Multiple fates of non‐mature lumenal proteins in thylakoids

Most proteins found in the thylakoid lumen are synthesized in the cytosol with an N–terminal extension consisting of transient signals for chloroplast import and thylakoid transfer in tandem. The thylakoid‐transfer signal is required for protein sorting from the stroma to thylakoids, mainly via the cpSEC or cpTAT pathway, and is removed by the thylakoidal processing peptidase in the lumen. An Arabidopsis mutant lacking one of the thylakoidal processing peptidase homologs, Plsp1, contains plastids with anomalous thylakoids and is seedling‐lethal. Furthermore, the mutant plastids accumulate two cpSEC substrates (PsbO and PetE) and one cpTAT substrate (PsbP) as intermediate forms. These properties of plsp1‐null plastids suggest that complete maturation of lumenal proteins is a critical step for proper thylakoid assembly. Here we tested the effects of inhibition of thylakoid‐transfer signal removal on protein targeting and accumulation by examining the localization of non‐mature lumenal proteins in the Arabidopsis plsp1‐null mutant and performing a protein import assay using pea chloroplasts. In plsp1‐null plastids, the two cpSEC substrates were shown to be tightly associated with the membrane, while non‐mature PsbP was found in the stroma. The import assay revealed that inhibition of thylakoid‐transfer signal removal did not disrupt cpSEC‐ and cpTAT‐dependent translocation, but prevented release of proteins from the membrane. Interestingly, non‐mature PetE2 was quickly degraded under light, and unprocessed PsbO1 and PsbP1 were found in a 440‐kDa complex and as a monomer, respectively. These results indicate that the cpTAT pathway may be disrupted in the plsp1‐null mutant, and that there are multiple mechanisms to control unprocessed lumenal proteins in thylakoids.     

Ultrastructural characterization of the reversible differentiation of chloroplasts in cucumber fruit

The changes in plastid ultrastructure in the pericarp of cucumber (Cucumis sativus L) fruit were studied during fruit yellowing (which accompanied maturation) and regreening. In the course of fruit maturation, the thylakoid system was progressively reduced, and only a small number of membranes remained in the plastids of mature fruit. At the same time, the plastoglobules increased in size, often remaining in close proximity to the degrading thylakoids. In pericarp tissue which turned green again, the thylakoid network in the plastids was gradually reconstituted. Morphological similarities between the plastids in mature and regreening fruit indicated that the chloroplasts in regreened tissue were redifferentiated from the plastids of mature fruit. Reconstitution of the thylakoid system appeared to start from two morphologically distinct types of membranes: from double membranes which resembled thylakoids and from membrane-bound bodies (MBBs). The latter appeared to form thylakoids by two mechanisms: by detachment of extensions from their surfaces and by fragmentation. The plastoglobules remained in the plastids during thylakoid system reconstitution and were often observed in close proximity to developing thylakoids. In the course of chloroplast redifferentiation, several types of membraneous structures were found to be associated with the plastid envelope: (i) vesicles which appeared to separate from the envelope and to fuse subsequently with the developing thylakoids, (ii) tubules, and (iii) double-membrane sheets which appeared asde novo forming thylakoids.     

Characterization of chloroplast division using the Arabidopsis mutant arc5.

arc5 is a chloroplast division mutant of Arabidopsis thaliana. To identify the role of ARC5 in the chloroplast replication process we have followed the changes in arc5 chloroplasts during their perturbed division. ARC5 does not affect proplastid division but functions at a later stage in chloroplast development. Chloroplasts in developing mesophyll cells of arc5 leaves do not increase in number and all of the chloroplasts in mature leaf cells show a central constriction. Young arc5 chloroplasts are capable of initiating the division process but fail to complete daughter-plastid separation. Wild-type plastids increase in number to a mean of 121 after completing the division process, but in the mutant arc5 the approximately 13 plastids per cell are still centrally constricted but much enlarged. As the arc5 chloroplasts expand and elongate without dividing, the internal thylakoid membrane structure becomes flexed into an undulating ribbon. We conclude that the ARC5 gene is necessary for the completion of the last stage of chloroplast division when the narrow isthmus breaks, causing the separation of the daughter plastids.     

Über das Vorkommen kleiner netzartiger Strukturen in den Plastiden

Summary In the chlorotic leaves of the Oenothera hybrid Oe. (lamarckiana x hookeri) velans· ^h hookeri with lamarckiana plastids the differentiation of the chloroplasts is disordered in different ways because of a disharmony between genom and plastom. Some of the plastids possess numerous vesicles and plastoglobuli but only a few isolated grana instead of the normal thylakoid system. Furthermore, the plastids contain lattice-like structures consisting of fibrils with a thickness of approximately 5 to 11 nm. These networks are connected with thylakoids, vesicles or plastoglobuli. They are interpreted as fragments of prolamellar bodies. Sometimes prolamellar bodies are distinctly recognizable in the chloroplasts even though the thylakoid system is rather well differentiated.     

Seasonal rhythmics of the leucoplast structure in bulb scales of Scilla sibirica L

A study was made of seasonal changes in plastids of ground tissue cells of bulb scales in early-spring ephemeroid Scilla sibirica L. In summer, plastids are represented by typical amyloplasts, with their main volume (97.0 +/- 4.3%) being occupied by one large starch grain. The volume fraction of plastid stroma is at its minimum. The stroma contains small plastoglobuli and no thylakoids. The same structure is characteristic of plastids in October. However, no starch is found in December, when some thylakoids are seen at the plastid periphery. In the early spring (March), when leaves still remain below the ground, the volume fraction of starch grains is 53.0 +/- 2.2%. In the stroma some structures superficially similar to those of microtubuli are revealed. The thylakoid system is fairly well developed, some of thylakoids being concentrically arranged. Some electron-opaque material is seen in the thylakoid lumen. Many plastids are sheathed with elements of the smooth endoplasmic reticulum. Based on the analysis of these and literature data, a conclusion is made that plastids of bulb scales not only store starch, but also seemingly participate in phytohormone biosynthesis.     

Fine structural observations of embryo development inPelargonium XHortorum bailey: with normal and mutant plastids

Summary The ultrastructural changes in the cotyledon, radicle and suspensor haustorium ofPelargonium, containing either normal or mutant plastids, are investigated from the heart stage of embryogenesis to the mature seed. The fine structure of parenchymatous cells from the cotyledon and radicle is essentially similar whereas that of the suspensor haustorium is very different.The cotyledon and radicle develop into one massive storage tissue possessing numerous lipid and several protein bodies per cell, and well developed starch grains. The suspensor haustorium has no storage function, rather it acts as a transitory tissue which dies off as the seed matures. The extensive chloroplast development suggests that, in addition to its traditional role, the suspensor haustorium also acts as a photosynthetic booster for the developing embryo.The development of surviving mutant embryos is similar to normal ones except that in cotyledon and radicle cells plastids develop only to vesicles, which associate into loose prolamellar bodies and sometimes small fenestrated thylakoids, and in the suspensor haustorium cells, only to small compact grana.     

PLASTID DEVELOPMENT IN IOJAP- AND CHLOROPLAST MUTATOR-AFFECTED MAIZE PLANTS

Plastids affected by either iojap or chloroplast mutator fail to green, and altered plastids are maternally transmitted to subsequent generations. The ultrastructure of iojap-affected plastids indicates that these plastids contain no ribosomes and are capable of supporting little internal membrane organization in either light or dark-grown plants. Chloroplast mutator-affected plastids of light-grown plants contain some organized internal membrane structures. In dark-grown plants, chloroplast mutator-aftected plastids contain a crystalline prolamellar body, numerous vesicles, and osmiophilic granules. The chloroplast mutator-affecled etioplasts display an abnormal distribution of lamellar membranes; these membranes, rather than radiating in a spokelike pattern from the prolamellar body, are condensed into a portion of the organelle. Light causes disruption of the prolamellar body in chloroplast mutator-affected plastids without promoting the organization of a normal thylakoid membrane system. The effects of iojap and chloroplast mutator are cell autonomous and apparently influence the individual plastid, as evidenced by the persistence of heteroplastidic cells containing normal and affected plastids.     

Comparative ultrastructural properties of pallisade tissue and spongy tissue chloroplasts from normal and mutant leaves of Pisum sativum L.*

Abstract. The ultrastructure of chloroplasts from palisade and spongy tissue was studied in order to analyse the adaptation of chloroplasts to the light gradient within the bifacial leaves of pea. Chloroplasts of two nuclear gene mutants of Pisum sativum (chlorotica-29 and chlorophyll b-less 130A), grown under normal light conditions, were compared with the wild type (WT) garden-pea cv. ‘Dippes Gelbe Viktoria’. The differentiation of the thylakoid membrane system of plastids from normal pea leaves exhibited nearly the same degree of grana formation in palisade and in spongy tissue. Using morphometrical measurements, only a slight increase in grana stacking capacity was found in chloroplasts of spongy tissue. In contrast, chloroplasts of mutant leaves differed in grana development in palisade and spongy tissue, respectively. Their thylakoid systems appeared to be disorganized and not developed as much as in chloroplasts from normal pea leaves. Grana contained fewer lamellae per granum, the number of grana per chloroplast section was reduced and the length of appressed thylakoid regions was decreased. Nevertheless, chloroplasts of the mutants were always differentiated into grana and stroma thylakoids. The structural changes observed and the reduction of the total chlorophyll content correlated with alterations in the polypeptide composition of thylakoid membrane preparations from mutant chloroplasts. In sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), polypeptide bands with a relative molecular mass of 27 and 26 kilodalton (kD) were markedly reduced in mutant chloroplasts. These two polypeptides represented the major apoproteins of the light harvesting chlorophyll a/b complex from photosystem II (LHC-II) as inferred from a comparison with the electrophoretic mobility of polypeptides isolated from the LHC-II.     

Assembly of the barley light-harvesting chlorophyll a/b proteins in barley etiochloroplasts involves processing of the precursor on thylakoids

A barley gene encoding the major light-harvesting chlorophyll a/b-binding protein (LHCP) has been sequenced and then expressed in vitro to produce a labelled LHCP precursor (pLHCP). When barley etiochloroplasts are incubated with this pLHCP, both labelled pLHCP and LHCP are found as integral thylakoid membrane proteins, incorporated into the major pigment-protein complex of the thylakoids. The presence of pLHCP in thylakoids and its proportion with respect to labelled LHCP depends on the developmental stage of the plastids used to study the import of pLHCP. The reduced amounts of chlorophyll in a chlorophyll b-less mutant of barley does not affect the proportion of pLHCP to LHCP found in the thylakoids when import of pLHCP into plastids isolated from the mutant plants is examined. Therefore, insufficient chlorophyll during early stages of plastid development does not seem to be responsible for their relative inefficiency in assembling pLHCP. A chase of labelled pLHCP that has been incorporated into the thylakoids of intact plastids, by further incubation of the plastids with unlabelled pLHCP, reveals that the pLHCP incorporated into the thylakoids can be processed to its mature size. Our observations strongly support the hypothesis that after import into plastids, pLHCP is inserted into thylakoids and then processed to its mature size under in vivo conditions.     

Effect of cytokinins on plastid development and photosynthetic polypeptides during organogenesis ofPinus ponderosa Dougl. cotyledons culturedin vitro

InPinus ponderosa Dougl., application of the cytokinins, benzyladenine and 2-isopentenyl adenine, to excised cotyledons, promoted thein vitro formation of meristematic centers which led to bud and shoot production. Meristematic cells showed plastids with poorly developed thylakoid membranes and rudimentary grana, whereas cells in non-meristematic tissues and in growth regulator free medium, had chloroplasts with well developed inner membranes, and more thylakoid membranes and grana than plastids of meristematic cells. Chlorophyll and six polypeptides associated with photosynthesis were present in lower concentrations in cytokinin-treated cotyledons than in those cultured in growth regulator free medium. Both benzyladenine and 2-isopentenyl adenine are effective in inhibiting the accumulation of at least two photosynthetic polypeptides in the first 24 h in culture. The ability of cotyledons to respond in this way to cytokinins is lost after three days in culture in growth regulator free medium prior to treatment with cytokinin.     

MFP1 is a thylakoid-associated,nucleoid-binding protein with a coiled-coil structure

Plastid DNA, like bacterial and mitochondrial DNA, is organized into protein–DNA complexes called nucleoids. Plastid nucleoids are believed to be associated with the inner envelope in developing plastids and the thylakoid membranes in mature chloroplasts, but the mechanism for this re-localization is unknown. Here, we present the further characterization of the coiled-coil DNA-binding protein MFP1 as a protein associated with nucleoids and with the thylakoid membranes in mature chloroplasts. MFP1 is located in plastids in both suspension culture cells and leaves and is attached to the thylakoid membranes with its C-terminal DNA-binding domain oriented towards the stroma. It has a major DNA-binding activity in mature Arabidopsis chloroplasts and binds to all tested chloroplast DNA fragments without detectable sequence specificity. Its expression is tightly correlated with the accumulation of thylakoid membranes. Importantly, it is associated in vivo with nucleoids, suggesting a function for MFP1 at the interface between chloroplast nucleoids and the developing thylakoid membrane system.     

Chromoplast development in a carotenoid mutant of maize

Summary The lethal recessive mutantlycopenic in maize is characterized by the synthesis of lycopene instead of the normal carotenoids. At normal conditions of illumination it loses chlorophyll by photo-oxidation. Seedlings of this mutant and of normal maize were grown at light intensities of 25–30 lux and 500–30,000 lux. Their plastid development was studied by electron microscopy.At low light intensities a kind of mesophyll chloroplast with elongated grana, long unpaired thylakoid segments, and sometimes prolamellar bodies is formed in mutant plants. In corresponding bleached plants the plastids are transformed into chromoplasts containing characteristic lycopene crystalloids similar to those found in tomato fruits. Various stages in this chromoplast development are described and illustrated. Also bundle-sheath plastids were found to develop into chromoplasts.It is concluded that the ultrastructure of plastids in a tissue is influenced by the nature of their pigments and that an altered carotenoid composition therefore can give rise to development of chromoplasts in plants which normally lack such organelles.     

Division and DNA distribution in ribosome-deficient plastids of the barley mutant “albostrians”

The ribsome-deficient plastids of the albino leaves of the barley mutant albostrians divide at about the same rate as normal plastids and contain similar levels of plastids DNA to the normal plastids. Double-ring structures were observed around the neck of constricting dumbbell-shaped, ribosome-deficient plastids in the basal intercalary meristem of albino leaves. In the distal region of albino leaves the ribosome-deficient plastids contain a rudimentary thylakoid system often closely associated with DNA nucleoids. It is suggested that nuclear coded proteins synthesized within the cytoplasm are responsible for the formation of the double-ring structures and the rudimentary thylakoids of albino plastids.     

Origins of the secondary plastids of Euglenophyta and Chlorarachniophyta as revealed by an analysis of the plastid‐targeting,nuclear‐encoded gene psbO1

Because the secondary plastids of the Euglenophyta and Chlorarachniophyta are very similar to green plant plastids in their pigment composition, it is generally considered that ancestral green algae were engulfed by other eukaryotic host cells to become the plastids of these two algal divisions. Recent molecular phylogenetic studies have attempted to resolve the phylogenetic positions of these plastids; however, almost all of the studies analyzed only plastid‐encoded genes. This limitation may affect the results of comparisons between genes from primary and secondary plastids, because genes in endosymbionts have a higher mutation rate than the genes of their host cells. Thus, the phylogeny of these secondary plastids must be elucidated using other molecular markers. Here, we compared the plastid‐targeting, nuclear‐encoded, oxygen‐evolving enhancer (psbO) genes from various green plants, the Euglenophyta and Chlorarachniophyta. A phylogenetic analysis based on the PsbO amino acid sequences indicated that the chlorarachniophyte plastids are positioned within the Chlorophyta (including Ulvophyceae, Chlorophyceae, and Prasinophyceae, but excluding Mesostigma). In contrast, plastids of the Euglenophyta and Mesostigma are positioned outside the Chlorophyta and Streptophyta. The relationship of these three phylogenetic groups was consistent with the grouping of the primary structures of the thylakoid‐targeting domain and its adjacent amino acids in the PsbO N‐terminal sequences. Furthermore, the serine‐X‐alanine (SXA) motif of PsbO was exactly the same in the Chlorarachniophyta and the prasinophycean Tetraselmis. Therefore, the chlorarachniophyte secondary plastids likely evolved from the ancestral Tetraselmis‐like alga within the Chlorophyta, whereas the Euglenophyte plastids may have originated from the unknown basal lineage of green plants.     

Ultrastructure of Lycopersicon peruvianum leaf explants and streptomycin-resistant shoots on medium containing streptomycin

The effect of streptomycin on morphogenic explants of Lycopersicon peruvianum Mill. was examined microscopically at both the light and ultrastructural level. Early stages in shoot regeneration from leaf explants were distinguished as meristematic tissue at both levels. Small starch grains were observed in the plastids in this tissue but not in plastids in regenerated shoots. In the presence of streptomycin, adventitious shoot regeneration from sensitive leaf strips was inhibited. Large layered bodies were observed within the plastids of sensitive leaf tissue, suggesting the disruption of thylakoid membrane formation. Streptomycin resistant L. peruvianum lines, as well as a chlorophyll-deficient line, were also examined microscopically. The chloroplasts of newly regenerated streptomycin resistant shoots contained well developed internal membranes and conspicuous starch grains. Cells containing a mixture of resistant and sensitive plastids were not observed. The plastids in chlorophyll-deficient tissue completely lacked thylakoid membranes, although small vesicles and intraplastid bodies were seen within the stroma.Abbreviations NMU N-methyl-N-nitrosourea     

Characterization of a cytoplasmically inherited yellow foliar mutant (cyt-Y 3) in soybean

Summary Genetic analysis of a yellow foliar mutant in soybean (Glycine max L. Merr.) showed maternal inheritance of the mutant phenotype designatedcyt-Y ₃. The mutant was grown beside normal green sibs (cyt-G ₃) under three different photosynthetic photon flux densities (PPFD), and samples were collected to determine pigment content and for electron microscopy analyses of plastid ultrastructure. The plastid ultrastructure ofcyt-Y ₃ appeared normal at low PPFD and the carotenoid level ofcyt-Y ₃ was also normal, but the chlorophyll content was only approximately one-third that ofcyt-G ₃. Under medium and high PPFD,cyt-Y ₃ plastids lacked a structured thylakoid, and total chlorophyll content was only 28% and 1% of normal, respectively; the carotenoid levels ofcyt-Y ₃ also dropped to 33% and 2% of normal, respectively. These data indicate that the effect of high PPFD oncyt-Y ₃ might result from a deficiency in a plastid membrane protein. The resulting changes in membrane configuration could then interfere with the accumulation or stabilization of chlorophylls and carotenoids, thereby resulting in the subsequent photooxidation of both at medium and high PPFD. This mutant could be useful in the study of thylakoid biosynthesis and pigment stabilization, or could provide a source of conditionally identifiable plastids for organelle segregation studies.This is a joint contribution of North Central Region, USDA-ARS., and Journal Paper No. J-11124 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011, Project 2471. Mention of a trademark of proprietary product by Iowa State University or the USDA does not imply its approval to the exclusion of other products that may also be suitable     

ULTRASTRUCTURE OF CHLOROPLASTS AND CHROMOPLASTS IN CAPSICUM ANNUUM I. THYLAKOID MEMBRANE CHANGES DURING FRUIT RIPENING

Developing chromoplasts in the fruit of Capsicum annuum were examined by electron microscopy. Special attention was given to changes in the thylakoid system. All grana and some intergranal thylakoids in the mature chromoplasts of the seven cultivars studied underwent lysis. The particulate nature of the granal membranes disappeared during lysis before the relationship between the partitions and locules was obscured. The changes during lysis support the globular concept of membrane structure. The selective lysis of the synaptic membranes of the granal partitions may be attributed to their distinctive composition and structure. Lipid globules (osmio-philic) did not accumulate in the immediate region of granal lysis, indicating that they are not directly derived from membranes undergoing degradation. During and following granal lysis a profuse development of intergranal thylakoid membranes occurred in several cultivars. In some instances a thylakoid plexus (prolamellar body) was formed. This specialized structure of the thylakoid system occurs in the chromoplasts of other species as well as in other types of plastids. Extensive, concentrically arranged thylakoid sheets with specific interspaced membrane relationships were frequently associated with the plexus. Several types of membrane associations and interrelationships in the plastid are described. An analysis of one type of membrane configuration, the thylakoid sheets, indicated that one method of growth may be through intussusception into the original membrane. The development of thylakoid plexes and of extensive thylakoid sheets during or after granal lysis indicates that dynamic synthetic activities occur in the chromoplasts of some cultivars of pepper during fruit ripening.     

Zea mays Fe deficiency‐related 4 (ZmFDR4) functions as an iron transporter in the plastids of monocots

Iron (Fe)‐homeostasis in the plastids is closely associated with Fe transport proteins that prevent Fe from occurring in its toxic free ionic forms. However, the number of known protein families related to Fe transport in the plastids (about five) and the function of iron in non‐green plastids is limited. In the present study, we report the functional characterization of Zea mays Fe deficiency‐related 4 (ZmFDR4), which was isolated from a differentially expressed clone of a cDNA library of Fe deficiency‐induced maize roots. ZmFDR4 is homologous to the bacterial FliP superfamily, coexisted in both algae and terrestrial plants, and capable of restoring the normal growth of the yeast mutant fet3fet4, which possesses defective Fe uptake systems. ZmFDR4 mRNA is ubiquitous in maize and is inducible by iron deficiency in wheat. Transient expression of the 35S:ZmFDR4–eGFP fusion protein in rice protoplasts indicated that ZmFDR4 maybe localizes to the plastids envelope and thylakoid. In 35S:c‐Myc‐ZmFDR4 transgenic tobacco, immunohistochemistry and immunoblotting confirmed that ZmFDR4 is targeted to both the chloroplast envelope and thylakoid. Meanwhile, ultrastructure analysis indicates that ZmFDR4 promotes the density of plastids and accumulation of starch grains. Moreover, Bathophenanthroline disulfonate (BPDS) colorimetry and inductively coupled plasma mass spectrometry (ICP‐MS) indicate that ZmFDR4 is related to Fe uptake by plastids and increases seed Fe content. Finally, 35S:c‐Myc‐ZmFDR4 transgenic tobacco show enhanced photosynthetic efficiency. Therefore, the results of the present study demonstrate that ZmFDR4 functions as an iron transporter in monocot plastids and provide insight into the process of Fe uptake by plastids.     

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.     

Chloroplast protein translocon components atToc159 and atToc33 are not essential for chloroplast biogenesis in guard cells and root cells

Protein import into chloroplasts is mediated by a protein import apparatus located in the chloroplast envelope. Previous results indicate that there may be multiple import complexes in Arabidopsis. To gain further insight into the nature of this multiplicity, we analyzed the Arabidopsis ppi1 and ppi2 mutants, which are null mutants of the atToc33 and atToc159 translocon proteins, respectively. In the ppi2 mutant, in contrast to the extremely defective plastids in mesophyll cells, chloroplasts in guard cells still contained starch granules and thylakoid membranes. The morphology of root plastids in both mutants was similar to that in wild type. After prolonged light treatments, root plastids of both mutants and the wild type differentiated into chloroplasts. Enzymatic assays indicated that the activity of a plastid enzyme was reduced only in leaves but not in roots. These results indicated that both the ppi1 and ppi2 mutants had functional root and guard cell plastids. Therefore, we propose that import complexes are cell type specific rather than substrate or plastid specific.