Chloroplast Doublets and Differential Staining of the Cell Walls in Photosynthetic Tissues of Amaranthus dubius Mart

Ultrastructural studies of the leaves of Amaranthus dubius revealpresence of chloroplast doublets in mature leaf tissues. Theyalso highlight the difference in the structural organisationof the bundle sheath and the mesophyll cell walls, a featurewhich may have functional significance. Amaranthus dubius Mart., Calaloo, mesophyll, bundle sheath, chloroplast doublets, plastid fusion, plastid division, differential staining of the cell walls     

The proteomics of senescence in leaves of white clover,Trifolium repens (L.)

A proteomics approach has been used to study changes in protein abundance during leaf senescence in white clover. Changes in cell ultrastructure were also examined using transmission electron microscopy. The most obvious ultrastructural changes during senescence occurred in chloroplasts, with progressive loss of thylakoid integrity and accumulation of osmiophilic globules in the stroma. Quantitative analysis of 590 leaf protein spots separated by two-dimensional electrophoresis indicated that approximately 40% of the spots showed significant senescence related changes in abundance. Approximately one-third of the protein spots present in mature green leaves were also visible by two-dimensional electrophoresis of an isolated chloroplast fraction, and these spots represented a major proportion of the proteins showing senescence related declines in abundance. Chloroplast proteins that were identified by matrix-assisted laser desorption/ionization-time of flight mass fingerprinting included rubisco large and small subunits, a rubisco activase and the 33 kDa protein of the photosystem II oxygen-evolving complex. These proteins declined in abundance late in senescence, indicating that the photosynthetic apparatus was being degraded. A chloroplast glutamine synthetase showed partial decline in abundance during late senescence but was maintained at levels that may support provision of glutamine for export to other tissues. The results emphasise the importance of proteolysis, chloroplast degradation and remobilisation of nitrogen in leaf senescence.     

Optimization of the Leaf Mesophyll Structure in Alloploid and Diploid Wheat Species

Comparative analysis of the indices of plant growth and mesostructure of the photosynthetic apparatus was carried out with ten wheat (Triticum L.) species of various origins. Wheat alloploid forms (tetra- and hexaploids with the chromosome numbers of 28 and 42) exceeded the diploid forms (the chromosome number of 14) 2.3–2.4-fold by their absolute growth rate (AGR). As a result, the alloploid species developed a larger assimilation area; this change involved the internal reorganization of leaf phototrophic tissues and an increase in the cumulative internal assimilation area. In addition, the alloploid species manifested a higher correlation between the surface areas of cell and chloroplast membranes caused by a decrease in the cell number per the unit leaf area, a relative increase of the number of composite multifaveolate cells, a considerable expansion (in volume and surface area) of mesophyll cells, and an increase in chloroplast size and numbers. The decreased ratio between the characteristics of the cell membrane and chloroplast envelope presumes that CO₂ diffusion via cell and chloroplast membranes in the leaves was better balanced in the alloploid wheat species than in the diploid forms. All wheat species did not notably differ in their plastid–cytoplasm ratio (cell volume corresponding to one chloroplast and cell surface area per one chloroplast) and the ratio of surface area of cells to cell volume. The discriminant analysis revealed the indices of leaf growth and mesophyll structure instrumental in distinguishing between the diploid and alloploid species: leaf area, AGR, and cell size and number. The change in the latter indices optimized the structure of leaf phototrophic tissues in tetraploid and hexaploid species; as a result, the internal assimilation area was expanded and, consequently, leaf CO₂ conductance was increased.     

Effect of N6-benzyladenine and indole-3-butyric acid on photosynthetic apparatus of Orthosiphon stamineus plants grown in vitro

The leaf structure and chloroplast ultrastructure of kidney tea (Orthosiphon stamineus Benth.) was studied in in vitro culture on standard MS medium supplemented with or without plant growth regulators (PGRs). The cytokinin N⁶-benzyladenine (BA) negatively affected the structure of the palisade parenchyma and chloroplast ultrastructure and increased the stomatal frequency of the adaxial epidermis. The auxin indole-3-butyric acid (IBA) did not modify the morphology of regenerated leaf tissues as well as the chloroplast ultrastructure. The effect of both PGRs applied in combination was manifested in well-differentiated mesophyll parenchyma, typical chloroplast ultrastructure and increased stomatal frequency on both leaf surfaces. This protocol can be suggested for further ex vitro propagation.     

油茶叶肿病变态叶片的形态特征和超微结构观察

利用扫描电镜(SEM)对油茶叶肿病变态叶叶片表面和横切面进行观察,利用透射电镜(TEM)对其细胞超微结构进行观察,以期探明油茶叶肿病变态叶的形态特征和细胞学特征。结果表明:(1)变态叶是受感染油茶幼叶组织增生形成的,肿大的叶片厚度比正常叶片厚度增加3~5倍,细胞体积增大3~8倍,细胞数增加1~2倍,叶片细胞形态和结构发生了变化。(2)叶片受细丽外担菌侵染后,菌丝存在于下表皮向内的4~7层细胞间隙中,感染后期叶片下表面脱落露出子实层。(3)变态叶细胞出现叶绿体膜破裂、类囊体片层膜数目减少及细胞器成分被破坏等异常现象。     

Photosynthesis and Other Traits in Relation to Chloroplast Number during Soybean Leaf Senescence

Soon after attaining full expansion, soybean (Glycine max [L.] Merr.) leaves enter a senescence phase marked by decline in photosynthetic rate and the progressive loss of chloroplast activity and composition. Our primary goal was to determine if this loss could be accounted for by sequential degradation of whole chloroplasts or by simultaneous degeneration of all chloroplasts. Total photosynthesis (TPs) measured as ¹⁴CO₂ uptake, chloroplast number, ribulose 1,5-bisphosphate carboxylase activity, uncoupled photosynthetic electron transport activity, soluble protein content, and chlorophyll content declined progressively during the 37 days after full leaf expansion. During this period, chloroplast number per unit leaf area was constant for all genotypes studied. We conclude that leaf senescence may be a two-stage process wherein the first stage chloroplast activity and composition declines, but chloroplast numbers do not change. During a brief terminal stage (11 days in our experiment), whole chloroplasts may be lost as well. As a second objective we wished to determine if variation in single-leaf total photosynthetic rate among soybean cultivars is related to corresponding variation in chloroplast number and/or chloroplast activity/composition. By comparing the means for three cultivars known to have rapid leaf TPs and for the three known to have slow TPs, we found the former group to be superior to the latter for all the previously mentioned leaf physiological traits. This superiority was related primarily to differences in chloroplast number and only secondarily to differences in activity and composition per chloroplast.     

Is Chloroplast Movement in Tobacco Plants Influenced Systemically after Local Illumination or Burning Stress?

Chloroplast movement has been studied In many plants mainly in relation to the local light, mechanical or stress effects. Here we investigated possible systemic responses of chloroplast movement to local light or burning stress in tobacco plants (Nicotiana tabacum cv. Samsun). Chloroplast movement was measured using two independent methods: one with a SPAD 502 Chlorophyll meter and another by collimated transmittance at a selected wavelength (676 nm). A sensitive pedodic movement of chloroplasts was used in high or low (2 000 or 50 μmol/m2 per s photosynthetically active radiation, respectively) cold white light with periods of 50 or 130 min. Measurements were carried out in the irradiated area, in the non-irradiated area of the same leaf or in the leaf located on the stem below the irradiated or burned one. No significant changes in systemic chloroplast movement in non-irradiated parts of the leaf and in the non-treated leaf were detected. Our data indicate that chloroplast movement in tobacco is dependent dominantly on the intensity and spectral composition of the incident light and on the local stimulation and state of the target tissue. No systemic signal was strong enough tovoke a detectable systemic response in chloroplast movement in distant untreated tissues of tobacco plants.     

Fatty Acid Desaturation during Chilling Acclimation Is One of the Factors Involved in Conferring Low-Temperature Tolerance to Young Tobacco Leaves

The FAD7 gene, a gene for a chloroplast [omega]-3 fatty acid desaturase, is responsible for the trienoic fatty acid (TA) formation in leaf tissues. The TA content of the leaf tissue of the 25[deg]C-grown transgenic tobacco (Nicotiana tabacum cv SR1) plants, in which the FAD7 gene from Arabidopsis thaliana was overexpressed, increased uniformly by about 10%. Fatty acid unsaturation in all major leaf polar lipid species increased in the 25[deg]C-grown FAD7 transformants but was approximately the same between the control plants and the FAD7 transformants when grown at 15[deg]C. Therefore, the overexpression of the exogenous FAD7 gene leads to the same consequence in the tobacco plants as the low-temperature-induced TA production that may be catalyzed by an endogenous, temperature-regulated chloroplast [omega]-3 fatty acid desaturase. In the 25[deg]C-grown control plants, the chilling treatment caused symptoms of leaf chlorosis and suppression of leaf growth. The 25[deg]C-grown FAD7 transgenic plants conferred alleviation of these chilling-induced symptoms. A reductions of the chilling injury similar to that of the FAD7 transformants was also observed in the 15[deg]C-preincubated control plants. These results indicate that the increased TA production during chilling acclimation is one of the prerequisites for the normal leaf development at low, nonfreezing temperatures.     

Changes in the activities of chloroplast and cytosolic isoenzymes of glutamine synthetase during normal leaf growth and plastid development in wheat

Soluble protein extracts and chloroplasts from a serial sequence of transverse sections of a 7-d-old wheat leaf (Triticum aestivum cv. Maris Huntsman) were used to study changes in the activity of glutamine synthetase (GS; EC 6.3.1.2) during cell and chloroplast development. Glutamine synthetase activity increased more than 50-fold per cell from the base to the tip of the wheat leaf. Two isoenzymes of GS were separated using fast protein liquid chromatography (FPLC). Glutamine synthetase localized in the cytoplasm (GS1) eluted at about 0.21 M NaCl, and the isoenzyme localized in the chloroplast (GS2) eluted at about 0.33 M NaCl. The increase in GS activity during leaf development was found to be caused primarily by an increase in the activity of the chloroplast GS2. The activity of the cytoplasmic GS1 remained constant as the cells were displaced from the base to the tip of the leaf, whereas GS2 activity increased within the chloroplast throughout development. At the base of the leaf, 26% of total GS activity was cytoplasmic; the remaining 74% was in the chloroplast. At 10 cm from the base, only 4% of the activity was cytoplasmic, and 96% was in the chloroplast. The results indicate that the chloroplast GS2 is probably responsible for most of the ammonia assimilation in the mature wheat leaf, whereas cytoplasmic GS1 may serve a role in immature developing leaf cells.Abbreviations FPLC fast protein liquid chromatography - GS glutamine synthetase - GS1 cytoplasmic glutamine synthetase - GS2 chloroplast glutamine synthetase     

Evidence for modification of protein phosphorylation by cytokinins

Kinetin stimulated phosphorylation of protein in floated Chinese-cabbage leaf discs, but inhibited protein phosphorylation in nuclei+chloroplast extracts from Chinese-cabbage or tobacco leaves. Kinetin also inhibited protein phosphorylation in isolated tobacco nuclei or nuclei from carrot secondary-phloem tissue. Purified Chinese-cabbage leaf ribosomes exhibited protein kinase activity which was inhibited by kinetin and zeatin. The ribosome-associated kinase responded to kinetin and zeatin differently from that associated with nuclei+chloroplast preparations. Protein phosphorylation in vitro was not affected by adenosine 3':5'-cyclic monophosphate, indol-3-ylacetic acid or gibberellic acid. It was only inhibited by N(9)-unsubstituted purines, among which the known cytokinins were the most effective inhibitors. The results are discussed in relation to possible similarities between the effects of cytokinins in plant tissues and the effects of adenosine 3':5'-cyclic monophosphate in animal tissues. Both compounds appear to modify the activity of protein kinases and both affect many different cellular processes.     

Expression of the B Subunit of E. coli Heat-labile Enterotoxin in the Chloroplasts of Plants and its Characterization

Transgenic chloroplasts have become attractive systems for heterologous gene expressions because of unique advantages. Here, we report a feasibility study for producing the nontoxic B subunit of Escherichia coli heat-labile enterotoxin (LTB) via chloroplast transformation of tobacco. Stable site-specific integration of the LTB gene into chloroplast genome was confirmed by PCR and genomic Southern blot analysis in transformed plants. Immunoblot analysis indicated that plant-derived LTB protein was oligomeric, and dissociated after boiling. Pentameric LTB molecules were the dominant molecular species in LTB isolated from transgenic tobacco leaf tissues. The amount of LTB protein detected in transplastomic tobacco leaf was approximately 2.5% of the total soluble plant protein, approximately 250-fold higher than in plants generated via nuclear transformation. The GM1-ELISA binding assay indicated that chloroplast-synthesized LTB protein bound to GM1-ganglioside receptors. LTB protein with biochemical properties identical to native LTB protein in the chloroplast of edible plants opens the way for inexpensive, safe, and effective plant-based edible vaccines for humans and animals.     

ULTRASTRUCTURAL AND PALEOBIOCHEMICAL CORRELATIONS AMONG FOSSIL LEAF TISSUES FROM THE ST. MARIES RIVER (CLARKIA) AREA,NORTHERN IDAHO,USA

Miocene angiosperm leaf tissues of Betula, Hydrangea, Platanus, and Quercus, and of Castanea, Persea, and gymnospermous tissues from offshore and onshore lacustrine sediments, respectively, reveal various states of ultrastructural detail in preservation which correlate with their paleobiochemical profiles. Excellent cytologic preservation of membrane-bound organelles in offshore tissue samples is correlated with relatively mild chlorophyll and fatty acid degradation (chlorins, unsaturated hydrocarbons), while progressive deterioration of ultrastructure seen in onshore specimens is concomitant with extensive chlorophyll degradation and saturation of hydrocarbons. Comparative studies between fossils found in pyroclastic (Succor Creek Flora, Miocene) and lacustrine (Clarkia Flora) environments indicate that the degree of pre- and post-depositional hydration of tissues is a less significant factor in determining preservation than redox potential and secondary metabolites (tannic acids, chlorophyll derivatives). A sequence of organelle degradation in leaf tissues is given, and it is suggested that chloroplasts and cell walls are the most stable cellular constituents, while the endoplasmic reticulum, nuclei, and mitochondria are the most labile.     

Genome-Dependent Factors in the Development of Leaf Phototrophic Tissues in Diploid and Alloploid Wheat Species

Growth and mesostructure of the photosynthetic apparatus were studied in leaves of ten Triticum L. species. Plants with the A^u genome were shown to develop larger leaf assimilation areas due to expanding areas of individual leaves and an increase in the absolute growth rate. Leaf and mesophyll thickness and mesophyll cell size decreased in the G-genome species. Leaf compactness, which depended on cell size and number per unit leaf area and leaf folding, determined the specific patterns of internal leaf organization in wheat species with diverse genotypes. These patterns did not affect cell plastid-to-cytoplasm ratio as shown by the stable indices of cell surface area/cell volume, cell surface area per chloroplast, and cell volume per chloroplast. The structural indices of leaf phototrophic tissues, mesophyll density, and mesophyll CO₂ conductance in alloploids, as compared to diploid species, depended on both ploidy and genome constitution.     

A role for membrane lipid polyunsaturation in chloroplast biogenesis at low temperature

Two different mutants of Arabidopsis thaliana deficient in chloroplast membrane lipid polyunsaturation were indistinguishable in appearance from the wild-type when grown at 22°C. By contrast, leaf tissues of the mutants that developed during growth at 5°C were chlorotic, whereas the wild type was not. This is the first direct evidence that chloroplast lipid polyunsaturation contributes to low-temperature fitness. Chloroplasts from mutant lines grown at 5°C were much smaller than those of the wild-type, and the thylakoid membrane content was reduced by up to 70%. However, there was no discernible effect of low temperature on chloroplasts that developed prior to exposure to low temperatures. These and related observations suggest that the high degree of chloroplast membrane lipid polyunsaturation is required for some aspect of chloroplast biogenesis.     

金边红苞凤梨叶片的超微观察和AbGLK1的克隆与表达分析

为了解Golden2-like (GLK)转录因子在金边红苞凤梨(Ananas comosus var. bracteatus ‘Chiyan’)绿白嵌合叶片形成中的作用,采用RT-PCR技术克隆得到了AbGLK1基因, 其开放阅读框全长1 371 bp,编码456个氨基酸,含有1个GARP-DNA结合域和1个C末端结构域GCT box (GOLDEN2 C-terminal box),属于GLK转录因子家族,在酵母中具有转录因子的转录激活活性。烟草亚细胞定位表明AbGLK1蛋白定位在细胞核。RT-qPCR分析表明,AbGLK1基因在金边红苞凤梨的根、茎、叶中均有表达,但具有组织器官差异性,在叶片中的表达量显著高于根和茎(P < 0.05)。AbGLK1基因在叶片边缘白化组织中的表达量显著低于绿色组织,约为绿色组织的1/3 (P < 0.05)。叶片白化组织叶绿体内膜系统模糊,无类囊体存在,含有大量囊状小泡,质体小球数量多且体积较大。因此,推测AbGLK1基因可能参与了金边红苞凤梨中的叶绿体发育,其下调表达可能导致叶片白化组织中叶绿体发育不成熟。     

The impact of photosynthesis on initiation of leaf senescence

Senescence is the last stage of leaf development preceding the death of the organ, and it is important for nutrient remobilization and for feeding sink tissues. There are many reports on leaf senescence, but the mechanisms initiating leaf senescence are still poorly understood. Leaf senescence is affected by many environmental factors and seems to vary in different species and even varieties of plants, which makes it difficult to generalize the mechanism. Here, we give an overview on studies reporting about alterations in the composition of the photosynthetic electron transport chain in chloroplasts during senescence. We hypothesize that alternative electron flow and related generation of the proton motive force required for ATP synthesis become increasingly important during progression of senescence. We address the generation of reactive oxygen species (ROS) in chloroplasts in the initiation of senescence, retrograde signaling from the chloroplast to the nucleus and ROS‐dependent signaling associated with leaf senescence. Finally, a few ideas for increasing crop yields by increasing the chloroplast lifespan are presented.     

Ultrastructure of Chloroplasts in Phloem Companion Cells and Mesophyll Cells as Related to the Stimulation of Sink Activity by Cytokinins

Changes in the chloroplast ultrastructure and starch and lipid content in the mesophyll and phloem companion cells of the phloem were studied after induction of source and sink functions in leaf tissues. A detached sugar-beet leaf, one half of which was treated with water (source part) and the other half of which was treated with 10^–4 M benzyladenine (BA) (acceptor part), was used as a model. After 65-h exposure to diffuse light, starch disappeared and lipid content increased in the source part of the leaf, with simultaneous disorganization of the chloroplast structure, which was most pronounced in the companion cells. Changeover from the source to sink function, induced by BA treatment, did not lead to marked destructive changes in the chloroplast structure of companion cells and resulted in the appearance of starch and in further increase in the level of lipids. Smaller amounts of starch also appeared in the mesophyll chloroplasts in the sink part of the leaf. We suppose that: (1) BA promotes the storage of assimilates, which are imported from the source part of the leaf to the companion cells, in the form of starch and lipids within chloroplasts; and this storage contributes to the maintenance of the sucrose concentration gradient in the conducting system between donor and sink parts of the leaf and, thus, activates metabolite inflow and (2) a barrier exists in the sink part of the leaf for assimilates destined to mesophyll cells, which restricts their export from the phloem.     

Effects of delta-aminolevulinic acid on pigment formation and chlorophyllase activity in French bean leaf

Chloroplast development and chlorophyll biosynthesis are co-regulated. Treatment by levulinic acid resulted in a linear relation in both chlorophyll and carotenoid contents, during greening of etiolated French bean leaf discs. Chlorophyll biosynthesis appeared to control that of caroteins. In the presence of levulinic acid; at different levels, photosystem II (PS II) activity decreased when expressed on a chlorophyll basis. Chlorophyllase activity was increased progressively by increasing levulinic acid concentration. Thus, levulinic acid could be used to arrest the light-induced chloroplast development at a desired phase of greening and acts as determinator of chloroplast development in green tissues.