘sink’ i.e. spikelets from wheat at anthesis delays the rate of flag leaf senescence. In this work, the antioxidant defense was studied in the flag leaf of Triticum aestivum cv. Kalyansona plants showing normal (S + plants) and delayed senescence via removal of spikelets (S? plants). This was done by measurement of metabolites and activities of enzymes such as superoxide dismutase, catalase, guaiacol peroxidase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase. S? plants had higher reduced glutathione/oxidized glutathione (GSH/GSSG) ratio and antioxidant enzyme activities than the control plants and the differences were apparent from 21 days after anthesis (DAA). The removal of the reproductive sink led to an increased antioxidant defense which may be contributing towards the delayed flag leaf senescence in wheat. Chloroplasts and mitochondria, important sources of ROS, were isolated at two stages representing early (7 DAA) and late (21 DAA) senescence. Oxidative damage to proteins was studied in these organelles in relation to SOD and APX. Mitochondria had higher levels of damaged proteins than chloroplasts at 7 DAA in both S+ and S? plants. Higher damage was related to the lower antioxidant enzyme levels of SOD and APX in mitochondria as compared to chloroplasts.     

Lipid globules on the plastid surface in Iris tepal epidermis cells during tepal maturation and senescence

Epidermis cells in the outer tepals of Iris flowers (Iris × hollandica, cv. Blue Magic) start programmed cell death (PCD) prior to floral opening. The tepals show visible senescence symptoms three days after full opening. Visible senescence coincides with collapse (death) of the upper epidermis cells. In these cells, electron-dense particles (plastoglobuli), membranes, and oil bodies were observed in the plastid interior. Electron-dense globules similar to plastoglobuli, thus apparently mainly consisting of lipids, were found on the plastid surface, from before flower opening until cell death. Such electron-dense globules were also present in the cytosol. The size of some of the globules on the plastid surface increased with time. The globules are likely involved in transfer of lipidic/proteinaceous material from the plastid to the cytosol. As the plastids contained ample oil bodies, up to the time of cell death, cell death was likely not due to lack of reserves. Mitochondrial ultrastructure also remained the same until cell death. The role of mitochondria in PCD is discussed.