Structure and distribution of chloroplasts and other organelles in leaves with various rates of photosynthesis

The ultrastructure and distribution of chloroplasts, mitochondria, peroxisomes, and other cellular constituents have been examined in cross sections of leaves from plants with either high or low photosynthetic capacity. Photosynthetic capacity of a given plant cannot be correlated with the presence or absence of grana in bundle sheath cell chloroplasts, the presence or absence of starch grains in bundle sheath or mesophyll cell chloroplasts, the chloroplast size in bundle sheath or mesophyll cells, or the location of chloroplasts within bundle sheath cells. We conclude that the number and concentration of chloroplasts, mitochondria, and peroxisomes in bundle sheath cells is the most reliable anatomical criterion presently available for determining the photosynthetic capacity of a given plant.     

Effect of Elevated CO2 Concentration on the Starch Grain Accumulation in Chloroplasts from Soybean Leaves at Different Nodes

Soybean ( Glycine max (L.) Merr. ) plants were grown under ambient and elevated CO2 (plus 350 μL/L) concentration in cylindrical open-top chamber to examine their effects on the ultra- structure of chloroplasts. The upper, lower and mid-node leaves were harvested after 7 days full expansion under different CO2 concentrations and ultrathin section were prepared for transmission electron microscopy. In general, the average content of starch grains and thylakoid membranes in the chloroplasts under the elevated CO2 concentration were always higher than the control. Under higher CO2 concentration, there were smaller and less starch grains in the chloroplasts from upper-node leaves than those from mid-node leaves. The shape of their starch grains changed from elliptical to oval,and their thylakoid membranes and grana remained normal. At lower-node leaves, one or two oval, or three timer starch grains accumulated in the chloroplasts. In the mid-node leaves,however, some chloroplasts under higher CO2 concentration had rather large tim elliptical starch grains which could consequently cause disruption of grana and stroama thylakoids in the chloroplasts, whereas in other chloroplasts, the thylakoid membranes and grana were not deformed as the starch grains were smaller and elliptical. On the other hand, under higher CO2 concentration, the stacking degree of thylakoid membranes and starch grains accumulation in the mid-node leaves were significantly higher than those in the lower-node leaves,and slightly higher than the upper-node leaves. These results, in agreement with the chlorophyll contents and photosynthetic rate which reported by other authors in the past, indicated that the ultrastmcture response of the chloroplasts from different leaf nodes of soybeen under elevated CO2 coneentration were different. The seed yield of soybean at different nodes was decreased gradually from mid-nodes towards both upper- and lower-nodes. The greatest effect of elevated CO2 eoneentrafion on seed yeild was at the mid-node leaves. The variation of seed yields of soybean at different nodes under elevated CO2 concentration was in eoneert with the change in the ultrastmcture of chloroplasts and in turn the change in their photosynthetic rates of leaves at different nodes.     

高CO_2浓度对大豆不同叶位叶片叶绿体淀粉粒积累的效应

对高CO_2浓度下生长的大豆(Glycine max(L.)Merr.)不同叶位的叶片进行了电镜观察,揭示出大豆不同叶位叶片的叶绿体对倍增的CO_2浓度反应不一。其显著的超微结构差异特征是:1.叶位居中的叶片叶绿体积累的淀粉粒不仅很大,而且最多,有的叶绿体中的淀粉粒可达20个,几乎充满着叶绿体的基质空间。2.下位叶叶绿体的淀粉粒积累较多,通常为2～5个;3.上位叶叶绿体所含淀粉粒既小又少,虽然有的叶绿体中也积累有3～4个淀粉粒,但大多数叶绿体中所含淀粉粒仅有1～2个。以上结果联系到大豆中位叶的光合作用速率较高及对籽粒产量起作用最大来讨论是很有意义的。     

Structural organization of chloroplast of tomato plants <Emphasis Type="Italic">Solanum lycopersicum</Emphasis> transformed by Fe-containing superoxide dismutase

In this work we analyzed the results of the transformation of tomato plants Solanum lycopersicum by gene FeSOD1 from Arabidopsis thaliana, equipped with the signal sequence for targeting into chloroplasts. PCR analysis showed that the gene was integrated into the genome of several tomato plants that underwent transformation followed by selection in the kanamycin-containing medium. Two lines, provisionally denoted as nos. 6 and 8, were selected from the independent transformants. Line 6 was characterized by a reduced growth rate and altered leaves and line 8, by normal growth and leaves typical for control plants. Both lines showed a significant increase in SOD activity. In line 8 the increase in SOD activity was accompanied by an increase of ascorbate peroxidase activity, and in line 6 this effect was not present. Electron microscopic analysis of parenchymal and guard cells of both lines was performed, with an emphasis on the ultrastructural organization of chloroplasts. It is shown that the chloroplasts of the two transgenic lines differ in the number and size of starch grains and deposited plastoglobules as well as in the organization of lamellae and grana. Taken together, the results indicate that the expression of the introduced gene FeSOD1 has a significant effect on metabolic processes in the plastids. The findings are discussed in relation to the hypothesis about the importance of low concentrations of ROS for the integration of structure and function of chloroplasts.     

Effects of low light stress on rubisco activity and the ultrastructure of chloroplast in functional leaves of peanut

Aims In recent years, intercropping system has become one of the major practice of peanut (Arachis hypogaea) cultivation in northern China because of the high land and energy utilization efficiency, to some extent compensating for the production loss caused by decreasing area of cultivation land. Intercropped peanut plants often have a lower pod yield compared with monoculture due to constraint on light availability. This study was conducted to explore the shade-tolerance mechanism in two peanut cultivars, ‘Huayu 22’ and ‘Baisha 1016’, that grew in an intercropping system, by studying chloroplast ultrastructure and rubisco activity under different levels of shading.
Methods A field experiment was conducted with three levels of light treatments, including full natural light (CK), 50% natural light indensity (NLI), and 15% NLI. The ‘Huayu 22’ was used as a shade-tolerant cultivar and the ‘Baisha 1016’ as a shade-susceptible cultivar based on previous studies. Experimental plants of both cultivars were shaded for 40 days from emergency in 2006. Rubisco activity, the number and shapes of chloroplasts and starch grains, and number of grana and granum lamella were investigated in functional leaves of plants in all treatments.
Important findings The functional leaves of peanut plants in the 50% and 15% NLI treatments had a lower rubisco activity than those in the CK treatment. In the ‘Baisha 1016’, the reduction in rubisco activity was 40.1% in the 50% NLI treatment and 59.4% in the 15% NLI treatment, respectively, compared to the CK treatment;whereas no significant differences were found among treatments in the ‘Huayu 22’ in the rubisco activity. Compared with the CK, the number of chloroplasts remained unchanged, the number of grana and lamella in grana increased, and the individual chloroplast was longer and in perfect development in the functional leaves of plants of the ‘Huayu 22’ grown in the 50% NLI treatment. In contrast, the number of chloroplasts, grana and starch grains of the ‘Huayu 22’ plants decreased significantly, the chloroplast membrane and grana lamella were damaged, the number of granum lamella increased, and the individual chloroplast became longer in the 15% NLI treatment. The number and ultrastructure of chloroplasts in the ‘Baisha 1016’ plants followed similar patterns of changes as those of the ‘Huayu 22’ in the 50% NLI treatment. For plants of the ‘Baisha 1016’ in the 15% NLI treatment, their chloroplasts became more roundly shaped, with decreasing number of grana lamella and increasing number of starch grains, compared with the CK. There were a greater decrease in the grana number and more damage in the grana lamella in plants of the ‘Baisha 1016’ than those of the ‘Huayu 22’. In conclusion, the shade tolerance of the ‘Huayu 22’ resulted from lack of changes in rubisco activity and less damage in the ultrastructure of chloroplasts when under low light stress compared with the ‘Baisha 1016’.     

Over-expression of a pepper plastid lipid-associated protein in tobacco leads to changes in plastid ultrastructure and plant development upon stress

Proteins homologous to fibrillin, a pepper plastid lipid-associated protein involved in carotenoid storage in fruit chromoplasts, have been recently identified in leaf chloroplasts from several species and shown to be induced upon environmental stress. To further investigate the role of the protein, transgenic Nicotiana tabacum plants over-expressing fibrillin using a constitutive promoter were generated. Transgenics grown under standard light intensities (300 micromol photons m-2 sec-1) were found to contain substantial amounts of fibrillin in flowers and leaves. In leaves, the protein was immunolocalized within chloroplasts in both stromal and thylakoid subfractions. No change was noticed in thylakoid structures from transgenics, but chloroplasts contained an increased number of plastoglobules organized in clusters. In petals, leucoplasts were also found to contain more agglutinated plastoglobules. The effects of environmental factors on fibrillin gene expression and protein localization were studied in tobacco leaves. Less fibrillin was present in plants grown under low light intensities, which can be explained by the involvement of a light-dependent splicing step in the control of fibrillin gene expression in leaves. Analysis of protein subfractions from plants subjected to drought or high light showed that both stresses resulted in fibrillin association with thylakoids. Whereas no growth difference between wild-type (WT) and transgenic plants was noticed under low light conditions, transgenics exhibit a longer main stem, enhanced development of lateral stems and accelerated floral development under higher light intensities. These data suggest that fibrillin-related proteins fulfil an important function in plant development in relation to environmental constraints.     

Chloroplast Structure and Starch Grain Accumulation in Leaves That Received Different Red and Far-Red Levels during Development

An important step in understanding influence of growth environment on carbon metabolism in plants is to gain a better understanding of effects of light quality on the photosynthetic system. Electron microscopy was used to study chloroplast ultrastructure in developing and fully expanded leaves of tobacco (Nicotiana tabacum L. cv Burley 21). Brief exposures to red or far-red light at the end of each day during growth under controlled environments influenced granum size, granum number and starch grain accumulation in chloroplasts, and the concentration of sugars in leaf lamina. Far-red-treated leaves had chloroplasts with more but smaller grana than did red-treated leaves. Red light at the end of the photosynthetic period resulted in more and larger starch grains in the chloroplasts and a lower concentration of sugars in leaves. Chloroplast ultrastructure and starch grain accumulation patterns that were initiated in the expanding leaves were also evident in the fully expanded leaves that received the treatment during development. It appears that the phytochrome system in the developing leaves sensed the light environment and initiated events which influenced chloroplast development and partitioning of photosynthate to adapt the plant for better survival under those environmental conditions.     

Effects of Quantum Flux Density on Photosynthesis and Chloroplast Ultrastructure in Tissue-Cultured Plantlets and Seedlings of Liquidambar styraciflua L. towards Improved Acclimatization and Field Survival

Liquidambar styraciflua L. seedlings and tissue-cultured plantlets were grown under high, medium, or low (315, 155, or 50 microeinsteins per square meter per second photosynthetically active radiation) quantum flux densities. Net photosynthesis, chlorophyll content, and chloroplast ultrastructure of leaves differentiated from these conditions were investigated. Seedling photosynthetic rates at light saturation were positively related to light pretreatments, being 6.44, 4.73, and 2.75 milligrams CO₂ per square decimeter per hour for high, medium, and low light, respectively. Cultured plantlets under all light conditions had appreciably higher photosynthetic rates than noncultured seedlings; corresponding rates were 12.14, 13.55, and 11.36 milligrams CO₂ per square decimeter per hour. Chlorophyll in seedlings and plantlets was significantly higher in low light-treated plants. Seedling leaves had chloroplasts with abundant starch regardless of light pretreatment. In high light, starch granules were predominant and associated with disrupted granal structure. Low light seedling chloroplasts had smaller starch grains and well-formed grana. In contrast, tissue culture-differentiated leaves were devoid of starch; grana were well organized in higher quantum flux density treatments, but disorganized at low flux densities.     

弱光胁迫对花生功能叶片RuBP羧化酶活性及叶绿体超微结构的影响

间作套种是我国主要的花生(Arachis hypogaea)种植方式之一。然而, 与单作相比, 在间作套种体系中, 花生截获的光能较少, 生长发育差, 产量低, 研究不同品种耐阴机理对选择适宜间作套种的花生品种具有重要意义。该研究用耐阴性不同的两个花生品种‘花育22号’ (强耐阴性)和‘白沙1016’ (弱耐阴性)为材料, 在大田条件下采用不同透光率遮阴网设置50%自然光强(中度弱光胁迫)和15%自然光强(严重弱光胁迫) 2个弱光处理, 从出苗期开始遮阴40天, 以自然光强为对照, 研究了弱光胁迫对花生功能叶片RuBP羧化酶活性和叶绿体超微结构的影响。结果表明: 光强为自然光照50%和15%的处理, ‘花育22号’ RuBP羧化酶活性与对照相比虽有降低, 但差异不显著, 而‘白沙1016’分别比对照低40.1%和59.4%, 显著低于对照。与对照相比, 50%自然光强下‘花育22号’叶绿体数不变, 叶绿体基粒数和基粒片层数显著增多, 叶绿体变长且发育完好, 15%自然光强下, 叶绿体数、基粒数和淀粉粒数显著减少, 叶绿体膜和基粒片层出现破损, 但叶绿体变长, 基粒片层数增加; ‘白沙1016’在50%自然光强下, 叶绿体数目和超微结构变化同‘花育22号’相似, 在15%自然光强下叶绿体变圆, 基粒数的降幅和基粒片层破损程度大于‘花育22号’且基粒片层数减少, 淀粉粒数增多。因此, 弱光胁迫特别是严重弱光胁迫条件下, 功能叶RuBP羧化酶活性降低幅度小、叶绿体超微结构受损程度低是‘花育22号’耐阴的光合生理基础。     

Fine Chloroplast Structure in Cucumber and Pea Leaves Developed under Red Light

Photosynthetic activity, the content of various photosynthetic pigments, and the chloroplast ultrastructure were examined in the leaves of cucumber (Cucumis sativus L.) and pea (Pisum sativum L.) plants of different ages grown under red light (600–700 nm, 100 W/m²). In pea leaves tolerant to red-light irradiation, chloroplast ultrastructure did not essentially change. In the first true leaves of cucumber plants susceptible to red-light irradiation, we observed a considerable increase in the number and size of plastoglobules, the appearance of chloroplasts lacking grana or containing only infrequent grana, and stromal thylakoids. In the upper leaves of 22-day-old cucumber plants, the chloroplast structure was essentially similar to that of the control chloroplasts in white light, and we therefore suppose that these plants have acclimated to red light.     

Starch Accumulation and Photosynthetic Activity in Primary Leaves of Bean (Phaseolus vulgaris L.)

The effects of starch accumulation on photosynthesis and chloroplastultrastructure were studied in primary leaves of bean (Phaseolusvulgaris L. cv. Bulgarian). De-topping the shoot above the primaryleaf node, caused over an 8-day period, a considerable increasein the photosynthetic activity of the primary leaves, despitethe fact that a large quantity of starch had accumulated intheir chloroplasts. The accumulation of starch was greater inthe chloroplasts of spongy cells in comparison with that ofthe palisade cells. Initiation of starch grains was observedmainly in the peripheral part of the chloroplast, distant fromthe cell wall. As a result, most of the starch was accumulatedclose to the inner part of the cell, leaving a considerablemass of the chloroplast near the cell wall free of starch. Theaccumulation of starch was accompanied by the destruction, deformationand disorientation of grana and thylakoids. It is concludedthat the accumulation of starch is not inevitably a limitingfactor in photosynthesis and the results cast doubt on the hypothesisthat starch accumulation or dissipation is the main factor involvedin the regulation of photosynthesis. Phaseolus vulgaris L, bean, photosynthesis, starch accumulation, chloroplast ultrastructure     

Effect of Doubled-CO2 Concentration on the Ultrastructure of Chloroplasts from Medicago sativa and Setaria italica

It has been reported in quite a number of literatures that doubled CO2 concentration increased the photosynthetic rate and dry matter production of C3 plants, but substantially affected C4 plants little. However, why may CO2 enrichment promote growth and either no change or decrease reproductive allocation of the C3 species, but havinag no effects on growth characteristics of the C4 plants? So far, there has been no satisfactory explanation on that mentioned above, except the differences in their CO2 compensatory points. In the past, although some studies on ultrastructure of the chloroplasts under doubled CO2 concentration were limitedly conducted. Almost all the relevant experimental materials were only from C3 plants not from C4 plants, and even though the results were of inconsistancy. Thereby, it needs to verify whether the differences in photosynthesis of C3 and C4 plants at doubled CO2 level is caused by the difference in their chloroplast deterioration. Experiments to this subject were conducted at the Botanical Garden of Institute of Botany, Academia Sinica in 1993 and 1994. Both experimental materials from C3 plant alfalfa (Medicago sativa) and C4 plant foxtail millet (Setaria italica) were cultivated in the cylindrical open-top chambers (2.2 m in diameter × 2.4 m in height) with aluminum frames covered by polyethylene film. Natural air or air with 350× 10-6 CO2 were blown from the bottom of the chamber space with constant temperature between inside and outside of the chamber 〈0.2℃〉. Electron microscopic observation revealed that the ultrastructure of the chloroplasts from C3 plant Medicago sativa and C4 plant Seteria italica growing under the same doubled CO2 concentration were quite different from each other. The differential characteristics in ultrastructure of chloro plasts displayed mainly in the configuration of thylakoid membrances and the accumulation of starch grains. They were as follows: 1. The most striking feature was the building up of starch grains in the chloroplasts of the bundle sheath cells (BSCs) and the mesophyll cells (MCs) at doubled CO2 concentra tion. The starch grains appeared centrifugally first in the BSCs and then in the chloroplast of the other MCs. It was worthy to note that the starch grains in the chloroplasts of C4 plant Setaria ira/ica were much more than those of the C3 plant Medicago sativa . The decline of photosynthesis in the doubled CO2-grown C4 plants might be caused by an over accumulation of starch grains, that deformed the chloroplast even demaged the stroma thylakoids and grana. There might exsist a correlation between the comformation of thylakoid system and starch grain accumulation, namely conversion and transfer of starch need energy from ATP, and coupling factor (CF) for ATP formation distributed mainly on protoplastic surface (PSu) of stroma thylakoid membranes, as well as end and margin membranes of grana thylakoids. Thereby, these results could provide a conclusive evidence for the reason of non effectiveness on growth characteristics of C4 plant. 2. Under normal condition , the mature chlolroplats of higher plants usually develop complete and regularly arranged photosynthetic membrane systems . Chloroplasts from the C4 plant Setaria italica, however, exerted significant changes on stacking degree, grana width and stroma thylakoid length under doubled CO2 concentration; In these changes, the grana stacks were smaller and more numerous, and the number of thylakoids per granum was greatly increased, and the stroma thylakoid was greatly lengthened as compared to those of the control chloroplasts. But the grana were mutually intertwined by stroma thylakoid. The integrity of some of the grana were damaged due to the augmentation of the intrathylakoid space . Similarly, the stroma thylakoids were also expanded. In case. the plant was seriously effected by doubled CO2 concentration as observed in C4 plant Setaria italica , its chloroplasts contained merely the stroma (matrix) with abundant starch grains, while grana and stroma thylakoid membranes were unrecognizable, or occasionally a few residuous pieces of thylakoid membranes could be visualized, leaving a situation which appeared likely to be chloroplast deterioration. However, under the same condition the C3 plant Medicago sativa possessed normally developed chloroplasts, with intact grana and stroma thylakoid membranes. Its chloroplasts contained grana intertwined with stroma thylakoid membranes, and increased in stacking degree and granum width, in spite of more accumulated starch grains within the chloroplasts. These configuration changes of the thylakoid system were in consistant with the results of the authors another study on chloroplast function, viz. the increased capacity of chloroplasts for light absorption and efficiency of PSⅡ.     

CO<Subscript>2</Subscript> exchange and structural organization of chloroplasts under hypothermia in potato plants transformed with a gene for yeast invertase

Growth, CO₂ exchange, and the ultrastructure of chloroplasts were investigated in the leaves of potato plants (Solanum tuberosum L., cv. Désirée) of wild type and transformed with a gene for yeast invertase under the control of patatin class I B33 promoter (for apoplastic enzyme) grown in vitro on the Murashige and Skoog medium supplemented with 2% sucrose. At a temperature of 22°C optimal for growth, the transformed plants differed from the plants of wild type in retarded growth and a lower rate of photosynthesis as calculated per plant. On a leaf dry weight basis, photosynthesis of transformed plants was higher than in control plants. Under hypothermia (5°C), dark respiration and especially photosynthesis of transformed plants turned out to be more intense than in control material. After a prolonged exposure to low temperature (6 days at 5°C), in the plants of both genotypes, the ultrastructure of chloroplasts changed. Absolute areas of sections of chloroplasts and starch grains rose, and the area of plastoglobules decreased; in transformed plants, these changes were more pronounced. By some ultrastructural characteristics: a reduction in the cold of relative total area of sections of starch grains and plastoglobules (in percents of the chloroplast section area) and in the number of granal thylakoids (per a chloroplast section area), transformed plants turned out to be more cold resistant than wild-type plants. The obtained results are discussed in connection with changes in source-sink relations in transformed potato plants. These changes modify the balance between photosynthesis and retarded efflux of assimilates, causing an increase in the intracellular level of sugars and a rise in the tolerance to chilling.     

Seasonal dimorphism and winter chilling stress in Thymus sibthorpii

Seasonal dimorphism (summer/winter) has been so far studied only in a few plants and has been focused on summer drought stress. However, Thymus sibthorpii in the study area appears to be affected by winter chilling stress and not by summer drought stress. Thus, the winter leaves were thicker and more compact compared to the summer leaves and they had more stomata and peltate hairs, more sclerenchymatous fibers, vacuoles with phenolics, and chloroplasts than the summer leaves. In addition, their chloroplasts possessed large grana and starch grains. In the summer leaves, cell vacuoles in mesophyll did not contain phenolics, and chloroplasts were devoid of starch grains and had large plastoglobuli. Physiological measurements revealed higher net photosynthetic rate and chlorophyll content in the winter leaves than in the summer leaves. Proline and soluble sugar content along with antioxidative enzyme (superoxide dismutase, peroxidase, ascorbate peroxidase, glutathione reductase) activities were increased in the winter leaves.     

Fine structural quantification of drought-stressed Picea abies (L.) organelles based on 3D reconstructions

Ultrastructural investigations of cells and organelles by transmission electron microscopy (TEM) usually lead to two-dimensional information of cell structures without supplying exact quantitative data due to the limited number of investigated ultrathin sections. This can lead to misinterpretation of observed structures especially in context of their three-dimensional (3D) assembly. 3D investigations and quantitative morphometric analysis are therefore essential to get detailed information about the arrangement and the amount of subcellular structures inside a cell or organelle, respectively, especially when the plant sample was exposed to environmental stress. In the present research, serial sectioned chloroplasts, mitochondria, and peroxisomes from first year spruce needles (Picea abies (L.) Karst.) were 3D reconstructed and digitally measured using a computer-supported image analysis system in order to obtain a detailed quantitative characterization of complete cell organelles including precise morphological data of drought-induced fine structural changes. In control plants, chloroplast volume was composed of 56% stroma, 15% starch, 27% thylakoids, and 2% plastoglobules. In drought-stressed chloroplasts, the relative volume of both the thylakoids and the plastoglobules significantly increased to 37% and 12%, respectively. Chloroplasts of stressed plants differed from control plants not only in the mean thylakoid and plastoglobules content but also in the complete lack of starch grains. Mitochondria occurred in variable forms in both control and stressed samples. In stressed plants, mitochondria showed a significant smaller mean volume which was only 81% when compared with the control organelles. Peroxisomes were inconspicuous in both samples and their volume did not differ between control and drought-stressed samples. The present study shows that specific subcellular structures are subject to significant quantitative changes during drought stress of spruce needles giving a detailed insight in adaptation processes of the investigated cell organelles.     

Effects of elevated CO<Subscript>2</Subscript> applied to potato roots on the anatomy and ultrastructure of leaves

The root system of potato (Solanum tuberosum L. cv. Favorita) plants was treated with different O₂ and CO₂ concentrations for 35 d in aeroponic culture. Under 21 or 5 % O₂ in the root zones, the thickness of leaves and palisade parenchyma significantly increased at 3 600 μmol(CO₂) mol⁻¹ in the root zone, compared with CO₂ concentration 380 μmol mol⁻¹ or low CO₂ concentration (100 μmol mol⁻¹). In addition, smaller cells of palisade tissue, more intercellular air spaces and partially two layers of palisade cells were observed in the leaves with root-zone CO₂ enrichment. Furthermore, there was a significant increase in the size of chloroplasts and starch grains, and the number of starch grains per chloroplast due to elevated CO₂ only under 21 % O₂. In addition, a significant decline in the thickness of grana and the number of lamellas, but no significant differences in the number of grana per chloroplast were observed under elevated CO₂ concentration. The accumulation of starch grains in the chloroplast under elevated CO₂ concentration could change the arrangement of grana thylakoids and consequently inhibited the absorption of sun radiation and photosynthesis of potato plants.     

Lipid peroxidation and antioxidant enzyme activities of Euphorbia millii hyperhydric shoots

A large number of micropropagated Euphorbia millii shoots from temporary immersion bioreactor showed thick broad leaves that were translucent, wrinkled and/or curled and brittle, symptoms of hyperhydricity. The environment inside bioreactor normally used in plant micropropagation is characterised by high relative humidity, poor gaseous exchange between the internal atmosphere of the bioreactor and its surrounding environment, and the accumulation of ethylene, conditions that may induce physiological disorders. A comparison of hyperhydric shoots (HS) with normal plants shows marked increase in malondialdehyde (MDA) content in HS plants. MDA, a decomposition product of polyunsaturated fatty acids hydroperoxides, has been utilized very often as a suitable biomarker for lipid peroxidation, which is an effect of oxidative damage. This hypothesis is also confirmed by the higher lipoxygenase (LOX) activity in HS plants. The potential role of antioxidant enzymes in protecting hyperhydric shoots from oxidative injury was examined by analyzing enzyme activities and isozyme profiles of hyperhydric and non-hyperhydric leaves of E. millii. Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity were significantly higher in hyperhydric tissue as compared to non-hyperhydric normal leaf tissue. After native polyacrylamide gel electrophoresis (PAGE) analysis, seven SOD isoenzymes were detected and the increase in SOD activity observed in hyperhydric tissue seemed to be mainly due to Mn-SOD and Cu/Zn-SOD. The activity of ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) was proportionally increased in HS tissue compared to normal leaves indicating a crucial role in eliminating toxic H₂O₂ from plant cells. The depletion of GSH and total glutathione in spite of higher GR activities observed in HS tissue indicates that mechanism of antioxidant defense was by enhanced oxidation of GSH to GSSG by DHAR yielding ascorbate (AA). The antioxidant metabolism has been shown to be important in determining the ability of plants to survive in hyperhydric stress and the up regulation of these enzymes would help to reduce the build up of ROS.     

Systemic changes in photosynthesis and reactive oxygen species homeostasis in shoots of Arabidopsis thaliana infected with the beet cyst nematode Heterodera schachtii

Photosynthetic efficiency and redox homeostasis are important for plant physiological processes during regular development as well as defence responses. The second‐stage juveniles of Heterodera schachtii induce syncytial feeding sites in host roots. To ascertain whether the development of syncytia alters photosynthesis and the metabolism of reactive oxygen species (ROS), chlorophyll a fluorescence measurements and antioxidant responses were studied in Arabidopsis thaliana shoots on the day of inoculation and at 3, 7 and 15 days post‐inoculation (dpi). Nematode parasitism caused an accumulation of superoxide and hydrogen peroxide molecules in the shoots of infected plants at 3 dpi, probably as a result of the observed down‐regulation of antioxidant enzymes. These changes were accompanied by an increase in RNA and lipid oxidation markers. The activities of antioxidant enzymes were found to be enhanced on infection at 7 and 15 dpi, and the content of anthocyanins was elevated from 3 dpi. The fluorescence parameter R_fd, defining plant vitality and the photosynthetic capacity of leaves, decreased by 11% only at 7 dpi, and non‐photochemical quenching (NPQ), indicating the effectiveness of photoprotection mechanisms, was about 16% lower at 3 and 7 dpi. As a result of infection, the ultrastructure of chloroplasts was changed (large starch grains and plastoglobules), and more numerous and larger peroxisomes were observed in the mesophyll cells of leaves. We postulate that the joint action of antioxidant enzymes/molecules and photochemical mechanisms leading to the maintenance of photosynthetic efficiency promotes the fine‐tuning of the infected plants to oxidative stress induced by parasitic cyst nematodes.     

Ultrastructure of Chloroplasts and Their Storage Inclusions in the Primary Leaves of Mesembryanthemum crystallinum Affected by Putrescine and NaCl

The effects of salinity (300 mM NaCl), putrescine (Put), and the combination of two agents on the structure of chloroplasts and storage deposits were studied in the third leaf pair of a facultative halophyte Mesembryanthemum crystallinum. Within 6 days, the common ice plants responded to NaCl and Put treatments by diminished chloroplast volumes and swollen grana. Different effects of the experimental treatments were primarily manifested in the chloroplast storage inclusions. Under the salinity conditions, the starch content dropped down almost threefold as compared to untreated plants (control), whereas the number of plastoglobules did not change. Put and Put + NaCl treatments further decreased the starch content per unit section area; in contrast, the plastoglobule area per chloroplast section increased eightfold and tenfold in Put and Put + NaCl treatments, respectively. The morphology and electronic density of plastoglobules changed in all treatments. In both Put treatments there ware no destructive changes in the chloroplasts, and therefore the authors presume that the increase in the numbers plastoglobules was related to the redirection of cell metabolism towards the products of the higher reduction potential. The ferritin deposits in the chloroplasts were observed in all treatments they were more abundant in the vascular parenchyma cells, especially under salinity. The ability of the common ice plants to accumulate large Fe quantities in their chloroplasts and the characteristic pectin-filled pockets, which were observed earlier, and intercellular spaces are probably related to the genetically determined traits of plant adaptation to salinity and water deficit.     

Ultrastructural and Morphological Characteristics of Cultivated Wheat Growing on Copper-Polluted Fields

A cultivated Greek variety of wheat (Triticum aestivum L. cv. Vergina) growing in fields naturally polluted by outcrops of copper ores was investigated. Wheat plants show a negative response to increasing quantities of soil copper, including reduced growth and chlorosis. Copper toxicity was demonstrated in the laboratory by a rooting test; the frequency of mitoses declines sharply with increasing copper concentration in the nutrient solution. The mesophyll cells of polluted plants display a circular shape (in transverse sections) with a few chloroplasts parietally distributed, in contrast with the elongate or pleomorphic shape of control leaves that contain numerous chloroplasts crowded at the cell periphery. Ultrastructurally, the chloroplasts of polluted plants contain a poorly developed internal membrane system consisting of thylakoids arranged parallel to each other with only a few, rudimentary grana. In addition, a number of statistically significant differences were found, including the number of starch grains and plastoglobuli, chloroplast surface area, volume fraction of starch grains and, most important, the volume fraction of the internal membrane system. All ultrastructural changes are attributed to the toxic effect of high concentrations of soil copper.