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.     

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.     

Plastid Ontogeny during Petal Development in Arabidopsis

Imaging of chlorophyll autofluorescence by confocal microscopy in intact whole petals of Arabidopsis thaliana has been used to analyze chloroplast development and redifferentiation during petal development. Young petals dissected from unopened buds contained green chloroplasts throughout their structure, but as the upper part of the petal lamina developed and expanded, plastids lost their chlorophyll and redifferentiated into leukoplasts, resulting in a white petal blade. Normal green chloroplasts remained in the stalk of the mature petal. In epidermal cells the chloroplasts were normal and green, in stark contrast with leaf epidermal cell plastids. In addition, the majority of these chloroplasts had dumbbell shapes, typical of dividing chloroplasts, and we suggest that the rapid expansion of petal epidermal cells may be a trigger for the initiation of chloroplast division. In petals of the Arabidopsis plastid division mutant arc6, the conversion of chloroplasts into leukoplasts was unaffected in spite of the greatly enlarged size and reduced number of arc6 chloroplasts in cells in the petal base, resulting in few enlarged leukoplasts in cells from the white lamina of arc6 petals.     

Partial Restoration of the High Rate of Plastid Pigment Development and the Ultrastructure of Plastids in Detached Water-stressed Wheat Leaves

Detached etiolated wheat (Triticum aestivum L. cv. Chris) leaves accumulated plastid pigments at a high rate, developed chloroplasts with stacked thylakoids, and stored plastid starch when wetted on filter paper in light. A moderate water deficit of — 10 bars markedly reduced the accumulation of chlorophyll and carotenoids in the 8-day-old detached leaves during greening. δ-Aminolevulinic acid treatment of stressed leaf segments resulted in slightly increased pigment accumulations but benzyladenine application restored plastid pigment formation in stressed tissue to within 15% of the pigment content of the nonstressed detached leaves. The addition of δ-aminolevulinic acid to benzyladenine-treated stressed leaf segments improved both chlorophyll and carotenoid formation to nearly the amounts found in nonstressed leaf tissue. Stressed leaf sections developed plastids that were small, lacked starch, contained few thylakoids per granum, and possessed dilated thylakoids. Benzyladenine application to the stressed leaf segments did not restore normal plastid stacking but benzyladenine induced the formation of extended intergranal lamellae and stimulated pigment accumulations in both stressed and nonstressed detached leaves. Starch was absent in plastids of benzyladeninetreated leaf sections.     

Correlation of ribulose 1,5-diphosphate carboxylase activity with chlorophyll content and ultrastructure in induced mutants of Hordeum vulgare

Ribulose 1,5-diphosphate (RuDP) carboxylase activity was examined in barley mutants deficient in chlorophyll, and the results were correlated with chlorophyll content and ultrastructure of these mutants. The mutants were induced by diethyl sulfate (dES) or ethyl methane sulfonate (EMS) in the inbred barley variety Himalaya. Essentially no RuDP carboxylase activity was found in 15 albino mutants tested, but mutants with reduced chlorophyll content show large variations in RuDP carboxylase activity. Three general groups of mutants can be recognized. One group has reduced chlorophyll content, but no reduction in RuDP carboxylase activity (dES 7, dES 19, and 28-3398). A second group shows reduced chlorophyll content and proportionally reduced RuDP carboxylase activity (EMS 11, dES 18, and yv), and a third group shows RuDP carboxylase activity reduced more than chlorophyll content (Unk 3, dES 1, Coast V, dES 17, and dES 9). Thus, no strict correlation between RuDP carboxylase activity and chlorophyll content was found in the mutants tested. A reduction in stroma density was observed in the mutants having greatly reduced RuDP carboxylase activity.Scientific Paper No. 3256, College of Agriculture, Washington State University, Pullman, Projects 1920 and 1916. Supported in part by funds provided for medical and biological research by Washington State Initiative Measure 171.     

Ultrastructure of the plastids of 3 types of Chlamydomonas reinhardi mutants phenotypically yellow in the light or in darkness]

A study was made of the ultrastructure of plastids of three mutant types of Chlamydomonas reinhardi which are phenotypically revealed either in the light or in the darkness as yellow mutants. Characteristics of pigments for each mutant have been given. Mutant Y-4 unable to synthesize chlorophyll either in the light or in the darkness shows a complete reduction of photosynthesizing membranes. Mutant Y-1 capable of synthesizing chlorophyll develops a normal system of photosynthesizing membranes. The dark synthesis of chlorophyll in this mutant is broken, the mutant accumulates only carotenoids, the membrane system of its plastid being reduced. On the contrary, mutant Y-3 has in the darkness a complete set of pigments and a well developed membrane system. In the light this mutant yellows due to chlorophyll photodestruction that is followed by destruction of the membrane system of chloroplasts.     

The inhibition of kohlrabi chloroplast degeneration by kinetin

Summary Detached kohlrabi leaves of late autumn material yellowed completely after 6 days in weak light. This process was accompanied by a decrease in chlorophyll, protein, and ribonucleic acid levels. In the chloroplasts, degeneration symptoms such as reduction in chloroplast volume, the decay of grana, development of the long thylakoid system, disappearance of chloroplast ribosomes, increase in the volume of plastoglobuli, and finally a complete breakdown of plastids in the digesting vacuoles, were observed. The ultrastructural changes in degenerating kohlrabi chloroplasts resembled those described earlier for brussels sprouts (Dennis et al. 1967), which suggests that the plastid degeneration model may be specific for speciesBrassica oleracea L.Kinetin inhibited the fall in the level of chlorophyll, proteins, and RNA in relation to the control material, and even stimulated chlorophyll and protein synthesis to a level higher than that of the initial material. Treatment with kinetin also markedly delayed the loss of chloroplast ribosomes. The most evident effect of the kinetin influence on the plastid ultrastructure was the stimulation of the formation and maintenance of grana. A possible mechanism for these processes in the light of the recent studies on the chloroplast membranes is discussed.     

Changes in the Ability of Photophosphorylation and Activities of Surface-Bound Adenosine Triphosphatase and Ribulose Diphosphate Carboxylase of Chloroplasts Isolated from the Barley Leaves Senescing in Darkness

Dark-induced aging of detached primary leaves of 11-day-old barley seedlings brings about a significant decline in the rates of ferricyanide [Fe(CN)₆]^3? reduction and photophosphorylations of isolated chloroplasts. Ferricyanide-supported noncyclic photophosphorylation is somewhat more susceptible to leaf aging than phenazine methosulfate (PMS)-supported cyclic phosphorylation. Non-latent membrane-bound adenosine triphosphatase (ATPase) and ribulosediphosphate carboxylase (RuDPCase) lose about half of their initial activities after 24 h, while during this period the electron transport and photophosphorylation activities are much less affected. Also, the loss of RuDPCase is almost complete, while chloroplasts still exhibit a significant level of [Fe(CN)₆]^3? reduction and photophosphorylations after 7 days of dark incubation. This would suggest that the enzymatic dark reactions are more sensitive to aging stress than the primary photochemical reactions of chloroplasts. Studies on the effect of divalent cations such as Mg²⁺ and Ca²⁺ on non-latent ATPase activity revealed that the dark stressed aging of detached leaves brings about a time dependent alteration in the response of this enzyme to Mg²⁺, but not to Ca²⁺. The former showed inhibitory as well as stimulatory response, whereas the latter always caused the usual stimulation. Addition of kinetin (50 μM) ensured retention of [Fe(CN)₆]^3? reduction, photophosphorylations and RuDPCase activity in chloroplasts during leaf aging, but it failed to preserve the initial loss in the activity of the non-activated membrane-bound ATPase.     

THE ULTRASTRUCTURAL DEVELOPMENT OF THE DIMORPHIC PLASTIDS OF ZEA MAYS L.

The development of the dimorphic chloroplasts of Zea mays L. in adult foliage leaves is described, and a method of correlating ultrastructural stages by means of leaf chlorophyll is presented. In addition, the developmental changes in chlorophyll a/b ratio are discussed. Both the mesophyll and the bundle sheath plastids contain small grana at the earliest stages of plastid development. As the plastids enlarge, the mesophyll grana stacks increase in both length of the appressed membrane and in the number of thylakoids per granum. Initially, the grana stacks in the bundle sheath plastids also enlarge, but as the plastids approach full size, most of the membrane appression is lost. However, the remaining areas of appression in the bundle sheath plastids show an increase in the number of thylakoids in each small granum.     

Plastid Development and Ultrastructure in Yellowish Mutants of Sugar Cane

The comparisons of the ultrastructures of plastids in yellowish mutant and yellowish-green striped mutant and in normal green plant from tissue culture of sugar cane were made. There was no difference found in the structure and development of chloroplasts between the normal green leaves and the green tissue of striped leaves, but the plastid in the yellowish tissue of two kinds of mutants were anomalous. They had not a fully developed system of grana and stroma lameilae as in the normal green leaves. This aberrant plastid contained only some vesicles, a few lamellae and more or less clearly defined ribosome particles and DNA-like mierofibrils, while some stacking and swelling of thylakoids were often observed. In some sections of this aberrant plastid a bunchy lamellae and cross connective fibrils between parallel lamellae were often found too. However, the mixed cell which contains both of normal chloroplast and defective plastid together was never found in the leaves of the mutant plants. It was suggested that yellowish and yellowish-green striped leaves from tissue culture of sugar cane might be caused by nuclear gene mutation.     

Electron microscopic evidence for the reversible transformation ofEuonymus plastids

Contradictory concepts on whether the differentiation of plastids is monotropically directed or reversibly transformable with one another have been argued for a long time. In the present report, the evidence to support the latter concept, i.e. the reversible transformation, will be presented. The seasonal yellowing and regreening ofEuonymus leaves were observed by means of electron microscopic study. In the yellowing of chloroplasts during winter, plastoglobules appeared in the plastid stroma and increased in number according to the disintegration of lamellae; then the degenerated chloroplasts (chromoplasts) were filled up with these plastoglobules. In the next spring, however, regreening of the yellowed leaves took place; the lamellae were regenerated in the chromoplasts to again restore the normal chloroplast structure. Infolding of the inner membrane was never observed in these regreening plastids. The number of plastoglobules in the plastids decreased as the lamellae regenerated, and the chlorophyll content increased. These observations suggest that the plastoglobules in chromoplasts (plastids in yellowed leaves) are made of material of the disintegrating lamellae and are re-used as the source of supply for the reformation of lamellae in the spring reversal.     

Photosynthetic and Heterotrophic Ferredoxin Isoproteins Are Colocalized in Fruit Plastids of Tomato

Fruit tissues of tomato (Lycopersicon esculentum Mill.) contain both photosynthetic and heterotrophic ferredoxin (FdA and FdE, respectively) isoproteins, irrespective of their photosynthetic competence, but we did not previously determine whether these proteins were colocalized in the same plastids. In isolated fruit chloroplasts and chromoplasts, both FdA and FdE were detected by immunoblotting. Colocalization of FdA and FdE in the same plastids was demonstrated using double-staining immunofluorescence microscopy. We also found that FdA and FdE were colocalized in fruit chloroplasts and chloroamyloplasts irrespective of sink status of the plastid. Immunoelectron microscopy demonstrated that FdA and FdE were randomly distributed within the plastid stroma. To investigate the significance of the heterotrophic Fd in fruit plastids, Glucose 6-phosphate dehydrogenase (G6PDH) activity was measured in isolated fruit and leaf plastids. Fruit chloroplasts and chromoplasts showed much higher G6PDH activity than did leaf chloroplasts, suggesting that high G6PDH activity is linked with FdE to maintain nonphotosynthetic production of reducing power. This result suggested that, despite their morphological resemblance, fruit chloroplasts are functionally different from their leaf counterparts.     

Impact of the stringency of cell selection on plastid segregation in protoplast fusion-derived Nicotiana regenerates

Summary Vegetative segregation of a mixed plastid population in protoplast fusion-derived cell lines can be directed by a selection favouring the multiplication of one of the parental plastid types. This report defines some of the critical conditions leading to a homogeneous plastid population in cybrid plants generated by protoplast fusion between Nicotiana plumbaginifolia and an albino and streptomycin-resistant N. tabacum plastid mutant. Light (1,500 lx) conferred a strong selective advantage to chloroplasts versus albino plastids, while the lack of this effect in dim light (300 lx) indicated that a sufficient light intensity is essential to the phenomenon. Selection on streptomycin-containing medium in the dark, however, led to the preferential multiplication of resistant plastids. Streptomycin selection of resistant chloroplasts in the light, consequently, results in a plastid selection of doubled stringency. In another experiment a definite, but leaky, selection for chloroplast recombination (selection for greening on streptomycin-containing medium in dim light) was used to reveal various recombination products. Protoplast fusion in fact resulted in cybrid plants showing only simple chimeric segregation of unchanged parental plastids. These results demonstrate the essential requirement for stringent plastid selection, as defined by cell culture conditions, to precede the formation of shoots expected to possess the desired plastid genetic composition.     

Freeze-etched membrane faces and photosynthetic activity in normal and mutantTradescantia chloroplasts

Summary Membrane structure and photosynthetic activity was investigated in normal and mutant plastids ofTradescantia albiflora cv.aureo-vittata. In the stacked membrane regions (the macrograna) of mutant plastids, the B fracture faces lack both 170 Å particles and photosystem II (PS II) activity. The C face has the normal 110 Å particles, and photosystem I (PS I) activity is also similar to that in normal chloroplasts. In dilated macrograna the particle size on the C face significantly decreases, and as progressive plastid destruction occurs so PS I activity also disappears. It has been concluded that the integrity of B face particles is related to PS II activity, rather than for membrane stacking. A similar correlation seems to be valid for C face particles and PS I activity.     

EGY1 plays a role in regulation of endodermal plastid size and number that are involved in ethylene-dependent gravitropism of light-grown Arabidopsis hypocotyls

Egy1 was isolated as an ethylene-dependent gravitropism-deficient Arabidopsis mutant. Molecular studies reveal that EGY1 gene encodes a 59-kDa plastid-targeted metalloprotease. It is actively expressed in hypocotyl tissue and targets to endodermal and cortex plastid. Its protein level is up-regulated by both ethylene and light. CAB protein accumulation and chlorophyll level is severely reduced in hypocotyls and endodermal cells, respectively. Sucrose is able to restore the severely reduced starch and lipid contents as well as the deficient endodermal plastid size found in light-grown egy1 hypocotyls yet it fails to rescue the reduced plastid number and chlorophyll level in egy1 endodermal cells. The loss-of-function egy1 mutation results in a smaller size (1.9 ± 0.3 μm in diameter) and less number (5 ± 1) of plastids in endodermal cells, which are nearly 50% of the wild-type. EGY1 is specially required for the development of full-size endodermal plastid in seedlings that are grown on sucrose-free media under light. It plays a direct role in controlling the light-induced chlorophyll production, grana formation and plastid replication in endodermal cell. However, it plays an indirect role in regulation of endodermal plastid size. It is likely that the ethylene-dependent gravitropism-deficient phenotype of egy1 hypocotyls may result from the smaller size and less number of endodermal plastids. Gravicurvature assays performed on ethylene-insensitive mutants, etr1-1, etr2-1, ers2-1, ein4-1 and ein2-5, have clearly demonstrated the necessary role for ethylene in vigorous gravitropism of light-grown hypocotyls. The degree of ethylene-dependent gravicurvature is positively correlated with the combined state of endodermal plastid mass and number. Neither ethylene nor EGY1-regulated full-size endodermal plastid is sufficient for promotion of vigorous hypocotyl gravitropism. Presence of 4 full-size plastids per endodermal cell together with ethylene pretreatment of hypocotyls becomes sufficient to trigger vigorous gravicurvature in light-grown seedlings. A model is therefore proposed to address the role of EGY1 in regulation of endodermal plastid size and number as well as the stimulatory effect of ethylene on hypocotyl gravitropism.     

Environment Dependent Chlorosis in a Mutant Plant of Festuca pratensis, Huds

The chlorophyll content of the leaf tissue from a chlorotichomozygous recessive mutant plant of Festuca pratensis was affectedby variation in the light and temperature regimes to which theplants were exposed. Chlorophyll content was depressed whenthe plant grew rapidly, but was similar to that of normal greenplants when grown slowly under low-light and low-temperatureregimes. Chlorotic plants showed a reduction in dry weight,percentage dry matter, and plastid number but an increase inthe ratio of chlorophyll a : chlorophyll b compared to normalplants.The chloroplasts of the rapidly grown chlorotic plantsshowed a reduced lamellar structure.     

Chlorophyll biosynthesis from glutamate or 5-aminolevulinate in intact Euglena chloroplasts

Chloroplasts observed, by electron microscopy, to be intact and uncontaminated, with high rates of light-dependent protein synthesis and CO₂ fixation were isolated from cells grown on low-vitamin-B₁₂ medium in the light or from cells grown in the same medium in the dark and then exposed to light for 36 h. Both types of chloroplasts were active but less variability was encountered with developing chloroplasts from 36-h cells. The 36-h chloroplasts showed good light-dependent incorporation of 5-amino-levulinic acid (ALA) or l-glutamic acid into chlorophyll (Chl) a which was linear for approx. 1 h. The specific activity of the Chl a remained the same after conversion to pheophytin a, methylpheophorbide a or pyromethylpheophorbide a and rechromatography, indicating that the label was in the tetrapyrrole. Incorporation of ALA was inhibited by levulinic acid, and by chloramphenicol and other inhibitors of translation of 70S-type chloroplast ribosomes at concentrations which did not appreciably inhibit photosynthesis but which blocked plastid protein synthesis nearly completely. Cycloheximide, an inhibitor of translation on 87S cytoplasmic ribosomes of Euglena, was without effect. The 70S inhibitors did not block uptake of labeled ALA. Although labeled glycine was taken up by the plastids, no incorporation into Chl a was observed. Thus the developing chloroplasts appear to contain all of the enzymatic machinery necessary to convert glutamic acid to Chl via the C5 pathway of ALA formation but the Shemin pathway from succinyl coenzyme A and glycine to ALA appears to be absent. The requirement for plastid protein synthesis concomitant with Chl synthesis indicates a regulatory interaction and also indicates that at least one protein influencing Chl synthesis is synthesized on 70S-type plastid ribosomes and is subject to metabolic turnover.Abbreviations ALA 5-aminolevulinic acid - Chl chlorophyll     

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.     

Pigment composition and plastid structure in leaves of carotenoid mutants of maize

Summary In monogenic, recessive chloroplast mutants of maize which contain chlorophylls, and lycopene or -carotene but no normal carotenoids, great variability in the size of plastids was associated with a number of ultrastructural abnormalities. In the mutant accumulating lycopene some plastids contain dense bundles of lamellae, whereas the chloroplasts of the -carotene mutant show poor thylakoid development. Neither of the mutants was able to form normal grana.A comparison of chlorophyll/carotenoid ratios in different chloroplast fractions of normal and mutant leaves showed that plastids of small size and delicate structure contain relatively less chlorophyll than fully differentiated chloroplasts.