The ultrastructural development of plastids in leaves of maize plants exposed to continuous illumination

Summary Chloroplast differentiation in relation to increasing leaf age has been investigated in maize plants exposed to continuous illumination. In the young leaves the proplastids differentiate into chloroplasts containing well organized grana as well as prolamellar bodies. In the older leaves, while plastids differentiate, the prolamellar bodies are no longer detectable. Chloroplast ability to build up prolamellar bodies does not seems so much a light dependent process as it is affected by cell differentiation rate.Supported by a grant of C.N.R.     

MACROMOLECULAR PHYSIOLOGY OF PLASTIDS : VIII. Pigment and Membrane Formation in Plastids of Barley Greening under Low Light Intensity

Sequential changes occurring in the etioplasts of the primary leaf of 7-day-old dark-grown barley seedlings upon continuous illumination with 20 lux have been investigated by electron microscopy, in vivo spectrophotometry, and thin-layer chromatography. Following photoconversion of the protochlorophyllide pigment to chlorophyllide and the structural transformation of the crystalline prolamellar bodies, the tubules of the prolamellar bodies are dispersed into the primary lamellar layers. As both chlorophyll a and b accumulate, extensive formation of grana takes place. After 4 hr of greening, protochlorophyllide starts to reaccumulate, and concomitantly both large and small crystalline prolamellar bodies are formed. This protochlorophyllide is rapidly photoconverted upon exposure of the leaves to high light intensity, which also effects a rapid reorganization of the recrystallized prolamellar bodies into primary lamellar layers.     

Lipid composition of envelopes,prolamellar bodies and other plastid membranes in etiolated,green and greening wheat leaves

Summary Comparative studies of lipid composition were made on prolamellar bodies, envelopes and other plastid membranes separately extracted from etiolated, green or greening (intermittent or continuous light) wheat (Triticum sativum L.) leaves. The different membrane fractions were examined by electron microscopy.The major lipid was digalactosyldiglyceride in the envelopes and prolamellar bodies and monogalactosyldiglyceride in stroma lamellae and grana. Phosphatidylcholine represented 60% of total phospholipids in the envelopes, 30% in prolamellar bodies and 14% in grana. All types of envelopes had the same lipid proportions.For all lipids the lowest fatty acid unsaturation was always found in the envelope membranes. The relative amount of {ie193-1} acid in the phosphatidylglycerol of envelopes increased from 4% (etioplasts) to an average of 15% (etiochloroplasts and chloroplasts).Abbreviations DGDG digalactosyldiglyceride - MGDG monogalactosyldiglyceride - PC phosphatidylcholine - PE phosphatidylethanolamine - PG phosphatidylglycerol - PI phosphatidylinositol - PS phosphatidylserine - SL sulfolipid     

Tentoxin effects on infrastructure and greening of ivyleaf morningglory (Ipomoea hederacea var. hederacea) cotyledons

The effects of 20 μM tentoxin on mesophyll chloroplast ultra-structural development, chlorophyll organization and accumulation, and pigment transformations in cotyledons of dark-grown, 4-day-old ivyleaf morningglory [Ipomoea hederacea (L.) Jacq. var. hederacea]were monitored. After 6 h of white light (200 μEm^?2T.s^?1), many plastids of tentoxin-treated tissues contained prolamellar bodies or inconsistent internal membrane orientation in contrast to the uniform internal membrane orientation and absence of prolamellar bodies in controls. Grana stacking did not progress beyond three to four disc loculi in tentoxin-treatments, and fret membranes were usually discontinuous and reduced. Cylindrical or cupped grana appeared in many chloroplasts after 3 days of light, while other chloroplasts in which disruption was more pronounced had few grana except for remnants, but usually did possess vesicles or structures resembling prolamellar bodies. Tentoxin had no apparent effect on stroma density or plastoglobuli size and number. No starch grains appeared in any of the tentoxin treatments, whereas they appeared after 24 h in controls. Initial protochlorophyllide content and its photoconversion to chlorophyllide and subsequent Shibata shift were not affected by tentoxin. Chlorophyll accumulation rates in tentoxin-treated cotyledons were about 10% of control rates during the first 24 h of greening and about 20% of controls from 48 to 72 h of greening. Chlorophyll alb ratio and PSU size (total Chl/P700) were not significantly affected by tentoxin.     

CHANGES INDUCED BY LOW LIGHT INTENSITIES ON THE PROLAMELLAR BODY OF 8-DAY,DARK-GROWN SEEDLINGS

Etioplasts of 8-day-old, dark-grown seedlings of Phaseolus vulgaris contain large, crystalline prolamellar bodies. The basic structural unit within the prolamellar body is a six-pointed star (star module) with four tubules fusing at each of the nodes. With sufficient illumination some of the tubules are withdrawn and the crystalline prolamellar body transforms to a complex tangle of tubules, the reacted prolamellar body. In vivo spectrophotometry and electron microscopic observations were carried out on portions of the same leaves after varying periods of illumination with low light intensity. Protochlorophyllide transformation was normal. However, the structural changes are not closely tied to protochlorophyllide conversion. The pigment conversion is complete after 20 sec of illumination, but 80% of the prolamellar bodies are still in the crystalline form after 20 min of illumination. After 1 and 2 hr of illumination all prolamellar bodies are reacted. After 4 hr of continuous illumination 35%, and by 12 hr 60%, of the prolamellar bodies returned to the crystalline form. Spectrophotometric evidence and presence of grana show chlorophyll synthesis during this period. The coexistence of grana and the crystalline prolamellar body indicates that when insufficient photosynthetic membrane constituents are provided by the photo-reactions, under low light intensity, the membranes of the reacted prolamellar body will be forced to reform a crystalline prolamellar body.     

The effects of inhibitors of protein synthesis on the differentiation of plastids in etiolated bean seedlings

Summary It has been shown that inhibitors of protein synthesis do not influence the breakdown of the crystal-lattice-like structure of the prolamellar bodies in the plastids when etiolated plants are exposed to light. The formation of grana and the greening of leaves are however considerably inhibited, depending on the concentration of the inhibitor used.     

DEVELOPMENT OF THE DIMORPHIC CHLOROPLASTS OF SUGAR CANE

The vascular bundle sheath cells of sugar cane contain starch-storing chloroplasts lacking grana, whereas the adjacent mesophyll cells contain chloroplasts which store very little starch and possess abundant grana. This study was undertaken to determine the ontogeny of these dimorphic chloroplasts. Proplastids in the two cell types in the meristematic region of light-grown leaves cannot be distinguished morphologically. Bundle sheath cell chloroplasts in tissue with 50% of its future chlorophyll possess grana consisting of 2-8 thylakoids/granum. Mesophyll cell chloroplasts of the same age have better developed grana and large, well structured prolamellar bodies. A few grana are still present in bundle sheath cell chloroplasts when the leaf tissue has 75% of its eventual chlorophyll, and prolamellar bodies are also found in mesophyll cell chloroplasts at this stage. The two cell layers in mature dark-grown leaves contain morphologically distinct etio-plasts. The response of these two plastids to light treatment also differs. Plastids in tissue treated with light for short periods exhibit protrusions resembling mitochondria. Plastids in bundle sheath cells of dark-grown leaves do not go through a grana-forming stage. It is concluded that the structure of the specialized chloroplasts in bundle sheath cells of sugar cane is a result of reduction, and that the development of chloroplast dimorphism is related in some way to leaf cell differentiation.     

Changes in the surface properties of chloroplast membranes from dark-grown Pinus silvestris following illumination

The etiochloroplasts of dark-grown seedlings of Pinus silvestris contain regular prolamellar bodies, grana and stroma thylakoids, chlorophylls a and b but no protochlorophyllide or chlorophyllide. The behaviour of etioplast membranes from Pinus silvestris in polymer two-phase counter-current distribution before and after illumination is however similar to that of etioplast membranes from Pisum sativum and Avena sativum indicating that they share common properties with regard to surface activity although their pigment content varies.     

Ultrastructural changes in isolated plastids

Summary Etio-chloroplasts were isolated from greening maize leaves and their ultrastructure was investigated immediately after isolation, as well as at intervals of several hours after their exposure to light or darkness. The following ultrastructural changes have been observed:In plastids isolated from etiolated leaves illuminated for 1–2 hours, the crystalline structure of the prolamellar bodies is partly restored during the isolation. In some plastids, regions with a regular, crystalline structure of densely packed tubules are even observed. The prolamellar bodies do not change further, either in light or in darkness.In young chloroplasts—i.e., in plastids isolated from etiolated leaves, illuminated for 6 or 15 hours—many prolamellar bodies, usually lying between the grana, appearde novo during isolation. These prolamellar bodies do not disappear in light either. They do not develop at all, however, if the isolation is performed at low temperature (4 °C).The results of the present paper indicate that in isolated etio-chloroplasts some tubular structures are newly formed, but that the conversion of this material into the thylakoids is not possible under the experimental conditions used.     

Accumulation of delta-Aminolevulinic Acid and Its Relation to Chlorophyll Synthesis and Development of Plastid Structure in Greening Leaves

Levulinic acid inhibited the greening of etiolated maize (Zea mays) and bean (Phaseolus vulgaris) leaves and caused accumulation of δ-aminolevulinic acid (ALA). ALA accumulation in maize was equivalent to the decrease in chlorophyll, over a wide range of experimental conditions. It was saturated at low light intensities and was not limited by the supply of substrates during the early hours of greening. During 20 hours in light, levulinic acid had little effect on the structural development of thylakoids in bundle sheath chloroplasts but significantly reduced the number and size of thylakoids in grana of mesophyll chloroplasts. Recrystallization of prolamellar bodies and their reformation was inhibited. Mitochondria appeared not to be affected.     

Changes induced on the prolamellar body of pea seedlings by white,red and blue low intensity light

Summary We analyzed transformation, recrystallization, splitting and dispersion of prolamellar bodies during chloroplast development in pea seedlings illuminated by white, red and blue light of low intensity. With the help of a stereometric method we determined that there was a significant increase of prolamellar body number and a sharp decrease of their volume in differentiating chloroplast even in the first 2 hours of illumination. Decrease of prolamellar body dimensions was due both to gradual dispersion of its elements into primary thylakoids (indicated by the decrease of total volume of prolamellar bodies in plastid) and to splitting of prolamellar bodies (indicated by the increase of number of promellar bodies in plastid). Red light was more effective in transformation, splitting and dispersion of prolamellar bodies than blue light during the first 8–12 hours. Longer treatment with blue light had a stronger influence on these processes and on complete recrystallization than other light treatments.     

Electron microscope studies on the morphogenesis of plastids

Streptomycin sulphate (2 mg/ml) did not affect the formation of proplastids or the elaboration of prolamellar bodies. The plastids of the streptomycin (SM)-treated cotyledons contained both crystalline prolamellar bodies and ribosomes, and were undistinguishable from the plastids of the water-grown cotyledon. However, plastids from dark-grown SM-treated cotyledons were no longer able to differentiate to more advanced stages of development, even after exposure to light. The plastids of light and dark-grown SM-treated cotyledons often contained prolamellar bodies and abnormal giant grana. Variegation developed in the cotyledons germinated in Hoagland's solution plus SM. The plastids in pale green tissue contained stroma-lamellae and one or two giant grana, whereas in those of pale yellow tissue, many osmiophilic globules, large vacuoles and crystal bodies were observed. It is suggested that the formation of prolamellar bodies may depend on cytoplasmic protein synthesis whereas functional stroma- and grana-lamellae may depend on protein synthesis within the plastids. The inhibitory effects of SM on protein synthesis were used as a tool to test this hypothesis. This work was carried out in the Department of Botany, University of California, Davis, by Grant-GB-11906 from National Science Foundation of U.S.A.     

Ultrastructural changes in isolated plastids

Summary Etioplasts were isolated from maize leaves and the changes in their ultrastructure were followed in light and in darkness for several hours. It has been shown that the regular crystalline structures of prolamellar bodies, present after the isolation in darkness, disappear after 30 to 60 minutes of illumination, and long straight tubules appear within prolamellar bodies. Their appearance is influenced by the molarity of the isolation medium used, by light intensity, duration of illumination and by the temperature at which the isolates are kept. Long tubules appear, however, also in isolated etioplasts incubated for several hours in complete darkness.In isolates illuminated for 2–3 hours long tubules disappear again, and prolamellar bodies produced eventually consist of irregularly connected short tubules. In prolamellar bodies, regions with regular and very dense arrangement of tubules sometimes develop at this stage. The thylakoids (usually perforated) are now arranged concentrically in the plastids. True grana or poly-thylakoids can never be found in isolated etioplasts, not even when the etioplasts have been illuminated for 6 hours or more (up to 24).The present investigations have indicated that in isolated etioplasts in light, tubular elements, which build up the prolamellar bodies, cannot normally be transformed into thylakoids as is the case with intact tissue.The survival of isolated etioplasts is limited at present, and for this reason changes in their fine structure could be followed successfully for as long as 6 hours (in light at 15 °C), although a certain percentage of plastids survive up to 24 hours.     

Tentoxin does not cause chlorosis in greening mung bean leaves by inhibiting photophosphorylation

Effects of the fungal toxin, tentotoxin, on development and chlorophyll accumulation of plastids of primary leaves of mung bean [ Vigna radiata (L.) Wilczek cv. Berken] were studied using spectrophotometric, electrophoretic, and microscopic procedures. In etioplasts of control tissues both prolamellar bodies and prothylakoids occurred, whereas small vesicles were associated with structurally distinct prolamellar bodies in tentoxin-affected etioplasts. As determined by in vivo spectrophotometry, tentoxin-affected etioplasts had 25% less phototransformable protochlorophyll(ide) and 35% less non-phototransformable protochlorophyll(ide) than had control etioplasts after 5 days of dark seedling growth. Tentoxin had no effect on the rate of the Shibita shift. Protochlorophyll(ide) resynthesis in the dark immediately after protochlorophyll(ide) phototransformation was five to six times slower in tentoxintreated than in control tissues. Effects on chlorophyll(ide) content were observed within 30 min of the beginning of continuous white light exposure. In vivo measurement of cytochrome f redox activity revealed that this cytochrome was linked to light-driven electron flow in control tissues within 20 min of the beginning of continuous white light, whereas in the tentoxin-treated tissues there was no linkage (despite the presence of cytochrome f ) at any time. Coupling factor 1 was present and had potential ATPase activity in both control and tentoxin-affected plastids. There was about sixteen times more chlorophyll in control than in tentoxin-treated tissues in continuous as well as in intermittent (2 min light/118 min dark) light. These data are consistent with the view that tentoxin disrupts normal etioplast and chloroplast development through a mechanism unrelated to photophosphorylation.     

Chlorophyll synthetase is latent in well preserved prolamellar bodies of etiolated wheat

Membrane fractions containing intact etioplasts, etioplast inner membranes, prolamellar bodies or prothylakoids from wheat ( Triticum aestivum L. cv. Walde) were assayed for chlorophyll synthetase activity. Calculated on a protein basis, the etioplast inner membrane fraction showed a higher activity than the intact etioplasts. The activity was higher in the prolamellar body fraction than in the prothylakoid fraction. However, when the fractions were incubated in isolation medium with 50% (w/w) sucrose and 0.3 m M NADPH, chlorophyll synthetase activity could not be detected in the prolamellar body fraction, while the prothylakoid fraction maintained a high activity. The spectral shift to a shorter wavelength of the newly formed endogenous chlorophyllide was very rapid in the prothylakoid fraction but slow in the prolamellar body fraction. The relation between the spectral shift of chlorophyllide and the esterification activity in the fractions is discussed. Even exogenous short-wavelength chlorophyllide could not be esterified in well preserved prolamellar bodies. This indicates that chlorophyll synthetase is present in an inactive state in the prolamellar body structure. A large-scale method for the synthesis of geranylgeranylpyrophosphate, one of the substrates of the chlorophyll synthetase reaction, is also presented.     

Etiolation symptoms in sunflower (Helianthus annuus) cotyledons partially covered by the pericarp of the achene

Background and Aims

Etiolation symptoms and the greening process are usually studied on dark-germinated seedlings and this raises the question – can these results be generalized for plants growing under field conditions? This work examines various aspects of the plastid differentiation under the covering of the achene wall, which often remains attached to the cotyledons of sunflower (Helianthus annuus) seedlings grown under light.

Methods

Cotyledons of 7- to 10-d-old sunflower seedlings grown in the dark and on light were examined. The partially covered cotyledons were sectioned into light-exposed, covered and transition zones. Pigment contents, 77 K fluorescence spectroscopy, electron microscopy and fluorescence imaging, along with fluorescence kinetic methods, were used.

Key Results

The light-exposed zone of the partially covered cotyledons was similar to cotyledons developed without achene covering. However, some of the plastids had prolamellar bodies among the granal thylakoid membranes; despite this no protochlorophyllide was detected. The fully covered, yellowish sections contained protochlorophyllide forms emitting at 633 and 655 nm and well-developed prolamellar bodies, similar to those of etiolated cotyledons. In addition, reduced amounts of chlorophyll a, chlorophyll b and stacked thylakoid membrane pairs were found in this region. The transitional sections showed a mixture of the characteristics of the covered and exposed sections. Various, but significantly different values of the photosynthetic activity parameters were found in each sector of the partially covered cotyledons.

Conclusions

The partial covering of the achene wall shades the cotyledon tissues effectively, enough to provoke the appearance of etiolation phenomena, i.e. the permanent presence of flash-photoactive protochlorophyllide complexes and prolamellar bodies (with or without protochlorophyllide), which proves that these phenomena may appear under natural illumination conditions.Key words: Cotyledon, etio-chloroplast, etioplast, etiolation, Helianthus annuus, photosynthetic activity, protochlorophyllide, prolamellar body, sunflower     

Phytochrome-mediated control of grana and stroma thylakoid formation in plastids of mustard cotyledons

The etioplast»chloroplast transition in the cotyledons of mustard seedlings (Sinapis alba L.) has been studied by electron microscopy. It was found that the active form of phytochrome, established by a red light pulse pretreatment, increases the initial rate and eliminates the lag of grana and stroma thylakoid formation after the onset of white light 60 h after sowing. The effect of a pretreatment with 15 s red light pulses is fully reversible by 756 nm light pulses. This reversibility is lost within 5 min. Evidence is presented which suggests that the time course of grana and stroma thylakoid formation is not correlated with the time course of the dispersal of the prolamellar body. The different functions of phytochrome and chlorophyll in controlling thylakoid formation are discussed.     

CHLOROPLAST DEVELOPMENT IN THE GERMINATING SAFFLOWER (CARTHAMUS TINCTORIUS) COTYLEDON

Proplastids in the mesophyll cells of the cotyledons of mature seeds of safflower are irregular in shape and compressed in narrow corners between the large inclusion bodies, oil vacuoles and protein bodies. The proplastids contain a few irregular internal membranes. During dark germination, sheets or sac-like membranes are produced by the activity of the inner component of the proplastid envelope. These continuous membranes become reticulate and aggregate to the center of the proplastid to form after seven days' germination a quasicrystalline prolamellar body. The membranes are at first irregularly arranged and are of two sorts: those found in the interior of the developing prolamellar body, composed of laterally connected spherical profiles, and those on the periphery of the prolamellar body, which are continuous smooth sheets. The prolamellar body in these dark-germinated proplastids reverts after 3 hr of illumination to the irregularly arranged membranous structure of the 5-day dark germination stage. After 6 hr of illumination membranes grow from the prolamellar body forming concentric loops which, in cross section, appear as concentric circles. These membranes must be nested semi-spheroids. Small grana appear immediately on these looped membranes close to the prolamellar body. With further illumination additional grana develop along the looped membranes in close proximity to the slowly disappearing prolamellar body. Grana increase in size and number along the looped intergranal membranes. The prolamellar body disappears after 15 hr of illumination. The interconnecting fret membranes, sparse at the 15-hr stage, increase and after 24-hr illumination result in the typical grana fretwork system of the mature chloroplast. Membranes are continuously being produced by the invagination of the inner member of the plastid envelope.     

Localization of NADPH-protochlorophyllide oxidoreductase in dark-grown wheat (Triticum aestivum) by immuno-electron microscopy before and after transformation of the prolamellar bodies

The localization of NADPH-protochlorophyllide oxidoreductase (PChlide reductase, EC 1.6.99.–) in dark-grown and in irradiated dark-grown leaves of wheat ( Triticum aestivum L. cv. Walde) was investigated by subjecting thin sections of Lowicryl K4M-embedded leaf pieces to a monospecific antiserum raised against PChlide reductase followed by protein A-gold. A well-preserved antigenicity of the tissue was achieved by polymerizing the resin under UV-light at low temperature. In dark-grown leaves PChlide reductase was found in prolamellar bodies only. In leaves irradiated for 30 min with white light PChlide reductase was found not only in the transformed prolamellar bodies but also to a large extent in connection with the prothylakoids. The localization of PChlide reductase is discussed in relation to fluorescence emission spectra of the dark-grown and greening leaves. We conclude that the light-dependent transformation of protochlorophyllide to chlorophyllide initiates a translocation of PChlide reductase from the prolamellar bodies to the prothylakoids.     

Steroidal Saponins are not Main Building Units of the Prolamellar Body in Etioplasts

Sugar-containing lipids were analyzed by thin layer chromatographyin various cell fractions of etiolated Aoena leaves, Auena intactetioplasts and prolamellar bodies isolated from Cucurbita etioplasts.We confirmed the presence of steroidal saponins, avenacosidesA and B in etiolated leaves and crude etioplast fraction ofAvena, but scarcely detected them in Avena intact etioplastspurified by Percoll density gradient centrifugation. Saponinswere hardly detected in the paracrystalline prolamellar bodiesfrom Cucurbita etioplasts. We concluded that steroidal saponinsare not main building units of the prolamellar body in the etioplasts. (Received August 5, 1982; Accepted February 15, 1983)