ent-Kaurene biosynthesis in a cell-free system from wheat (Triticum aestivum L.) seedlings and the localisation of ent-kaurene synthetase in plastids of three species

A cell-free system capable of converting [¹⁴C]geranylgeranyl diphosphate to ent-[¹⁴C]kaurene and to an unidentified acid-hydrolysable compound was obtained from the basal portions of 5-d-old shoots of wheat seedlings (Triticum aestivum L.). By means of marker enzyme activities, the synthesis of ent-kaurene and the unknown compound could be quantitatively assigned to a plastid fraction obtained by Percoll-gradient centrifugation of the homogenate. The enzyme activities were located within the plastids, probably in the stroma, because they withstood trypsin treatment of the intact plastids, and the plastids had to be broken to release the activity, which was then obtained in soluble form. Plastid membranes had no activity. Plastid stroma preparations obtained from pea (Pisum sativum L.) shoot tips and pumpkin (Cucurbita maxima L.) endosperm also yielded ent-kaurene synthetase activity, but did not form the unknown compound. The exact nature of the active plastids was not ascertained, but the use of methods for proplastid isolation was essential for full activity, and the active tissues are all known to contain high proportions of proplastids, developing chloroplasts or leucoplasts. We therefore believe that ent-kaurene synthesis may be limited to these categories. Mature chloroplasts from the wheat leaves did not contain ent-kaurene synthetase activity and did not yield the unknown component. Incorporation of [¹⁴C]geranylgeranyl diphosphate into ent-[¹⁴C]kaurene and the unknown component was assayed by high-performance liquid chromatography with on-line radiocounting. ent-[¹⁴C]Kaurene was identified by Kovats retention index and full mass spectra obtained by combined gas chromatography-mass spectrometry. The unknown component was first believed to be copalyl diphosphate, because it yielded a compound on acid hydrolysis, which migrated like copalol on high-performance liquid chromatography and gave a mass spectrum very similar to that of authentic copalol. However, differences in the mass spectrum and in retention time on capillary gas chromatography excluded identity with copalol. Furthermore, the unhydrolysed compound was not converted to ent-kaurene by a cell-free system from C. maxima endosperm as copalyl diphosphate would have been.Abbreviations ADH alcohol dehydrogenase - AMO 1618 2isopropyl-4-(trimethylammoniumchloride)-5-methylphenyl piperi-dine-1-carboxylate - BSA bovine serum albumin - DTT dithioth-reitol - GA_n gibberellin A_n - GAPDH NADP⁺-glyceraldehyde 3-phosphate dehydrogenase - GC-MS combined gas chromatography-mass spectrometry - GGPP all trans-isomer of geranyl-geranyl diphosphate - KS ent-kaurene synthetase - MDH malate dehydrogenase - MAA mevalonate activating activity - SOR shikimate oxidoreductase We thank Mrs. Gudrun Bodtke and Mrs. Dorothee Dasbach for able technical assistance, Prof. L.N. Mander (Australian National University, Canberra, Australia) for ent-[²H₂]kaurene and Dr. Yuji Kamiya (RIKEN, Saitama, Japan) for geranylgeraniol and copalol. The work was supported by the Deutsche Forschungsgemeinschaft.     

Regreening of senescent Nicotiana leaves. II. Redifferentiation of plastids

Single senescent leaves attached to decapitated shoots of Nicotiana rustica L. regreened, especially when treated with cytokinin. Regreening caused an increase in leaf thickness, due to cell expansion. Senescent leaf plastids (gerontoplasts) were smaller than green chloroplasts, with degenerated membrane systems and stroma, and larger plastoglobuli. At advanced senescence, micrographs showed disintegrating gerontoplasts, reduced numbers of plastids were counted, and regreening became variable. The redevelopment of grana and stroma in regreening plastids was accelerated by cytokinin. All plastids in regreening leaves were identifiable as redifferentiating gerontoplasts because of their content of plastoglobuli and starch. Immunogold labelling showed significant association of POR with etioplasts in cotyledons, but with mature plastids in regreening leaves. No proplastids or dividing chloroplasts were observed in regreening leaves. Plastids numbers declined during senescence and did not increase again during regreening. It is concluded that the chloroplasts of regreening leaves arose by redifferentiation of gerontoplasts.Keywords: Chloroplasts, cytokinin, Nicotiana, senescence, regreening.      

甜菊组织培养物中叶绿体的超微结构与脱分代

含有叶绿体的甜菊（Ｓｔｅｖｉａｒｅｂａｕｄｉａｎａ）愈伤组织细胞转移至新鲜培养基后,导致光合片层的逐渐减少或消失,最后叶绿体脱分化形成原质体样的结构。超微结构观察表明,光合片层的减少或消失与降解及叶绿体分裂特别是不均等缢缩分裂而致基质组分和类囊体膜稀释有关。这一过程并不完全同步,一些质体含有少量正常的片展而另一些质体含有退化的片层甚至片展结构完全消失。细胞的一个明显特点是细胞器大多聚集在细胞核附近,细胞质增加并向细胞中央伸出细胞质丝。同时可观察到原质体。培养７ｄ后,许多细胞呈分生状态,细胞质富含细胞器,充满了细胞的大部分空间。此时细胞中的质体大多呈原质体状态。在细胞生长的稳定期,质体内膜组织成基质基粒片层,同时质体核糖体增加。文中讨论了高度液泡化细胞脱分化与细胞中叶绿体脱分化的关系。     

THE ULTRASTRUCTURAL MORPHOLOGY AND ONTOGENY OF OAT COLEOPTILE PLASTIDS

Structurally similar proplastids occur in the shoot, scutellum, and root of the oat embryo at the start of germination. These proplastids follow several pathways of differentiation, depending on their location within an organ and on previous exposure to light. During the first 24 hr of germination morphologically similar amyloplasts are formed from the preexisting proplastids in most of the cells of the seedling. After about 24 hr in the light, unique chloroplasts begin to develop in a subepidermal ring of small cortical parenchyma cells in the coleoptile and give the organ a pale green color. At 48 and 72 hr the coleoptile chloroplasts and etioplasts are conspicuously different from the corresponding leaf plastids in morphology and ontogeny but contain typical photosynthetic grana and prolamellar bodies. Study of the ontogeny of plastids in the epidermal and nongreening parenchymal regions of dark grown coleoptiles shows that these plastids undergo significant losses in starch content, and some increase of membranes within the plastid, related to the age of the cell. Light has little effect on the structure of these plastids. It is suggested that the ontogeny of all the plastid types of the oat seedling begins with a common precursor—a relatively simple proplastid that is present at the time of germination. Starch grains showing two distinct types of erosion, apparently enzymatic, were observed in oat coleoptile plastids. In one type (grooved appearance) the starch grains are consistently associated with plastid membranes, while in the other type (irregular, spiny appearance) the starch grains are associated with the plastid stroma only. We suggest that there are two enzyme systems for metabolizing starch in oat plastids—one membrane-bound and the other free in the stroma.     

南湖菱苗端茎轴质体起源与发育的超微结构研究

南湖菱苗端茎轴质体的发育经历了变形、内吞、出芽等变化过程。在苗端茎轴的边缘部位以及幼叶中 ,前质体将发育成为具发达类囊体片层的叶绿体。而在茎轴的中央部位 ,前质体将发生消亡 ,自身水解及液泡内吞是原质体消亡的重要原因。南湖菱苗端的自然发育过程为研究南湖菱质体的起源与分化提供了重要素材。     

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.     

Ultrastructural localization of photosynthetic activity in thylakoids during chloroplast development in maize

The localization of photosynthetic activity in developing maize (Zea mays L.) chloroplasts was studied in situ by two electron-microscopic-cytochemical methods. The activity of photosystem I was detected by photooxidation of 3,3-diaminobenzidine (DAB) and the activity of the photosystem II by photoreduction of thiocarbamyl nitrotetrazolium blue (TCNBT). During the transformation of proplastids into chloroplasts, at the base of the leaf blade the DAB reaction appeared before the TCNBT reaction. A positive DAB reaction was observed in the single thylakoids of plastids in cells located only about 0.5 mm above the base. Dark, osmiophilic DAB polymers accumulated in the lumina of the thylakoids. Plastid envelopes and tubules of the prolamellar bodies in immature chloroplasts were DAB-negative. In fully differentiated leaf tissue the DAB reaction was intense in the thylakoids of bundle-sheath chloroplasts, as well as in the stroma thylakoids and the peripheral grana thylakoids of mesophyll chloroplats. The photoreduction of TCNBT started in leaf tissue about 1 mm above the base. Dark granular material of reduced TCNBT appeared mostly in the partitions of grana, i.e. interthylakoidally, but some granules were also attached to the stroma thylakoids. The membranes of plastid envelopes and the tubules of prolamellar bodies showed a negative TCNBT reaction. Young bundle-sheath chloroplasts contained some reduced TCNBT in their grana; these deposits largely disappeared in the course of further differentiation. In mature leaf tissue the photoreduction of TCNBT was conspicuous in the grana of mesophyll chloroplasts, but very weak in the single thylakoids and in the granal rudiments of bundle-sheath chloroplasts.Abbreviations DAB 3,3-diaminobenzidine·4 HCl - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PS(I,II) photosystem (I,II) - TCNBT thiocarbamyl nitrotetrazolium blue chloride     

Protein crystalloids in ribosome-deficient plastids of Aeonium domesticum cv. variegatum (Crassulaceae)

Protein crystalloids are typical constituents of Aeonium domesticum plastids. They are composed of hexagonally arranged tube-like elements situated in the stroma without a bordering membrane. The single tubule has an external diameter of about 20 nm and an internal one of about 10 nm. The green-white-green mesochimera Ae. domesticum cv. variegatum contains normal chloroplasts in the green tissue and colourless plastids in the pale tissue. The defective plastids have a double-layered envelope, scarce internal membrane structures and contain, in the mature stage, a large vacuole. Plastid ribosomes can be detected only rarely in proplastids. They lose their ribosome complement entirely in the course of development. Polyacrylamide gel electrophoresis of total nucleic acids extracted from white tissue revealed the absence of the 23S and 16S rRNA normally present in plastids. Despite the loss of ribosomes, the plastids contain large protein crystalloids, which are structurally identical with those of normal green chloroplasts. Consequences concerning problems of encoding and transport of crystalloid protein(s) are briefly discussed.Abbreviations CAM crassulacean acid metabolism - FIP fraction I protein - L I epidermis - L II subepidermal layer - L III leaf core - SPC succulent protein crystalloid This is the first part of a series on the crystalloid-forming succulent protein     

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.     

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     

Lipid biosynthesis by chloroplasts isolated from developing Zea mays

Fatty acid biosynthesis by isolated plastids has been examined in relation to chloroplast development and differentiation in leaves of maize plants grown in light for 7 days. Biosynthesis of fatty acids from acetate by proplastids prepared from the basal regions of the leaf was low and mainly palmitate was synthesized. The greatly increased utilization of acetate for fatty acid biosynthesis as the plastids increased in size was due to an increased synthesis of oleate. The maximum synthesis of total fatty acids and monoenoic fatty acids was obtained in chloroplasts prepared from leaf tissue 6–8 cm from the base of the plant where granal formation was most active. Fully-developed chloroplasts prepared from distal regions of the leaf were less active in fatty acid biosynthesis. Maize chloroplasts failed to synthesize fatty acids when isolated by methods commonly used to prepare active spinach chloroplasts. The method of isolation which included a density gradient gave a high proportion of Class I chloroplasts from maize leaves and incorporated up to about 10% of the acetate used. Biosynthesis of unsaturated fatty acids, especially with chloroplasts prepared from the most mature tissue, was increased by the addition of both mitochondrial and microsomal fractions. Increases in polyunsaturated fatty acids were also obtained but the proportions in the newly-synthesized fatty acids were well below the endogenous levels. Monoenoic synthesis was greatly stimulated by increasing the pH in the range 7·0–8·0 and also the highest proportions of unsaturated fatty acids were obtained at short incubation times.     

Ulfrastructure of the histological zones in growing vegetative buds of Salix spp.

Ultrastructural differentiation in the shoot apex of growing vegetative buds of Salix was studied, and some micrographs analysed morphometrically. The distribution of inorganic phospahte (P;) was analysed cytochemically. A distinct histological zona–tion was observed in the apex. The relative volumes of nuclei and plastids were significantly higher in the central tunica zone than in the peripheral one. The corpus differed from the central tunica zone by significantly lower volume density of nuclei and higher of vacuoles and mitochondria. During differentiation of the rib meristem vacuole volume increased significantly, while the relative volumes of nuclei, mitochondria, nucleoli, and heterochromatin decreased. It was not possible to decide whether the vacuoles originate from ER or GERL. Morphogenesis of chloroplasts with large starch grains and grana from proplastids was evident in the rib meristem; dedifferentiation to S–plastids was found in the protophloem. Prolamellar bodies were observed in the procambium plastids. The protophloem was characterized by P–protein and spiny vesicles. P_i was found in the nucleoli of most epidermis cells, several procambium cells, and a few chlorencyma cells, but never in the tunica of the growing apical and developing lateral buds. P_i also occurred in some plasmalemma–somes and occasionally in the walls in connection with plasmodesmata.     

Gain and loss of photosynthetic membranes during plastid differentiation in the shoot apex of Arabidopsis

Chloroplasts of higher plants develop from proplastids, which are undifferentiated plastids that lack photosynthetic (thylakoid) membranes. In flowering plants, the proplastid-chloroplast transition takes place at the shoot apex, which consists of the shoot apical meristem (SAM) and the flanking leaf primordia. It has been believed that the SAM contains only proplastids and that these become chloroplasts only in the primordial leaves. Here, we show that plastids of the SAM are neither homogeneous nor necessarily null. Rather, their developmental state varies with the specific region and/or layer of the SAM in which they are found. Plastids throughout the L1 and L3 layers of the SAM possess fairly developed thylakoid networks. However, many of these plastids eventually lose their thylakoids during leaf maturation. By contrast, plastids at the central, stem cell-harboring region of the L2 layer of the SAM lack thylakoid membranes; these appear only at the periphery, near the leaf primordia. Thus, plastids in the SAM undergo distinct differentiation processes that, depending on their lineage and position, lead to either development or loss of thylakoid membranes. These processes continue along the course of leaf maturation.     

Subcellular localization of chorismate-mutase isoenzymes in protoplasts from mesophyll and suspension-cultured cells of Nicotiana silvestris

The subcellular locations of two readily discriminated chorismate-mutase (EC 5.4.99.5) isoenzymes from Nicotiana silvestris Speg. et Comes were determined in protoplasts prepared from both leaf tissue and isogenic suspension-cultured cells. Differential centrifugation was used to obtain fractions containing plastids, a mixture of mitochondria and microbodies, and soluble cytosolic proteins. Isoenzyme CM-1 is sensitive to feedback inhibition by l-tyrosine and comprises the major fraction of total chorismate mutase in suspension-cultured cells. Isoenzyme CM-2 is not inhibited by l-tyrosine and its expression is maximal in organismal (leaf) tissue. Isoenzyme CM-1 is located in the plastid compartment since (i) proplastids contained more CM-1 activity than chloroplasts, (ii) both chloroplast and proplastid fractions possessed the tyrosine-sensitive isoenzyme, and (iii) latency determinations on washed chloroplast preparations confirmed the internal location of a tyrosine-sensitive isoenzyme. Isoenzyme CM-2 is located in the cytosol since (i) the supernatant fractions were heavily enriched for the tyrosineinsensitive activity, and (ii) a relatively greater amount of tyrosine-insensitive enzyme was present in the supernatant fraction derived from organismal tissue.     

Study of amino acid composition and biosynthesis of protein components of the membrane system of corn plastids during biogenesis of chloroplasts]

The biosynthesis of membrane proteins in maize plastids at different stages of differentiation of the chloroplast lamellar system was studied. Prolamellar and lamellar system preparations were isolated from maize plastids, disintegrated by osmotic shock under hypotonic conditions. Changes in the amino acid composition of 14C membrane proteins were observed at all stages of chloroplast ultrastructure formation. The maximal level of the apolar amino acids was observed in the membrane fraction of chloroplasts. Washed membranes from maize proplastids and chloroplasts can be resolved into at least 14 protein bands on formic acid--urea polyacrylamide gel. It is pointed out that biogenesis process leads to the increase of lipophylic protein content in the chloroplast lamellae fraction.     

Immunogold localization of glutamine synthetase in soybean leaves,roots, and nodules

Summary Immunogold labelling was used to detect the cellular and sub-cellular distribution of glutamine synthetase (GS) in nodulatedGlycine max var. maple arrow. The protein was detected in thin sections of tissue embedded in LR white acrylic resin by employing two polyclonal antibody preparations, one active chloroplastic GS, the other against the cytosolic form of the enzyme. In the mature leaf tissue, GS was visualized only in the chloroplasts, exclusively within the stroma matrix; in the root cortical tissue, the enzyme was distributed homogenously in the cytosol but with a slight preferential localization associated with certain endomembranes, whereas in the root nodules both cytosolic and plastidial compartments were labelled in infected and uninfected cells. Particular to the infected cells, the bacteroids' inner matrix reacted slightly to the GS antibody and a strong signal was preferentially localized on the bacteroids' outer envelope membranes. In general, GS was more concentrated in nodules as estimated by gold particle distribution, whether in the cytosol, plastids or on the bacteroid envelope membranes, than in either root tissue or leaf tissue. Although the cytoplasmic labelling density in nodules was similar in uninfected and infected cells, certain structural features in the latter (abundant cytosol, numerous GS-positive bacteroids and GS-reactive proplastids) contribute to a more enzyme-rich type than its uninfected counterpart.Abbrevation GS glutamine synthetase     

Subcellular localization of beta-glucosidase in rye, maize and wheat seedlings

The subcellular compartmentation of β -glucosidase was studied in rye, maize and wheat seedlings by immunocytochemical methods. For detection, we used a 10 nm gold-labeled secondary antibody, and results were observed using transmission electron microscopy. In all three species, β -glucosidase was found in plastids, cytoplasm and cell walls. In rye, gold particles were seen on cell walls and cytoplasm in epidermal cells of the root tip and shoot, in bundle sheath cells of the shoot and in all cells, except the vascular bundle cells of the coleoptile. Gold labeling was also observed in plastids of the bundle sheath cells of rye shoot tips and in cortical cells of root tips. In wheat, gold labeling was observed on cell walls and cytoplasm of epidermal cells in the shoot base and coleoptile, and on cell walls and plastids in epidermal cells of the root tip. In maize, gold labeling was mainly found in plastids or proplastids in vascular bundle cells and bundle sheath cells of the shoot, in bundle sheath cells of the coleoptile and in epidermal cells of the root. Some gold particles were also found in cell walls and cytoplasm of stomatal guard cells of the shoot base and vascular bundle cells of the shoot tip and in the cell walls of bundle sheath cells of the shoot tip and root tip epidermal cells. Results are discussed in relation to the role of β -glucosidase in hydroxamic acid release and overall defense mechanism of monocotyledons.     

DAG, a gene required for chloroplast differentiation and palisade development in Antirrhinum majus.

We have identified a mutation at the DAG locus of Antirrhinum majus which blocks the development of chloroplasts to give white leaves with green revertant sectors. The green areas contain normal chloroplasts whereas the white areas have small plastids that resemble proplastids. The cotyledons of dark-grown dag mutant seedlings have plastids which also resemble proplastids. The palisade cells in the white areas of dag mutant leaves also lack their characteristic columnar shape. The DAG locus was cloned by transposon tagging: DAG encodes a novel protein with a predicted Mr of 26k, which is targeted to the plastids. Cleavage of its predicted transit peptide gives a mature protein of Mr 20k. Screening of databases and analysis of Southern blots gave evidence that DAG belongs to a protein family with homology to several proteins of unknown function from plants. Expression of DAG is required for expression of nuclear genes affecting the chloroplasts, such as CAB and RBCS, and also for expression of the plastidial gene RPOB encoding the plastidial RNA polymerase beta subunit, indicating that it functions very early in chloroplast development.     

Purification and Properties of an NADPH-Aldose Reductase (Aldehyde Reductase) from Euonymus japonica Leaves

Intact chloroplasts isolated from Euglena gracilis exhibit high rates of light-driven protein synthesis, whereas protein synthesis by isolated proplastids is absolutely dependent upon the addition of an exogenous energy source in the form of equimolar ATP and Mg²⁺. ATP and Mg²⁺ also stimulate translation by chloroplasts. The greatly increased rates of protein synthesis obtained by supplementing proplastids with ATP and Mg²⁺ have allowed the first clear characterization of proplastid translation products. Two-dimensional polyacrylamide gel electrophoretic analysis of proteins synthesized in organello shows that, while many translation products are common to both plastid types, most are unique to either the proplastid or the chloroplast. Pulse-chase experiments using both proplastids and chloroplasts indicate similar rates of turnover of newly synthesized proteins in both types of plastids. Thus, the differences seen between proplastid and chloroplast translation products are apparently not due to turnover. Immunoprecipitation of large subunit of ribulose-1,5-bisphosphate carboxylase (LS) from pulse-chase experiments indicates that LS is made in both proplastids and in chloroplasts and that the rate of LS turnover is similar in both types of plastids.