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.     

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.     

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.     

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.     

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 DEVELOPMENT IN NICOTIANA TABACUM ‘MARYLAND MAMMOTH’

The development of chloroplasts in light-grown and in previously etiolated tissues of tobacco has been studied. A single membrane-bound body is found in the developing plastids of both light- and dark-grown tissue. The contents of the body appear homogeneous, becoming progressively granular as the chloroplast develops. In the mature chloroplast the body contains a fibrillar network resembling strands shown to be DNA by other workers. The prolamellar body persists even in moderately well developed chloroplasts in light-grown plants. Frequently the prolamellar body is connected to the membrane-bound body as well as to the grana. Relatively mature chloroplasts are seen to divide in this tissue. The membrane-bound body may have a role in the formation of lamellae, but the nature of its contents is yet to be determined.     

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.     

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.     

Electron microscope studies on the morphogenesis of plastids

Comparisons of the ultrastructure of plastids in three kinds of variegated leaves of tomato plants were made. No difference in the structure and development of chloroplasts in normal green leaves and in the green tissue of variegated leaves was found. The albescent tissues of chromosomal genetic variegated leaves contained only aberrant plastids, which were amoeboid or cup-shaped and had large vacuoles in the stroma. Ribosomes were absent from all plastids in this kind of variegated leaves. Three types of plastids, i.e. chloroplasts containing grana, chloroplasts lacking grana, and plastids lacking internal membranes, were present in the pale green tissues of the variegated leaves of extrachromosomal genetic tomato mutants. Depending on the distribution of these plastids, five cell types were observed in these tissues. Ribosomes were present in all plastids in this type of variegated leaves. In the albescent tissues of variegated leaves induced by streptomycin treatment, two kinds of plastids were observed, one containing giant grana and the other lacking organized internal membranes. A common feature of plastids in this albescent tissue was the presence of light stainable ribosomes. It was suggested that the development of variegated leaves may be caused by blocking an early stage of plastid development. 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 Development of Chloroplasts in Sacred Lotus Embryo Bud under Invisible Light

The present paper reports that the development ultrastructural observations of chloroplasts from sacred lotus (Nelumbo nucifera) embryo buds under invisible light. Embryo bud of sacred lotus is enclosed by three layers of thick integument (pericap, seed coat and thick fleshy cotyledons). During the period of the formation of embryo bud, it remained in dark condition, but turned from pale yellow to bluish-green. It was noteworthy that chloroplasts of the embryo bud had well developed giant grana under invisible light. Their developmental pathway in sacred lotus, however, was different from those of other higher plants grown under sunlight, intermittent light, or even in dark conditions (Fig. 1). The chloroplast development of embryo buds in Sacred lotus seeds in invisible light underwent only in the following three stages: (1) In the first stage the development was similar to that from other higher plants, the inner envelope membranes of the proplastids were invaginating. (2) In the second stage, a proplastid centre composed of prolamellar bodies (PLB)with semicrystalline structure was formed, and was accompanied by one or two huge starch grains in almost each proplastid. In the meantime, prothylakoid membranes extended parallelly from the plastid centre in three forms: (a) One plastid centre extending parallelly prothylakoid membranes from itself in one direction; (b) The same to (a), but extending in two directions; (c) Two plastid centres extending parallelly prothylakoid membranes between the centres. (3) In the third stage, grana and stroma thylakoid membranes of chloroplasts were formed. It is to be noted that most of chloroplasts had only one or two giant grana which often extended across the entire chloroplast body, and the length of the grana thylakoid membranes of the chloroplasts from embryo bud in Sacred lotus is 3 to 5 times as many as that in other higher plants. However, their stromatic thylakoid membranes were rather rare and very short. The giant grana were squeezed to the margin of the chloroplast envelope by one or two huge starch grains.     

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.     

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.     

Ultrastructure of Protein Bodies and Plastids in Cotyledon of Nelumbo nucifera Gaertn.at Seed Germination

The present article deals mainly with the formation and dissolution of protein bodies and development of plastids in cotyledon cells of Nelumbo nucifera during seed germination. Electron microscopic studies reveal that protein bodies are formed after imbibition of the cotyledons before germination. They are produced through accumulation of protein material in small vacuoles delivered from the exudates of endoplasmic reticulum or by fragmentation of endoplasmic reticulum itself. In the period of germination, most of the material in the protein bodies dissolute and they coalesce with each other forming large vacuoles. The protein residue of the vacuoles condenses into small blocks with high electron density adhering to the tonoplast or freely floating in the vacuole. Thus, it suggests that the protein bodies of the germinating N. nucifera cotyledons are originated from vacuoles formed by endoplasmic reticulum. Part of the plastids found in cotyledonous cells of mature N. nucifera seeds exists as proplastids. They develop continuously after imbibition of the cotyledons. During the period of seed germination, many concentric lamellae are developed along the plastid membrane on which they later coalesce with the neighboring concentric lameUae forming loosely organized prolamellar bodies which condense into paracrystalline lattices. No ribosomes are present in the inter spaces of paracrystatline lattice. One to several prolamellar bodies can be developed in one plastid.     

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.     

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.     

THE EFFECT OF 3-AMINO-1,2,4-TRIAZOLE ON THE ULTRASTRUCTURE OF PLASTIDS OF TRITICUM VULGARE SEEDLINGS

The application of sublethal doses of 3-amino-1,2,4-triazole (AT) to germinating, light-grown wheat grains causes chlorosis of the resulting leaves. An ultrastructural examination of the leaf tissue reveals that the plastids lack normal grana-fret membrane systems and chloroplast ribosomes. A few disorganized membranes are always present in these chloroplasts. However, AT-treated, dark-grown seedlings contain proplastids with non-crystalline prolamellar bodies and ribosomes. When these etiolated, treated plants are exposed to 600 ft-c light for various periods of time, the proplastids fail to develop into normal, grana-containing chloroplasts.     

FORMATION OF THE PROLAMELLAR BODY IN 8-DAY,DARK-GROWN SEEDLINGS

The development of the prolamellar body in etioplasts of dark-grown seedlings of Phaseolus vulgaris is followed through the 8th day. From 2 to 6 days there is an increase in plastid size and starch content and synthesis of a system of porous lamellae which appear to arise, as such, from the inner component of the plastid envelope. From 6 to 8 days much of the starch disappears accompanied by rapid membrane synthesis resulting in an extensive prolamellar body. A model of the prolamellar body is discussed in which the basic structural unit is a six-pointed star with four tubules joining at each node. Observation of face views of the porous peripheral lamellae at their juncture with the prolamellar body suggests the origin of the prolamellar body by the continued contraction of the porous lamellae and the formation of interconnecting tubules between adjacent lamellae. The pores of the peripheral lamellae appear to correspond to the areas of stroma within each star module. Short lengths of membranes of individual peripheral lamellae fuse, forming short overlaps which resemble small, two-compartmented grana. It is postulated that this is the initial step in grana formation.     

DEVELOPMENTAL ANATOMY OF EPIDERMAL AND MESOPHYLL CHLOROPLASTS IN OPUNTIA BASILARIS LEAVES

Developmental studies of Opuntia basilaris Engelm. & Bigel. leaves revealed the presence of three morphologically distinct types of plastids. All epidermal cells examined contained chloroplasts. After 13 days of growth in the dark the plastids of epidermal and mesophyll cells were characterized by the presence of a prolamellar body and fibril inclusions. Epidermal plastids which developed under light conditions contained large stromacentres and a limited grana-fret membrane system. Guard cell plastids developed under similar conditions had a much poorer developed grana system with fibril inclusions apparent only during the developmental stages. At maturity these plastids appeared swollen or dilated. Mesophyll plastids had fibril inclusions during all stages of development and at maturity contained a very extensive grana-fret membrane system. Microbodies were found in association with the mesophyll plastids. Starch accumulation was common in subsidiary cell and guard cell plastids.     

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.     

THE ETIOPLAST–CHLOROPLAST TRANSFORMATION IN TOBACCO: CORRELATION OF ULTRASTRUCTURE,REPLICATION, AND CHLOROPHYLL SYNTHESIS

Plastid ultrastructure was correlated with plastid replication and growth during the greening of etiolated tobacco tissue. The plastids grow rapidly, and this growth is correlated with a period of rapid chlorophyll synthesis. Chlorophyll synthesis is correlated with the formation of grana. The plastids divide by constriction and continue to do so after most of the grana have been formed. They contain single membrane-bound bodies, which are observed less frequently in the last stages of chloroplast maturation. No internal morphological features of the plastids appear to be related to causal aspects of replication.