Senescence induced structural reorganization of thylakoid membranes in Cucumis sativus cotyledons; LHC II involvement in reorganization of thylakoid membranes

We report the formation and appearance of loosely stacked extended grana like structures along with plastoglobuli in the chloroplasts isolated from 27-day old senescing cucumber cotyledons. The origin and the nature of these extended grana structures have not been elucidated earlier. We isolated Photosystem I complexes from 6-day-old control and 27-day-old senescing cotyledons. The chlorophyll a/b ratio of the isolated Photosystem I complex obtained from 6-day cotyledons was 5–5.5 as against a ratio of 2.9 was found in Photosystem I complexes obtained from 27-day-old senescing cotyledons. We also found that the presence of LHC II in the Photosystem I complexes isolated from 27-day cotyledonary chloroplasts. The presence of LHC II in Photosystem I complexes in senescing and not in control samples, clearly suggest the detachment and diffusion of LHC II complexes from stacked grana region to Photosystem I enriched stroma lamellar region thereby, forming loose disorganized extended grana structures seen in the transmission electron microscope. Furthermore, we show that under in vitro condition the senescing cotyledon chloroplasts exhibited lower extent of light induced phosphorylation of LHC II than the control samples suggesting a possible irreversible phosphorylation and diffusion of LHC II in vivo during the progress of senescence in Cucumis cotyledons. From these findings, we suggest that the senescence induced phosphorylation of LHC II and its migration towards Photosystem I may be a programmed one some how causing the destruction of the thylakoid membrane. The released membrane components may be stored in the plastoglobuli prior to their mobilization to the younger plant parts. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plastid Development in Pisum sativum Leaves during Greening : II. Post-Translational Uptake by Plastids as an Indicator System to Monitor Changes in Translatable mRNA for Nuclear-Encoded Plastid Polypeptides

When isolated pea plastids are incubated with translation products of poly(A⁺) mRNA they specifically import precursor molecules of plastid polypeptides. Etioplasts and chloroplasts import the same polypeptides from identical translation products, and, the imported polypeptides can be well resolved by two-dimensional gel electrophoresis. Therefore, the posttranslational uptake system using isolated chloroplasts can monitor changes in the abundance of translatable plastid-targeted messages. Poly(A⁺) mRNA was isolated from peas at various times during greening and analyzed by this technique. (a) After 48 hours of illumination of dark-grown plants, the relative portion of nuclear encoded messages for plastid targeted proteins had increased by a factor of 2. The percentage of polypeptides recovered in the stroma fraction increased from about 50 to 65%. (b) More than 140 imported polypeptide species could be detected in fluorograms of two-dimensional gels, most of which could be identified throughout the time course of greening. At least 37 imported polypeptides decreased and 36 increased in relative abundance during greening of darkgreen plants. (c) In most cases, where differences in translatable messages were seen between dark- and light-grown plants, they were accompanied by parallel changes in polypeptide abundance.     

ULTRASTRUCTURAL CHANGES IN SUNFLOWER CHLOROPLASTS FOLLOWING INOCULATION WITH PSEUDOMONAS SYRINGAE PV. TAGETIS

Severe chlorosis and ultrastructural modifications of chloroplasts occur in sunflower in response to infection by Pseudomonas syringae pv. tagetis. Chlorosis became apparent within 2 days after the cotyledons of 10-day-old sunflower seedlings were inoculated with the bacteria. The first symptoms generally appeared in the center of leaves at the second node above the cotyledons. Leaves above the second node lost essentially all of their pigmentation but remained turgid and continued to expand. Grana thylakoids became dilated and separated from the granal stacks. These thylakoid membranes did not chemically breakdown as in the case in chromoplast formation or normal chloroplast senescence. Both grana and stroma thylakoid membranes coalesced to form a large membrane sheet within the plastid. The ultrastructural changes are unlike those reported to be caused by other chlorosis-inducing bacteria or chlorosis associated with normal senescence.     

Localization and integration of thylakoid protein translocase subunit cpTatC

Thylakoid membranes have a unique complement of proteins, most of which are nuclear encoded synthesized in the cytosol, imported into the stroma and translocated into thylakoid membranes by specific thylakoid translocases. Known thylakoid translocases contain core multi-spanning, membrane-integrated subunits that are also nuclear-encoded and imported into chloroplasts before being integrated into thylakoid membranes. Thylakoid translocases play a central role in determining the composition of thylakoids, yet the manner by which the core translocase subunits are integrated into the membrane is not known. We used biochemical and genetic approaches to investigate the integration of the core subunit of the chloroplast Tat translocase, cpTatC, into thylakoid membranes. In vitro import assays show that cpTatC correctly localizes to thylakoids if imported into intact chloroplasts, but that it does not integrate into isolated thylakoids. In vitro transit peptide processing and chimeric precursor import experiments suggest that cpTatC possesses a stroma-targeting transit peptide. Import time-course and chase assays confirmed that cpTatC targets to thylakoids via a stromal intermediate, suggesting that it might integrate through one of the known thylakoid translocation pathways. However, chemical inhibitors to the cpSecA-cpSecY and cpTat pathways did not impede cpTatC localization to thylakoids when used in import assays. Analysis of membranes isolated from Arabidopsis thaliana mutants lacking cpSecY or Alb3 showed that neither is necessary for cpTatC membrane integration or assembly into the cpTat receptor complex. These data suggest the existence of another translocase, possibly one dedicated to the integration of chloroplast translocases.     

Chromoplast-Targeted Proteins in Tomato (Lycopersicon esculentum Mill.) Fruit

The chloroplast to chromoplast transition during tomato (Lycopersicon esculentum Mill.) fruit ripening is characterized by a dramatic change in plastid structure and function. We have asked whether this process is mediated by an increase in the steady-state level of RNA for plastid targeted proteins. Assays for import of radiolabeled translation products into isolated pea (Pisum sativum L.) chloroplasts were used to monitor levels of chromoplast-targeted proteins at four stages of tomato fruit development. We have found striking increases during development in levels of translatable RNA for two such proteins. Additionally, the import of in vitro translation products was examined for seven individual cDNA clones known to encode RNA that increase during fruit ripening. Three of these clones produced in vitro translation products that were imported into pea chloroplasts. This implies that there is synthesis and import of new proteins during the transition from chloroplast to chromoplast and that the plastid conversion is an active developmental program rather than a simple decline in synthesis of the photosynthetic apparatus. Furthermore, our results demonstrate the utility of this method for identification of structural genes involved in plastid morphogenesis.     

Sorting of precursor proteins between isolated spinach leaf mitochondria and chloroplasts

The precursors of the F₁-ATPase -subunits fromNicotiana plumbaginifolia andNeurospora crassa were imported into isolated spinach (Spinacia oleracea L.) leaf mitochondria. Both F₁ precursors were imported and processed to mature size products. No import of the mitochondrial precursor proteins into isolated intact spinach chloroplasts was seen. Moreover, the precursor of the 33 kDa protein of photosynthetic water-splitting enzyme was not imported into the leaf mitochondria. This study provides the first experimental report ofin vitro import of precursor proteins into plant mitochondria isolated from photosynthetic tissue and enables studies of protein sorting between mitochondria and chloroplasts in a system which is homologous with respect to organelles. The results suggest a high organellar specificity in the plant cell for the cytoplasmically synthesized precursor proteins.     

A proton gradient is required for the transport of two lumenal oxygen-evolving proteins across the thylakoid membrane.

The 33- and 23-kDa proteins of the photosynthetic oxygen-evolving complex are synthesized in the cytosol as larger precursors and transported into the thylakoid lumen via stromal intermediate forms. We have investigated the energetics of protein transport across the thylakoid membrane using import assays that utilize either intact chloroplasts or isolated thylakoids. We have found that the light-driven import of the 23-kDa protein into isolated thylakoids is almost completely inhibited by electron transport inhibitors or by the ionophore nigericin but not by valinomycin. These compounds have similar effects in chloroplast import assays: precursors of both the 33- and 23-kDa proteins are imported and processed to intermediate forms in the stroma, but transport into the thylakoid lumen is blocked when electron transport is inhibited or nigericin is present. These results indicate that the transport of these proteins across the thylakoid membrane requires a protonmotive force and that the dominant component in this respect is the proton gradient and not the electrical potential.     

Maternal inheritance,cytology and macromolecular composition of defective chloroplasts in a variegated mutant of Nicotiana tabacum

Summary By phase microscopy of living cells the cause of a maternally-inherited variegated, spontaneous mutation of Nicotiana tabacum L. cv. Turkish Samsun was shown to be the presence of defective chloroplasts. These were intermingled with normal chloroplasts in some of the cells of the mesophyll tissue. In young, expanding leaves, the defective chloroplasts contain traces of chlorophylls a and b in the same ratio as found in normal chloroplasts, but only one-thirtieth of the quantity. As the defective chloroplasts mature, the green pigments disappear. The defective chloroplasts thus appear to be greatly deficient in thylakoid membranes. From their dynamic changes in shape, the defective chloroplasts appear to consist almost entirely of mobile phase, the structure which surrounds the thylakoid system of membranes of normal chloroplasts of higher plants. Consistent with this idea, two constitutents located in the mobile phase of normal chloroplasts—70S ribosomes and Fraction I protein—were detected in defective chloroplasts. The Fraction I protein was unchanged in specific ribulose diphosphate carboxylase activity from enzyme isolated from normal chloroplasts. Speculations are presented that the mutation in chloroplast DNA responsible for the formation of defective chloroplasts cannot be attributed to cistrons coding for the protein of Photosystem II, chloroplast ribosomal RNA or proteins, Fraction I protein, or the DNA-dependent RNA polymerase of chloroplasts.     

切除上胚轴对绿豆子叶衰老逆转及其核酸和蛋白质代谢的效应

在绿豆子叶衰老达到不归点(萌发后5～6d)前切除上胚轴可使开始衰老的子叶中核酸和蛋白质含量回升,衰老短期逆转。衰老不归点后的子叶中核酸和蛋白质变化的主要特征是:丧失了较多的核主带DNA、25S、18S rRNA以及大部分可溶性蛋白质组分,一种小分子DNA 组分完全消失。不归点前切除上胚轴可使上述核酸和蛋白质组分明显增加,表明子叶衰老的逆转可能与这些重要功能物质的回升有关。在切除上胚轴的茎顶涂抹IAA,能阻止子叶核酸和蛋白质回升,也消除了切除上胚轴对子叶裹老的逆转作用。     

Change in Gene Expression in Radish Cotyledons during Dark-Induced Senescence

Cotyledons detached from light-grown radish (Raphanus sativusL. cv. Comet) seedlings were used as a model system to studythe changes in nuclear gene expression during dark-induced senescenceof green leaves. Polyadenylated RNA was prepared from the cotyledonsat different times and then translated in a wheat germ system.Approximately 1,000 different polypeptides of the translationproducts were separated from each other by two-dimensional gelelectrophoresis. As judged from the density of autoradiographicspots of the translation products, the induction of senescenceby dark treatment involved an increase in 26 species, a decreasein 11 species, and a temporary increase and subsequent decreasein 8 species of translatable mRNA. A similar pattern of changein protein synthesis was also observed in the dark-treated cotyledonswhen the cotyledons were pulse-labeled with ³⁵S-methionine andthe soluble proteins separated by two-dimensional gel electrophoresis,though the polypeptide pattern on the gel did not coincide exactlywith those of the cell-free translation products. These findingsstrongly suggest that the process of leaf senescence is notsimply a passive and gradual death of the tissue, but involvesa drastic and sequential response of the cells to environmentalstimuli with respect to the gene expression of the cells. (Received July 21, 1987; Accepted September 30, 1987)     

Lipid composition and molecular organization in plasma membrane-enriched fractions from senescing cotyledons

Plasma membrane fractions isolated from cotyledons of Phaseolus vulgaris L. cv. Kinghorn at various stages of senescence showed no significant change in fatty acid saturation with advancing senescence. However, the steroliphospholipid ratio increased by about 400% as senescence intensified. The lipid phase transition temperature of the membranes, which was measured by wide-angle x-ray diffraction, also rose from a point well below the growing temperature for young tissue to about 50°C for membrane from extensively senescent 9-day-old tissue. This means that by day 4 of germination there was a mixture of liquid-crystalline and gel phase phospholipid in the membrane matrices. Crystallinity attributable to sterol-sterol interaction was also apparent in the diffraction patterns for senescent membranes. The co-existence of gel and liquid-crystalline phase phospholipid in the aging membranes as well as the crystalline sterol aggregates presumably render the storage cells of cotyledons leaky and may thus facilitate the translocation of hydrolyzed food reserves into the vascular network.     

Quantification of mRNA in Chloroplasts of Chlamydomonas reinhardii: Equal Distribution of mRNA for a Soluble and a Membrane Polypeptide in Stroma and Thylakoids

The relative contents of the mRNAs were analyzed for the 32kDa herbicide-binding protein and for the large subunit of ribulose-l,5-bisphosphatecarboxylase in the membrane fraction and in the soluble fractionof chloroplasts from Chlamydomonas reinhardii. The presenceof mRNA for the two proteins in both subchloroplast fractionswas demonstrated by in vitro translation of isolated RNA inthe reticulocyte lysate. The relative amounts of the two mRNAswere measured by hybridizations with cloned chloroplast DNAprobes at two stages of the cell cycle. Both mRNAs were distributedin the same ratio between membrane and soluble fractions, about75% of both mRNAs being in the membrane and 25% in the solublefraction. Therefore, in chloroplasts the accumulation of mRNAson thylakoid membranes does not reflect the final localizationof soluble and membrane proteins. ¹Present address: Department of Biology, Ben Gurion University,Beer-Sheva, Israel. (Received April 28, 1987; Accepted September 29, 1987)     

Molecular changes of Arabidopsis thaliana plastoglobules facilitate thylakoid membrane remodeling under high light stress

Plants require rapid responses to adapt to environmental stresses. This includes dramatic changes in the size and number of plastoglobule lipid droplets within chloroplasts. Although the morphological changes of plastoglobules are well documented, little is known about the corresponding molecular changes. To address this gap, we have compared the quantitative proteome, oligomeric state, prenyl-lipid content and kinase activities of Arabidopsis thaliana plastoglobules under unstressed and 5-day light-stressed conditions. Our results show a specific recruitment of proteins related to leaf senescence and jasmonic acid biosynthesis under light stress, and identify nearly half of the plastoglobule proteins in high native molecular weight masses. Additionally, a specific increase in plastoglobule carotenoid abundance under the light stress was consistent with enhanced thylakoid disassembly and leaf senescence, supporting a specific role for plastoglobules in senescence and thylakoid remodeling as an intermediate storage site for photosynthetic pigments. In vitro kinase assays of isolated plastoglobules demonstrated kinase activity towards multiple target proteins, which was more pronounced in the plastoglobules of unstressed than light-stressed leaf tissue, and which was diminished in plastoglobules of the abc1k1/abc1k3 double-mutant. These results strongly suggest that plastoglobule-localized ABC1 kinases hold endogenous kinase activity, as these were the only known or putative kinases identified in the isolated plastoglobules by deep bottom-up proteomics. Collectively, our study reveals targeted changes to the protein and prenyl-lipid composition of plastoglobules under light stress that present strategies by which plastoglobules appear to facilitate stress adaptation within chloroplasts.     

Protein translocation within chloroplast is similar in Euglena and higher plants

It is currently thought that chloroplasts of higher plants were derived from endosymbiont oxygenic photosynthetic bacteria (primary endosymbiosis), while Euglena, a photosynthetic protista, gained chloroplasts by secondary endosymbiosis (i.e., incorporation of a photosynthetic eukaryote into heterotrophic eukaryotic host). To examine if the protein transport inside chloroplasts is similar between these organisms, we carried out heterologous protein import experiments with Euglena precursor proteins and spinach chloroplasts. The precursor of a 30-kDa subunit of the oxygen-evolving complex (OEC30) from the thylakoid lumen of Euglena chloroplasts contained the N-terminal signal, stroma targeting, and thylakoid transfer domains. Truncated preOEC30s lacking the N-terminal domain were post-translationally imported into spinach chloroplasts, transported into the thylakoid lumen, and processed to a mature protein. These results showed that protein translocations within chloroplasts in Euglena and higher plants are similar and supported the hypothesis that Euglena chloroplasts are derived from the ancestral Chlorophyta.     

Detection of a Precursor Polypeptide of the Rapidly-Synthesized 32,000-Dalton Thylakoid Protein in Spinach Chloroplasts

Spinach chloroplast RNA was translated in a wheat germ cell-freesystem in the presence of [³⁵S]methionine or [³H]lysine, andthe products were analyzed by SDS polyacrylamide gel electrophoresisand fluorography. A polypeptide with molecular mass of 2,000-Dalarger than the 32,000-Da thylakoid protein was detected asa major product labeled by [³⁵S]methionine but not by [³H]lysine.Peptide mapping of this polypeptide showed a pattern very closeto that of the 32,000-Da protein synthesized in isolated chloroplasts.A better separation of this polypeptide from the 32,000-Da proteinwas observed in the electrophoresis on polyacrylamide gel includingurea at 8 M. Pulse-labeling of the isolated chloroplasts showedthe occurrence of the larger molecular weight form, which wasconverted to the mature size by a chasing incubation with coldmethionine. These results suggested that the 32,000-Da proteinof spinach is translated primarily as a high molecular weightprecursor in the chloroplasts, as has been reported for otherplant species. (Received March 30, 1985; Accepted April 23, 1985)     

The Phenotype of a Virescent Chloroplast Mutation in Tobacco Is Associated with the Absence of a 37.5 kD Thylakoid Polypeptide

The Vir-c mutation is a virescent chloroplast mutation found in a line of plants derived from protoplast fusions between a Nicotina tabacum line and a line containing N. tabacum nuclei with Nicotiana suaveolens cytoplasm. Vir-c displays a lag period in chlorophyll accumulation and granal stack formation in young leaves. We examined total chloroplast protein in young leaves and showed the mutant contains 1.3 to 2.1 times less stromal protein, and 2.9 to 4.3 times less thylakoid protein when compared to the N. tabacum var “Turkish Samsun” control. Electrophoretic patterns of total thylakoid proteins indicated three polypeptides were specifically decreased in amount within the context of the overall reduction in thylakoid protein. Electrophoresis of thylakoid proteins synthesized by chloroplasts isolated from half-expanded leaves demonstrated that mutant chloroplasts did not synthesize a 37.5 kilodalton polypeptide which was synthesized by “Samsun” chloroplasts. A polypeptide of this molecular weight was synthesized by Vir-c chloroplasts isolated from mature leaves which had recovered the normal phenotype. Restriction digestion and electrophoresis of the mutant's chloroplast DNA produced a pattern of restriction fragments different from either N. tabacum or N. suaveolens chloroplast DNA.     

Changes in Protein Content and in the Structure and Number of Chloroplasts during Leaf Senescence in Rice Seedlings

Two types of experiment were carried out to examine whetheror not the inactivation of photosynthesis is related to lossof chloroplasts during foliar senescence of rice seedlings.Levels of both soluble and insoluble leaf proteins decreasedduring senescence, the loss of the soluble proteins being fasterthan that of the insoluble ones. There was a good positive correlationbetween the rate of oxygen evolution and the level of solubleproteins. The inactivation of photosynthesis was also linearlyrelated to the loss of a major fraction of insoluble proteins.Thus, the loss of photosynthetic ability is ascribable to thedegradation of relevant proteins and enzymes during leaf senescence.Electron microscopy revealed that senescence caused the disorientationof the grana and stroma thylakoids, a decrease in the numberof starch granules, and an increase in the size and number ofplastoglobuli. Large grana consisting 20 to 30 thylakoids appearedin aged leaves. In addition to these changes in ultrastructure,there was a significant decrease in the size of chloroplasts.Furthermore, the number of chloroplasts in mesophyll cells wasalso notably reduced during senescence. Thus, the loss of leafproteins and inactivation of photosynthesis are both relatedto the decrease in the total mass of chloroplasts during senescenceof rice seedlings. ³Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113 Japan. (Received January 4, 1989; Accepted April 19, 1989)