Large Starch Grains Research Articles

The Ultrastructure of the Early Development of Schizaea pusilla Gametophytes

Schizaea pusilla is a fern that maintains a completely filamentous gametophyte throughout its development. In this study, we characterize young gametophytes by using light and electron microscopy. In order to gain more reliable structural information, the cryofixation techniques of high pressure freezing and propane jet freezing were used. Apical protonemal cells of young S. pusilla gametophytes are unlike apical cells of other ferns studied to date. The apical protonema was divided into four regions with zone 1 at the apex and zone 4 at the base. Zone 1 has apical vacuoles, numerous Golgi stacks, ER, and a high density of apical vesicles. Zone 2 consists of chloroplasts with large starch grains, small vacuoles, uncharacterized electron-dense inclusions, mitochondria, lipid bodies, Golgi stacks, and ER. The nucleus is located in zone 3, and zone 4 consists of a large vacuole with traversing cytoplasmic strands, which contain chloroplasts, mitochondria, Golgi stacks, and ER. The cytoskeleton and endomembrane system were particularly well-preserved in cryofixed protonemata. Novel structures observed in cryofixed cells include microfilament bundles, multivesicular bodies, and apical vesicles, which appeared to be fusing with the plasma membrane. This study is the first report to use cryofixation/freeze substitution to characterize fern gametophytes and demonstrates that these techniques can be useful in ultrastructural studies of fern development.

Ultrastructural studies on the embryo sac ofViscum minimum I. Megasporogenesis

Changes taking place during megasporogenesis of a mistletoe (Viscum minimum) were examined at both light and electron microscopy levels. No distinct ovules, integuments, or ovarian cavity are present at any stage of development. The multicellular archesporium originates in the center of a solid ovary. Several functional megasporocytes are developed from the archesporial cells, either adjacent to each other or separated by unspecialized cells. The megasporocyte is much larger than surrounding cells, is invested by a thick wall, and possesses a large nucleus and amyloplasts. Although plasmodesmata are absent even between the adjacent megasporocytes, cells enter meiosis simultaneously. Following meiosis a linear tetrad is formed. Double and treble linear tetrads are frequently observed. The development of the embryo sac conforms to the monosporic or Polygonum type of megasporogenesis. However, the bisporic or Allium type of development is occasionally observed in preparations. Factors determining the pattern of development are discussed. As in other plant species which follow the monosporic type of development, only one functional megaspore cell undergoes further development while others degenerate. Unlike the healthy functional megaspore cell, the degenerating cells have large starch grains and electron-dense cytoplasm. At a later stage of development, the degraded cells are absorbed by the surrounding tissue.

Effect of Potassium, Magnesium, and Calcium Deficiencies on Carbohydrate Pools and Metabolism in Citrus Leaves

Carbohydrate content and related enzyme activities were determined in leaves of rough-lemon (Citrus volkameriana Ten. & Pasq) plants grown under K, Mg, and Ca deficiencies. Starch content was lower and soluble sugar significantly higher in K-deficient than in control leaves. Magnesium- and Ca-deficient leaves, on the other hand, accumulated large amounts of starch. Electron micrographs also showed a reduction in the number and size of starch grains in chloroplasts of K-deficient leaves, while those of Mg- and Ca-deficient leaves were filled with large starch grains. Total amylase activity increased 3- to 7-fold in K-deficient leaves, with maltose as the major product and small amounts of maltotriose. Electrophoretic separation of amylase isozymes on native gels containing starch or amylopectin showed higher band intensities in K-deficient leaves. Examination of the capacity of leaf extracts to use amylopectin vs. β-limit dextrin indicated that mainly 8-amylase was involved. Acid invertase activity increased 7-fold in K-deficient leaves, while alkaline invertase showed little change.

Effects of drought and waterlogging on ultrastructure of Scots pine and Norway spruce needles

Effects of water stress on needle ultrastructure of 2-year-old Scots pine (Pinus sylvestris L.) and 5-year-old Norway spruce [Picea abies (L.) Karst.] seedlings were studied in greenhouse experiments. Drought stress was induced by leaving seedlings without watering, and waterlogging stress was produced by submerging the seedling containers in water. Needle samples for ultrastructural analyses were collected several times during the experiments, and samples for nutrient analyses at the end of the experiments. In drought stress, plasmolysis of mesophyll and transfusion parenchyma tissues, aggregation of chloroplast stroma and its separation from thylakoids and decreased size and abundance of starch grains in needles of both species were observed. The concentration of lipid bodies around the chloroplasts were detected in pine needles. Calcium and water concentrations in spruce needles were lower by the end of the experiments compared to controls. In waterlogging treatment, swelling of phloem cells in pine needles and large starch grains, slight swelling of thylakoids and increased translucency of plastoglobuli in chloroplasts of both species studied were observed. The phosphorus concentration in pine needles was higher while phosphorus, calcium and magnesium concentrations in spruce needles were lower in the waterlogging treatments compared to controls. Typical symptoms induced by drought stress, e. g. aggregation of chloroplast stroma and its separation from thylakoids, were detected, but, in waterlogging stress, ultrastructural symptoms appeared to be related to the developing nutrient imbalance of needles.

Structural changes in rapid-cycling Brassica rapa selected for differential waterlogging tolerance

Two populations of rapid-cycling Brassica rapa L. selected for differential waterlogging tolerance and a commercially available standard population were compared in their structural responses to waterlogging. Rates of dry matter accrual were similar under drained conditions but were lower after 6 days of waterlogging, especially in the sensitive population. Chlorophyll content of leaves from the sensitive population was significantly lower than the tolerant population. Examination of the foliage by TEM after 4 days of waterlogging revealed large starch grains that apparently disrupted the grana stacks in the sensitive population. Root morphology also distinguished the populations. After 8 days of waterlogging, numerous adventitious roots were visible in the standard and tolerant populations but not in the sensitive population. No aerenchyma was detected by examination of root cross sections. Mitochondrial morphology was affected by waterlogging, resulting in elongate, branched organelles. Isozyme analysis of malic enzyme, malate dehydrogenase, glucose-6-phosphate dehydrogenase, and pyruvate decarboxylase revealed no differences due to population, treatment, or duration. Isozymes of alcohol dehydrogenase varied with duration of waterlogging, and isozyme forms of phosphoglucomutase were distinct depending on the population. The appearance of stress-specific structural features in populations of B. rapa is discussed in relation to differences in waterlogging tolerance. Key words: hypoxia, isozymes, aerenchyma, adventitious roots, waterlogging, starch.

Chloroplast ultrastructure changes in Pisum sativum associated with supplementary ultraviolet (UV‐B) radiation

ABSTRACTPea plants (Pisum sativum L. cv. Greenfeast) were grown and exposed to supplementary UV‐B radiation from day 17 after planting under growth cabinet conditions. The effects of this exposure on the ultrastructure of chloroplasts and the total soluble sugar and starch concentrations were estimated. Supplementary UV‐B radiation was shown to damage the structure of chloroplasts, as manifested by dilation of thylakoid membranes, a progressive disruption of the thylakoid structure and disintegration of the double membrane envelope surrounding the chloroplast, accompanied by the accumulation of large starch grains. Diurnal changes observed in starch concentration suggest that the higher concentration of starch in supplementary UV‐B‐treated leaves is due to its immobilization, rather than to any increase in starch synthesis: soluble sugars accumulated and remained at a higher level and then later declined.

Cytoplasmic DNA apportionment and plastid differentiation during male gametophyte development in Pelargonium zonale

In the male gametophyte of Pelargonium zonale, generative and sperm cells contain cytoplasmic DNA in high density compared to vegetative cells. Cytoplasmic DNA was examined using the DNA fluorochrome DAPI (4'6-diamidino-2-phenylindole) and observed with epifluorescence and electron microscopy. The microspore cell contains a prominent central vacuole before mitosis; mitochondria and plastids are randomly distributed throughout the cytoplasm. Following the first pollen grain mitosis, neither the vegetative cell nor the early generative cell display a distributional difference in cytoplasmic DNA, nor is there in organelle content at this stage. During the maturation of the male gametophyte, however, a significant discrepancy in plastid abundance develops. Plastids in the generative cell return to proplastids and do not contain large starch grains, while those in the vegetative cell develop starch grains and differentiate into large amyloplasts. Plastid nucleoids in generative and sperm cells in a mature male gametophyte are easily discriminated after DAPI staining due to their compactness, while those in vegetative cells stained only weakly. The utility of the hydrophilic, non-autofluorescent resin Technovit 7100 in observing DAPI fluorescence is also demonstrated.

Plant regeneration from cultured protoplasts of the cooking banana cv. Bluggoe (Musa spp., ABB group)

Suspensions of embryogenic cells of a triploid banana (Musa spp., cv. Bluggoe) were initiated from the uppermost part of meristematic buds, and used as protoplast source. After 20 weeks in culture, the suspension contained a mixture of globular structures or globules and embryogenic cell clusters, as well as single cells. Two types of protoplasts were obtained from embryogenic suspension culture: small (20-30 μm) and larger (30-50 μm) protoplasts with a dense cytoplasm and large starch grains respectively. The small protoplasts probably originated from embryogenic cell clusters, and also from pseudocambial cells of globules, while larger protoplasts were probably released from oval starchy cells and those of the globule peripheral area. In co-culture with a suitable feeder, consisting of suspensions of diploid banana cells, the protoplasts of triploid banana reformed the cell wall within 24 h and underwent sustained divisions leading to the formation of small clusters of 2-3 cells within 7 days. The latter developed directly into embryos without passing through an apparent callus phase. 10% of such embryos gave rise to plantlets when subcultured in 2.2 μM 6-benzylaminopurine and 2 μM 4 amino-3,5,6-trichloropicolinic acid for 1 week, before transfer to MS medium containing 10 μM 6-benzylaminopurine. The rest of the embryos underwent intensive direct secondary embryogenesis which could lead to the formation of plantlets with a frequency of up to 50% upon further transfer to hormone-free medium.

Analysis of Two Rubisco-Deficient Tobacco Mutants, H7 and Sp25; Evidence for the Production of Rubisco Large Subunits in the Sp25 Mutant that Form Clusters and are Inactive

The basis for the lesions in the Sp25 and H7 ribulose-l,5-bisphosphate carboxylase/oxygenase (Rubisco) mutants of tobacco was studied in detail because these plants may be suitable hosts for transformation with the genes for Rubisco enzymes of various origins that have different substrate specificities. We show that the Sp25 mutant lacks active holoenzyme, but contains the large and small subunit polypeptides, Rubisco activase and the chloroplast chaperonin, Cpn 60. The large subunit polypeptides were not distributed uniformly in the stroma in the Sp25 mutant as they were in the wild-type plants, but had an anomalous distribution being present only in aggregated clusters notably in chloroplasts with large starch grains. Furthermore, these clusters were not uniformly distributed throughout the photosynthetic cells but were localized largely in the mesophyll cells surrounding the vascular tissue. In contrast to the Sp25 mutant, the H7 mutant contained the Rubisco holoenzyme, but in this case the enzyme was inactive. It is clear that in both these mutants the Rubisco holoenzyme fails to assemble correctly. In the Sp25 mutant assembly is lost completely while in the H7 mutant the holoenzyme is formed, but the assembly process fails to produce an active enzyme. We suggest that the flaw in assembly in the Sp25 mutant results from a defect in chloroplast encoded proteins.

The fine structure of aleurone cells in the soybean seed coat

The morphology and fine structure of aleurone cells of soybean [Glycine max (L.) Merr.] seed coats were analyzed with transmission electron microscopy for the period of rapid seed fill up to physiological maturity. Thin sections and freeze-fracture replicas were prepared for each stage. The aleurone is a tissue lining the embryo sac and consists of a single layer of cells attached to the aerenchyma of the seed coat proper. During seed fill, aleurone cells contained numerous Golgi-derived vesicles in the basal region of the cytoplasm that were either free or attached to the plasma membrane along the lateral and basal regions of the cell wall. Correspondingly, the Golgi apparatus were well developed with individual dictyosomes having 5 to 8, highly fenestrated stacked cisternae. The degree of fenestration along the periphery of each cisterna increased from the cis to trans region. Rough endoplasmic reticulum (RER) was also abundant, often consisting of up to 30, stacked swollen cisternae which occupied large regions of cytoplasm. Plasmodesmata which connected adjacent aleurone cells was not observed along the dorsal walls of aleurone cells that faced aerenchyma. At physiological maturity, dictyosome cisternae were less fenestrated and had fewer associated secretory vesicles. Stacked lamellae of RER were absent, being replaced by short tubular cisternae and small vesicles. At physiological maturity, the aleurone cells had thick walls, and contained numerous lipid bodies in apposition to the plasma membrane. The cytoplasm appeared densely stained in thin-sections and contained protein bodies and amyloplasts with large starch grains. We conclude that during the period of rapid seed fill aleurone cells produce, package, transport and secrete vesicular contents toward the embryo, that is followed at physiological maturity by the storage of lipid, protein and starch in the same cells. The embryo is the most likely destination for secretory products during the period of rapid seed fill. The fate of the stored food reserves in aleurone cells at physiological maturity may be analogous to that of aleurone tissue of grasses, being utilized during imbibition for processes important to germination.

Rising CO2 levels and their potential significance for carbon flow in photosynthetic cells

Abstract. In the first part of this review, I discuss how we can predict the direct short‐term effect of enhanced CO2 on photosynthetic rate in C3 terrestrial plants. To do this, I consider: (1) to what extent enhanced CO2 will stimulate or relieve demand on partial processes like carboxylation, light harvesting and electron transport, the Calvin cycle, and end‐product synthesis; and (2) the extent to which these various processes actually control the rate of photosynthesis. I conclude that control is usually shared between Rubisco (which responds sensitively to CO2) and other components (which respond less sensitively), and that photosynthesis will be stimulated by 25–75% when the CO2 concentration is doubled from 35 to 70 Pa. This is in good agreement with the published responses. In the next part of the review, I discuss the evidence that most plants undergo a gradual inhibition of photosynthesis during acclimation to enhanced CO2. I argue that this is related to an inadequate demand for carbohydrate in the remainder of the plant. Differences in the long‐term response to CO2 may be explained by differences in the sink‐source status of plants, depending upon the species, the developmental stage, and the developmental conditions. In the third part of the review, I consider the biochemical mechanisms which are involved in ‘sink’ regulation of photosynthesis. Accumulating carbohydrate could lead to a direct inhibition of photosynthesis, involving mechanical damage by large starch grains or Pi‐limitation due to inhibition of sucrose synthesis. I argue that Pi is important in the short‐term regulation of partitioning to sucrose and starch, but that its contribution to ‘sink’ regulation has not yet been conclusively demonstrated. Indirect or ‘adaptive’ regulation of photosynthesis is probably more important, involving decreases in amounts of key photosynthetic enzymes, including Rubisco. This decreases the rate of photosynthesis, and potentially would allow resources (e.g. amino acids) to be remobilized from the leaves and reinvested in sink growth to readjust the sink‐source balance. In the final part of the review, I argue that similar changes of Rubisco and, possibly, other proteins are probably also involved during acclimation to high CO2.

Molecular aspects of plastid envelope biochemistry

One of the most characteristic features of a plant cell is the existence of plastids representing a whole family of interrelated organelles [1, 2]. Meristematic cells contain small undifferentiated plastids or proplastids, which have little internal structure apart from a few flattened sacs which occasionally have continuity with the inner envelope. They ensure the continuance of plastids within a species from generation to generation and are capable of considerable structural and metabolic transformations which give rise to more mature plastids. During root cell differentiation, proplastids develop mostly into amyloplasts, which are storage plastids containing large starch grains. During leaf cell differentiation in the presence of light, proplastids develop into chloroplasts, which are the site of photosynthesis, but in the absence of light, they develop into etioplasts, containing a crystal-like structure called the prolamellar body. Etioplasts can then be transformed into chloroplasts upon illumination. Carotenoid-rich plastids, or chromoplasts, can develop from proplastids or chloroplasts during flower development, fruit ripening or senescence. Therefore, plastid differentiation within plant cells is both light- and tissue-specific, and mechanisms should exist that control nuclear and plastid gene expression.

Fluoride effects on leaf cell ultrastructure of olive trees growing in the vicinity of the Aluminium Factory of Greece

Leaves of olive trees growing in the vicinity of the Aluminium Factory of Greece were ultrastructurally investigated in order to determine any malformations caused by environmental air pollutants, especially hydrogen fluoride, in comparison with control samples and normal seasonal senescence. Estimation of some elements accumulated by these leaves showed that they contained high amounts of F and Al attributable to the operation of the nearby factory. The most seriously effected cell components were found to be the mesophyll chloroplasts that show a dilation of the intrathylakoid space, increase of the number of plastoglobuli, discoloration of plastoglobuli, accumulation of large starch grains and an overall disorganized appearance of the organelle. The nuclear crystalloid inclusions have unusual shapes, while the vacuoles contain a fibrillar/granular material that increases their electron density. It is concluded that the ultrastructural malformations are caused by a combination of environmental stresses and air pollutants.

Development and Structure of Pollen and Embryo Sac in Peanut (Arachis hypogaea L.)

Of the eight stamens in the flower of peanut (Arachis hypogaea L.), four have oblong anthers and four globose ones. Each oblong anther contains four loculi and each globose anther two. The anther wall development follows the basic pattern. Cytomictic channels between microsporocytes were found from pachytene to telophase II. The mature pollen is two-celled. The mature generative cell, which has a PAS positive wall, associates with the vegetative nucleus and may form a male germ unit. There are two ovules, both hemianatropous and bitegmic. Cells of both integuments and nucellus contain starch grains. The nucellar epidermal cells have a larger nucleus, larger starch grains, and thicker walls than other cells of the ovule. A few cells at the micropylar end persist as a nucellar cap. Embryo sac development is of the Polygonum type. The central cell contains many large starch grains, and the antipodal cells are ephemeral. The synergids possess a filiform apparatus. Wall ingrowths occur in the central cell; those at the micropylar end are more massive than those at the chalazal end. Meiosis in microsporocytes occurs earlier and proceeds faster than that in the megasporocyte. The time required for formation, differentiation, and maturation of the generative cell and the egg cell is the same.

Characteristics of plastids responsible for starch synthesis in developing pea embryos

The nature of the starch-synthesising plastids in developing pea (Pisum sativum L.) embryos has been investigated. Chlorophyll and starch were distributed throughout the cotyledon during development. Chlorophyll content increased initially, then showed little change up to the point of drying out of the embryo. Starch content per embryo increased dramatically throughout development. The chlorophyll content per unit volume was highest on the outer edge of the cotyledon, while the starch content was highest on inner face. Nycodenz gradients, which fractionated mechanically-prepared plastids according to their starch content, failed to achieve any significant separation of plastids rich in starch and ADP-glucose pyrophosphorylase from those rich in chlorophyll and a Calvin-cycle marker enzyme, NADP-glyceraldehyde-3-phosphate dehydrogenase. However, material that was not sufficiently dense to enter the gradients was enriched in activity of the Calvin-cycle marker enzyme relative to that of ADP-glucose pyrophosphorylase. Nomarski and epi-fluorescence microscopy showed that intact, isolated plastids, including those with very large starch grains, invariably contained chlorophyll in stromal structures peripheral to the starch grain. We suggest that the starch-storing plastids of developing pea embryos are derived directly from chloroplasts, and retain chloroplast-like characteristics throughout their development. Developing pea embryos also contain chloroplasts which store little or no starch. These are probably located primarily on the outer edge of the cotyledons where there is sufficient light for photosynthesis at some stages of development.

Stereological analysis of cotyledon tissue inVicia faba L. var. aquadulce during germination

Stereological analysis was carried out on cotyledon tissue at three different stages of germination. The tissue was selected by reference to adjacent sections which were assayed histochemically for protease activity. Day six tissue had no activity while eight and 14-day tissue did. Results show that during germination both tissue and cellular components undergo changes. The cells increase in size, the intercellular spaces increase, the cytoplasm increases in volume, the protein bodies swell and fuse, and small starch grains appear while the large starch grains do not undergo any major changes.

Importance of foliar nectaries in the physiology of tree of heaven (Ailanthus glandulosaDesf., Simaroubaceae)

SummaryOur knowledge of the role of extrafloral nectaries in the tree physiology is limited except the ecological function.Ailanthus glandulosa is characterized by a very diversified secretory system in which foliar nectaries are the major components. The initiation of these nectaries may be due to a carbohydrate accumulation during the ontogeny of leaves.With an important requirement of photosynthetic products (vegetative growth, flowering, fruit development), carbohydrate accumulation in leaf is no important. Normal plastids are observed and photosynthetic activity is maximum. Excess in carbohydrates is evacuated by nectaries. When the requirement for assimilates disappears (inflorescence abortion, fall of staminate flowers, cessation of vegetative growth) plastids of palisade cells accumulate large starch grains and numerous plastoglobuli. Starch and lipids increase in the lamina and photosynthetic activity and nectar secretion decrease. These transformations are irreversible. The ablation of foliar ne...

Basil Chlorosis: A Physiological Disorder in CO2-Enriched Atmospheres

(...)Attempts to transmit the disease mechanically or by grafting to unaffected, greenhouse-grown basil and certain other plant species failed. Affected basil that was transferred from the hydroponic facility to a greenhouse and grown in potting mix produced new, chlorosis-free leaves. Under experimental conditions, basil became chlorotic in 1,000 ppm CO 2 , but not when in ambient CO 2 . This was true whether the basil was grown hydroponically or in pots, or under fluorescent or high-pressure sodium lights. Electron microscopy revealed no detectable pathogens, but large starch grains were observed in chloroplasts of chlorotic leaves. Elevated CO 2 concentrations apparently induced basil chlorosis

Leaf ultrastructure, carbohydrates and protein of soybeans grown under CO 2 enrichment

Soybeans ( Glycine max [L.] Merr. cv Bragg) grown under 800 μl CO 2 per liter showed increases of 37, 205, 108, 33, 22 and 31% in specific leaf weight, starch, sucrose, reducing sugars, chlorophyll and soluble protein, respectively, over control plants at 330 μl CO 2 per liter. CO 2 enrichment caused an increase in leaf thickness, due to an increased number of palisade cells, but no major alteration in chloroplast ultrastructure. The extra starch formed under CO 2 enrichment was distributed as slightly larger starch grains among more and larger chloroplasts and more mesophyll cells, rather than as more starch grains per chloroplast. The K m (CO 2) of ribulose biphosphate carboxylase was unchanged by growth at high CO 2, similarly there was little effect on the activity of this enzyme. The increase in starch storage sites, together with the maintenance of ribulose bisphosphate carboxylase activity, enable soybeans to continue to exhibit high photosynthetic rates throughout the growth period under high CO 2 levels.

Host cell responses in susceptible hard pine tissue infected with Endocronartium harknessii

Compatible interactions between susceptible hypocotyl tissue of Pinus banksiana Lamb, and Endocronartium harknessii (J. P. Moore) Y. Hirat. were studied using light and transmission electron microscopy. Host endoplasmic reticulum was observed to be closely associated with the haustorial body, although staining with silver proteinate failed to show any similarity between the contents of the endoplasmic reticulum and extrahaustorial matrix. The haustorium was also commonly observed to be closely associated with the host nucleus, often indenting the latter, though never in direct contact. Chloroplasts in recently infected cells appeared similar to those in uninfected cells, but in more advanced infections large starch grains were observed in the chloroplasts of the outer cortex; such chloroplasts normally contained little starch. Collars were another common feature of infected cells. Collars were continuous with the host cell wall and reacted to silver proteinate in a similar manner to the cell wall; callose was not evident. Collars were associated with portions of the cell wall that were inwardly displaced by the fungus; however, cytoplasmic vesicles were also observed in association with the collar and possibly contributed to their development.