PHYSIOLOGICAL AND ULTRASTRUCTURAL RESPONSES OF CYCLOTELLA MENEGHINIANA (BACILLARIOPHYTA) TO LIGHT INTENSITY AND NUTRIENT LIMITATION1

Photosynthetic characteristics and chloroplast ultrastructure of Cyclotella meneghiniana Kütz. were quantified while the organism was simultaneously adjusting to light and nutrient stress. Cells were grown in batch culture at either low or high light intensity on medium with a nitrogen/phosphorus molar ratio of 2:1 as a control, or with nitrogen or phosphorus deleted from the medium to create nutrient deficiencies. Analysis of variance indicated that light intensity, nutrient deficiency and duration of nutrient deficiency all had significant effects on cell growth, chlorophyll (Chl) concentration/cell, cellular fluorescence capacity (CFC), chloroplast volume and thylakoid surface density. Because interactions existed among nutrient deficiency, extent of nutrient deficiency, and light intensity, all three must be considered together in order to describe accurately the physiology and chloroplast ultrastructure of the diatom. Significant correlations were found between the Chl/cell or CFC/cell and chloroplast volume and thylakoid surface density. Through an increase in Chi concentration, chloroplast volume and thylakoid surface density, the cells successfully adapted to the conditions of low light intensity even while under nutrient stress. In contrast, less Chl/cell, smaller chloroplast volume and less thylakoid surface density were found at high light intensity.     

Exposure to low levels of photosynthetically active radiation induces rapid increases in palisade cell chloroplast volume and thylakoid surface area in sunflower (Helianthus annuus L.)

Summary Sudden changes in photoactive radiation (PAR) (wavelength, 400–700 nm) induces rapid surface area changes in chloroplast thylakoid membranes. Although this response may have important photo-acclimative functions for the plant, little is known about the mechanisms by which changes in irradiance are detected or how thylakoid membranes actually increase or decrease surface area. Knowledge of the time required for significant changes in thylakoid area would help eliminate or support several possible mechanisms that may be involved in this aspect of photo-acclimation in plants. Leaf tissues were acclimated to a PAR of 500 mol quanta per m² per s then exposed to low irradiance (PAR, 50 mol quanta per m² per s) and sampled at 5, 15, 30, and 60 min post exposure. Tissue and cell structure were quantified and results showed a significant increase in the surface-to-volume ratio and surface area per unit of standard leaf volume for both appressed and nonappressed thylakoids within 5 min of exposure to low irradiance. On the basis of the ratios of appressed to nonappressed thylakoids, the surface area of the nonappressed thylakoids was found to increase faster than that of the appressed thylakoids throughout the sample period. The portion of the appressed thylakoids in contact with the stroma was defined as margin thylakoids. Margin thylakoid surface-to-volume ratio did not change relative to the high-irradiance control during the sample period but did remain significantly lower than the low-irradiance control during the sample period. The ratio of appressed to margin thylakoids indicated a broadening and shortening of the appressed thylakoid stack within the first 5 min of low-irradiance exposure. The rapidity of the shade response indicates that the early events in this response probably do not directly involve gene activation pathways.Abbreviations PAR photosynthetically active radiation - Sv surface to volume density - Vv volume density - UV-B ultraviolet B radiation     

Below-ambient levels of UV induce chloroplast structural change and alter starch metabolism

Summary. Electromagnetic radiation (EMR) in the 400–700 nm bandwidth of photosynthetically active radiation (PAR) has been established as an important source of energy for photosynthesis and environmental signals regulating many aspects of green-plant life. Above-ambient levels of UV-B radiation (290–320 nm) under high-PAR conditions have been shown to elicit responses in chloroplasts of Brassica napus similar to those of chloroplasts at low-PAR exposure (W. Fagerberg and J. Bornman, Physiol. Plant. 101: 833–844, 1997). The question arises as to whether UV at normal levels can also evoke similar responses. Here we provide evidence that even below-ambient levels of UV-B (1/28 ambient; Durham, N.H., U.S.A., 1200 hours, March) were capable of inducing an increase in thylakoid surface area relative to the chloroplast volume typical of a low-PAR response (shade response) in sunflowers. This response occurred even though leaves were concurrently exposed to PAR levels that normally induce a “sun” or high-PAR response in the absence of UV-B. Subambient levels of UV-B were also associated with a decrease in chloroplast and starch volume. Exposure to levels of UV-A 1/10 of ambient appeared to enhance the high-PAR response of the chloroplast, characterized by an increase in the amounts of stored starch, an increase in chloroplast volume density ratio values, and a decrease in thylakoid surface area density ratios relative to the high-light controls. These effects were opposite to those seen in UV-B-exposed tissue. In a general sense, subambient levels of UV-B evoked a response similar to that elicited by low-PAR irradiance, while subambient UV-A elicited responses similar to those typical of high-PAR irradiance. The fact that below-ambient levels of UV altered a normal chloroplast structural response to PAR provides evidence that UV may be an important environmental signal for plants. Correspondence and reprints: Department of Plant Biology, University of New Hampshire, Durham, NH 03824, U.S.A. This is Scientific Contribution number 2292 from the New Hampshire Agricultural Experiment Station.     

Isoelectric points of spinach thylakoid membrane surfaces as determined by cross partition

The isoelectric points of unbroken chloroplast lamellae and various subchloroplast fractions, including a preparation of inside-out thylakoids, have been determined using aqueous two-phase systems containing dextran and charged polyethylene glycol. When the amounts of material in the top phase in a phase system with the positively charged trimethylamino polyethylene glycol are plotted against pH the curve intersects the corresponding curve obtained from phase systems with the negatively charged polyethylene glycol sulfonate. This cross-point can be correlated with the isoelectric point of the material.The cross-point for unbroken chloroplast lamellae was found to be around pH 4.7. Mechanical disintegration lowered the cross-point to around pH 4.4, probably because of exposure of new membrane surfaces. The disintegrated chloroplasts were fractionated by differential centrifugation to separate the grana and stroma lamellae. The stroma lamellae vesicles showed the same isoelectric point as the unbroken lamellae, while a cross-point at pH 4.3 was obtained for the grana-enriched fraction. For thylakoid membranes destacked under low salt conditions the cross-point was 0.3 pH unit lower than for membranes originating exclusively from the stroma lamellae. The most acidic cross-point (pH 4.1) was observed for the fraction enriched in inside-out grana thylakoids. It is suggested that the differences in isoelectric point between various subchloroplast fractions reflect a heterogeneous arrangement of surface charge along and across the thylakoid membrane.     

Isoelectric points of spinach thylakoid membrane surfaces as determined by cross partition.

The isoelectric points of unbroken chloroplast lamellae and various subchloroplast fractions, including a preparation of inside-out thylakoids, have been determined using aqueous two-phase systems containing dextran and charged polyethylene glycol. When the amounts of material in the top phase in a phase system with the positively charged trimethylamino polyethylene glycol are plotted against pH the curve intersects the corresponding curve obtained from phase systems with the negatively charged polyethylene glycol sulfonate. This cross-point can be correlated with the isoelectric point of the material. The cross-point for unbroken chloroplast lamellae was found to be around pH 4.7. Mechanical disintegration lowered the cross-point to around pH 4.4, probably because of exposure of new membrane surfaces. The disintegrated chloroplasts were fractionated by differential centrifugation to separate the grana and stroma lamellae. The stroma lamellae vesicles showed the same isoelectric point as the unbroken lamellae, while a cross-point at pH 4.3 was obtained for the grana-enriched fraction. For thylakoid membranes destacked under low salt conditions the cross-point was 0.3 pH unit lower than for membranes originating exclusively from the stroma lamellae. The most acidic crosspoint (pH 4.1) was observed for the fraction enriched in inside-out granathylakoids. It is suggested that the differences in isoelectric point between various subchloroplast fractions reflect a heterogeneous arrangement of surface charge along and across the thylakoid membrane.     

LIGHT AND ELECTRON MICROSCOPIC CHARACTERIZATION OF TWO TYPES OF PYRENOIDS IN GONIUM (GONIACEAE,CHLOROPHYTA)1

The single, basal pyrenoids of Gonium quadratum Pringsheim ex Nozaki and G. pectorale Müller (Goniaceae, Chlorophyta) differed in appearance when vegetative colonies were cultured photoheterotrophically in medium containing sodium acetate. Chloroplasts of G. quadratum had distinct pyrenoids when grown in medium without major carbon compounds. However, the pyrenoids degenerated and were markedly reduced in size when such cells were inoculated into a medium containing 400 mg·L^?1 of sodium acetate. No pyrenoids were visible under the light microscope; however, with electron microscopy small pyrenoids and electron-dense bodies were visible within the degenerating chloroplasts, which had only single layers of thylakoid lamellae at the periphery. The chloroplasts subsequently developed distinct pyrenoids and several layers of thylakoid lamellae as the culture aged. In contrast, vegetative cells of G. pectorale always showed distinct pyrenoids when cells were inoculated into medium containing sodium acetate, sodium pyruvic acid, sodium lactate, and/or yeast extract. Therefore, we propose two terms, “unstable pyrenoids” and “stable pyrenoids,” for pyrenoids of G. quadratum and G. pectorale, respectively. Chloroplasts of the colonial green flagellates should thus be examined under various culture conditions in order to determine whether their pyrenoids are unstable or stable when pyrenoids are used as taxonomic indicators. Immunogold electron microscopy showed that the ratios of gold particle density of ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO) between pyrenoid matrix and chloroplast stroma in G. quadratum grown in medium with or without sodium acetate were lower than those of G. pectorale. Heavy labeling by anti-RuBisCO was observed in both the electron-dense bodies and pyrenoid matrix of G. quadratum. This is the first electron microscopic demonstration of degeneration and development of both pyrenoids and thylakoid lamellae in the chloroplast as a function of culture condition in green algae.     

Surface density and volume density measurements of chloroplast thylakoids in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) under chilling conditions

Measurements of ultrastructural characteristics of chloroplast thylakoids are important for studies of ontogenic or ecological limitations of leaf photosynthetic functions. Most frequently, volumetric proportion of thylakoids in the chloroplast is measured; however, such measurement does not provide a direct information about the surface area of thylakoids which is most important from the functional point of view. Therefore, we adapted the stereological method using “local vertical windows” for estimating thylakoid surface area in the chloroplast volume and compared thus obtained surface density results with results of conventional volume density measurements. The methods were tested in the study of chloroplast ultrastructure in the leaves of plants of two maize (Zea mays L.) hybrid combinations, 2013×CE810 and CE704×CE810, developing in control and chilling conditions. Correlation analysis revealed a tight relationship between the granal/intergranal thylakoid surface density and volume density results, both indicating that under chilling conditions the development of the system of thylakoids in maize leaves is suppressed, while the difference is more pronounced in CE704 than in CE810 genotype, known to have a better photosynthetic performance.     

Chloroplast Growth and Replication in Germinating Spinach Cotyledons following Massive gamma-Irradiation of the Seed

Spinach seeds (Spinacia oleracea L.) given massive doses of γ-irradiation (500 krad) germinate and form a seedling with two green cotyledons and a radicle, but develop no further. Irradiated cotyledons show no increase in cell number or total DNA over a 7-day period in the light, while in control cotyledons there is a small increase in cell number and large increases in total DNA and chloroplast number. The chloroplasts of irradiated cotyledons are delayed in their division, become greatly enlarged and contain large amounts of starch. The whole population of chloroplasts subsequently undergoes a wave of division. The daughter chloroplasts show normal thylakoid development, but have some abnormal structural features caused by the radiation stress. Information on the effect of X-irradiation, ultraviolet irradiation, and 5-fluorodeoxyuridine on chloroplast replication and on chloroplast and nuclear DNA synthesis was obtained from cultured spinach leaf discs. It appears that chloroplast replication is more resistant to ionizing radiation than cell division and can proceed in the absence of nuclear DNA synthesis and greatly reduced chloroplast DNA synthesis.     

PHOSPHORUS STATUS AND CYTOPLASMIC STRUCTURES IN SCENEDESMUS (CHLOROPHYCEAE) UNDER DIFFERENT METABOLIC REGIMES1

Phosphorus deficiency affects the anatomy of the unicellular green alga Scenedesmus obtusiusculus Chad. and influences the distribution of other inorganic elements in the cell in addition to phosphorus. Scenedesmus was grown under standard conditions with or without phosphorus. Cells were then cultured with phosphorus under conditions favouring glycolysis, respiration, or photophosphorylation for 2 h or photosynthesis for up to 8 h. The dominating features of phosphorus starvation, were loss of phosphorus and coions from polyphosphate bodies, accumulation of starch, decrease in the volume density of ribosomes both in the chloroplast and cytoplasm, and an increase in wall thickness. Under conditions favoring photosynthesis the mass fraction for phosphorus is low after 1 h, exceptionally high by 2 h, and diminishes by 8 h. High amounts of phosphorus are also regained under conditions favoring glycolysis and photophosphorylation but not respiration. After 2 h under photosynthetic conditions the volume densities of the chloroplast, cytoplasmic ribosomes, the vacuole, and the mitochondrion increased over controls. By 8 h the relative volume of the single ramified mitochondrion had decreased slightly and recognizable segments of it were sequestered within the vacuome. The autophagic nature of the vacuole was further evidenced by the presence of ribosomes and whorls of lamellae within it. Serial sections showed that all polyphosphate granules and sequestered materials were located within a continuous vacuolar cisterna.     

Effect of water stress on cotton leaves : I. An electron microscopic stereological study of the palisade cells

Palisade cells from fully expanded leaves from irrigated and nonirrigated, field grown cotton (Gossypium hirsutum L. cv. Paymaster 266) were subjected to a microscopic examination to evaluate the effect of water stress on subcellular structures. The water potential difference between the two treatments was 13 bars at the time of sampling. The dimensions of the palisade cells and their density per unit leaf area were determined by light microscopy. Palisade cells from stressed plants had the same diameter, but were taller than their counterparts in irrigated plants. The density of the palisade cells was the same in both treatments as was the fractional volume of the intercellular space. It was concluded that the reduced leaf area observed in the stressed plants resulted primarily from a mitotic sensitivity to water stress. Further, expansion of palisade cells was not inhibited by the stress imposed in this study.

Morphometric analysis of electron micrographs was used to evaluate the subcellular structure of palisade cells from nonstressed and stressed plants. The fractional volumes of cell walls, total cytoplasm, chloroplasts, starch granules, intrachloroplast bodies, mitochondria, peroxisomes, and central vacuoles were determined. The surface densities of grana and stroma lamellae, outer chloroplast membranes, mitochondrial cristae, endoplasmic reticulum and Golgi cisternae were also measured. The number of chloroplasts, mitochondria, and peroxisomes were determined. These data were expressed as actual volumes, areas, and numbers per palisade cell for each treatment. Palisade cells from stressed plants had thinner cell walls, larger central vacuoles and approximately the same amount of cytoplasm compared to cells from nonstressed plants. Within the cytoplasm, stressed plants had more but smaller chloroplasts with increased grana and stroma lamellae surfaces, larger mithchondria with reduced cristae surfaces, smaller peroxisomes and reduced membrane surfaces of endoplasmic reticulum and Golgi cisternae.

     

UV-B辐射增强对芦苇光合生理及叶绿体超微结构的影响

在UV-B辐照增强条件下,研究不同辐照梯度对芦苇光合特性、光合色素含量及叶绿体超微结构的影响. 结果表明: 与自然光照相比,UV-B辐照增强显著降低芦苇叶片净光合速率、气孔导度、蒸腾速率,且随辐照强度增大,降低程度加剧,胞间CO₂浓度升高,光合效率显著降低;与自然光照相比,UV-B辐照增强显著降低芦苇叶片光合色素含量(包括叶绿素a、叶绿素b、总叶绿素和类胡萝卜素),且随辐照强度增大,降低程度加剧;UV-B辐射增强条件下,叶绿体超微结构遭到破坏,表现为叶绿体结构变形,类囊体片层排列稀疏紊乱、膨胀甚至模糊不清,并且UV-B辐射强度越大,损伤越大,高强度UV-B辐射对叶绿体超微结构的影响大于低强度辐射.     

The degree of stacking and the average membrane area of thylakoids in lettuce chloroplasts

The surface density of stacked and total thylakoid membranes in chloroplasts was determined morphometrically using the method of vertical sections. The degree of stacking, defined as the fraction of the total membrane area which is involved in stacking, was calculated from the surface densities and found to be 0.70 for chloroplasts of lettuce grown under field conditions. An average membrane area of 500 microns 2 for the thylakoids in a chloroplast was obtained from the surface density and the average volume of the chloroplasts (11.6 microns 3, estimated by means of equivalent oblate spheroids). The advantage of the morphometric method over alternative techniques and the relevance of the results with respect to the topology of thylakoid membranes are discussed.     

Downregulation of ClpR2 leads to reduced accumulation of the ClpPRS protease complex and defects in chloroplast biogenesis in Arabidopsis

Plastids contain tetradecameric Clp protease core complexes, with five ClpP Ser-type proteases, four nonproteolytic ClpR, and two associated ClpS proteins. Accumulation of total ClpPRS complex decreased twofold to threefold in an Arabidopsis thaliana T-DNA insertion mutant in CLPR2 designated clpr2-1. Differential stable isotope labeling of the ClpPRS complex with iTRAQ revealed a fivefold reduction in assembled ClpR2 accumulation and twofold to fivefold reductions in the other subunits. A ClpR2:(his)(6) fusion protein that incorporated into the chloroplast ClpPRS complex fully complemented clpr2-1. The reduced accumulation of the ClpPRS protease complex led to a pale-green phenotype with delayed shoot development, smaller chloroplasts, decreased thylakoid accumulation, and increased plastoglobule accumulation. Stromal ClpC1 and 2 were both recruited to the thylakoid surface in clpr2-1. The thylakoid membrane of clpr2-1 showed increased carotenoid content, partial inactivation of photosystem II, and upregulated thylakoid proteases and stromal chaperones, suggesting an imbalance in chloroplast protein homeostasis and a well-coordinated network of proteolysis and chaperone activities. Interestingly, a subpopulation of PsaF and several light-harvesting complex II proteins accumulated in the thylakoid with unprocessed chloroplast transit peptides. We conclude that ClpR2 cannot be functionally replaced by other ClpP/R homologues and that the ClpPRS complex is central to chloroplast biogenesis, thylakoid protein homeostasis, and plant development.     

不同海拔火绒草叶绿体超微结构的比较

利用透射电镜对生长于青藏高原东北部3个不同海拔地区(2300m、2700m和3800m)的火绒草叶绿体超微结构进行了比较观察。结果发现,随着海拔的升高,叶绿体结构差异明显。海拔2300m处,叶绿体呈扁船形,沿细胞壁分布,基粒片层排列整齐,片层可达32层;海拔2700m处,叶绿体呈扁船形,沿细胞壁分布,基粒片层排列不规则,片层下降到十几层,类囊体出现轻微膨大;海拔3800m处,叶绿体呈圆形,位于细胞中央,基粒片层则严重扭曲,片层只有几层,类囊体膨大严重,出现脂质小球。研究表明,火绒草叶绿体结构的变化是对逆境的一种适应,是青藏高原特殊生态条件长期胁迫的结果。     

Morphological and quantitative data of plastids and mitochondria within drought-stressed spinach leaves

Summary. Selected cell organelles were investigated at a high level of resolution with the transmission electron microscope, using ultrathin serial sections to create three-dimensional reconstructions. On the basis of these reconstructions, morphological data of chloroplast fine structures, mitochondria, and peroxisomes from control and drought-stressed spinach leaves were evaluated and compared. Mesophyll cell chloroplasts of control plants contained 60% stroma, 23% thylakoids, and 16% starch. In drought-stressed plants, the volume of both the stroma and the thylakoids increased to 68% and 32%, respectively. The amount of plastoglobuli was about 0.3% in both samples. Chloroplasts of stressed plants differed from control plants not only in the thylakoid and stroma values but also in the lack of starch grains. Mitochondria occurred in variable forms in control and stressed samples. In stressed plants, mitochondria had only 65% of the volume compared with control plants. Peroxisomes were inconspicuous.     

达坂山蚤缀和裸茎金腰叶绿体超微结构的研究

对高山植物达坂山蚤缀和裸茎金腰叶绿体超微结构的研究表明,叶绿体和线粒体镶嵌很紧密;基粒和基质类囊体膨大,膨大的类囊体呈梭形或圆形,没有发现脂质小球的存在;基粒叠垛程度很低,平均每个基粒类囊体的片层数为5．4。这2种高山植物叶绿体超微结构上的特征是青藏高原特殊的生态条件,包括低温、低气压、强辐射影响的结果。     

Pinguiochrysis pyriformis gen. et sp. nov. (Pinguiophyceae), a new picoplanktonic alga isolated from the Pacific Ocean

Pinguiochrysis pyriformis gen. et sp. nov. is a brown, naked, non‐motile, marine picoplankton. A culture was established from a surface sample collected in 1991 from the tropical Western Pacific Ocean. Typical cells of P. pyriformis are distinctively pear‐shaped and have one ovoid chloroplast; these two features distinguish this species from the other picophytoplankton species. However, the pyriform morphology is not consistent and cells frequently change to a subspherical shape. The chloroplast and mitochondrion ultrastructure confirm that this species belongs to the photosynthetic stramenopiles (chromophytes). Additional distinctive ultrastructural characteristics of P. pyriformis include (i) a chloroplast envelope forming a tubular invagination that penetrates into the pyrenoid; (ii) thylakoid lamellae consisting of more than three layers in some cells; (iii) the lack of basal bodies and centrioles; and (iv) the lack of scales or other extracellular structures. Based on the morphological features, this picoplanktonic species was described as a new species and placed in the Pinguiophyceae on the basis of the molecular phylogenetic analysis and biochemical data published elsewhere.     

The stacking of chloroplast thylakoids: Effects of cation screening and binding,studied by the digitonin method

The differential action of digitonin on stacked and unstacked chloroplast thylakoids was used to investigate the molecular interactions between thylakoid membranes. The yield of the heavy fraction which is obtained from chloroplasts after digitonin incubation and differential centrifugation was taken as a measure of the degree or tightness of membrane appression. The effects of various mono-, di-, and trivalent cations on the yield of the heavy fraction were studied, and the results interpreted in terms either of electrostatic screening or ion binding to the thylakoid membrane surface: Although there was some degree of cation specificity in the degree of thylakoid appression indicative of cation binding, the nonspecific screening effect was much more important in determining the overall balance of forces. It is postulated that stacking occurs in regions of low net surface charge density, with a possible segregation of excess negative charges into nonstacked regions.     

Modification of leaf cytology and anatomy in Brassica napus grown under above ambient levels of supplemental UV-B radiation.

Plants exposed to natural solar radiation usually show acclimation responses on a daily and seasonal basis. Many of these responses are complex and modified by interactions with acclimation responses to other climatic factors. While changes in photosynthetically active radiation (PAR, 400-700 nm) are the driving force for many acclimation responses in plants, radiation outside the PAR range is also important. Recently, interest has increased in the potential role of UV-A (320-400 nm) and UV-B (280-320 nm) components of sunlight in plant developmental, physiological and daily acclimation processes. In order to explore the role of UV-B further, Brassica napus L. cv Paroll plants were grown to maturity under 13 kJ d(-1) of biologically effective ultraviolet-B radiation (UV-B(BE), 280-320 nm) plus 800 micromol photons m(-2) s(-1) photosynthetically active radiation (PAR, 400-700 nm) or PAR alone. Leaf anatomy and palisade cell structure were quantified using stereological techniques. The leaves of plants grown under UV-B radiation exhibited an increase in overall leaf width, although no change in leaf anatomy was discerned. Palisade cells in UV-B exposed leaves showed a significant decrease in chloroplast, mitochondrial, starch, and microbody volume density (Vv), while the vacuolar Vv increased compared to cells exposed to PAR only. In UV-B exposed leaves, there was an increase in the appressed and non-appressed thylakoid surface area density (Sv) within the chloroplasts. Since the relative proportion of appressed to non-appressed thylakoid surface area did not change, both thylakoid systems changed in concert with each other. Thylakoid stacks were broader and shorter in leaves subjected to UV-B. In general these responses were similar to those which occurred in plants moved from a high to low PAR environment and similar to mature plants exposed to 13 kJ d(-1) UV-B(BE) for only a short period of time. Although UV absorbing pigments increased by 21% in UV-B exposed leaves, there was no significant difference in chlorophyll a,b or carotenoid content compared to plants exposed to only PAR.