Subcellular volumes and metabolite concentrations in barley leaves

Metabolite concentrations in subcellular compartments from mature barley (Hordeum vulgare L. cv. Apex) leaves after 9 h of illumination and 5 h of darkness were determined by nonaqueous fractionation and by the stereological evaluation of cellular and subcellular volumes from light and electron micrographs. Twenty one-day-old primary leaves of barley with a total leaf volume of 902 μL per mg chlorophyll were found to be composed of 27% epidermis, 42% mesophyll cells, 6% veins, 4.5% apoplast and 23% gas space. While in epidermal cells 99% of the volume was occupied by the vacuole, mesophyll cells with an average volume of 31.3 pL consisted of 23 pL (73%) vacuole, 4.6 pL (19%) chloroplasts, 2.06 pL (6,7%) cytosol (including smaller organelles and vesicles), 0.34 pL (1%) mitochondria and 107 fL (0.34%) nucleus. The differences between leaves harvested after 9 h of illumination and after 5 h of darkness were in the size of the stromal compartment and the starch grains therein. Subcellular metabolite concentrations were calculated from the compartmental volumes and metabolite contents of the compartments as determined by nonaqueous fractionation. The amino-acid concentrations in stroma and cytosol were rather similar after 9 h of illumination and 5 h of darkness. In contrast, the vacuolar amino-acid concentrations were about one order of magnitude lower than the stroma and cytosol values, and there was a slight increase in concentration after 5 h of darkness.     

Subcellular Volumes and Metabolite Concentrations in Potato (Solanum tuberosum cv. Désirée) Leaves1

Cellular and subcellular volumes in mature leaves of potato (Solanum tuberosum cv. Désirée) were determined stereologically from light and electron micrographs. Leaves of ten-week-old plants with a total leaf volume of 623 μL per mg chlorophyll (Chl) were found to be composed of 12 % epidermis, 68 % mesophyll, 5 % vascular tissue, 3 % apoplast and 16 % gas space. In the epidermal cells 97 % of the volume was occupied by the vacuole. The mesophyll cells consisted (as expressed per mg Chl) of 323 μL (76 %) vacuole, 35 μL (8 %) chloroplast stroma, 22 μL (5 %) cytosol plus nucleus and peroxisomes, and 4μL, (1 %) mitochondria. A comparison of these values with subcellular volumes previously determined for spinach and barley leaves, shows that the relative sizes of the subcellular volumes are strikingly similar. Subcellular concentrations of carbohydrates, of the phosphorylated intermediates of carbohydrate metabolism, of malate, and of amino acids have been evaluated from measurements of the corresponding subcellular metabolite contents determined using the non-aqueous fractionation technique. Malate, glucose and fructose were found to accumulate in the vacuole, whereas the concentration of sucrose and amino acids in the cytosol were much higher than in the vacuole. The amino acid concentration in the chloroplast stroma is similar to that in the cytosol. Phosphorylated intermediates of carbohydrate metabolism are confined to the chloroplast stroma and the cytosol, confirming the validity of the fractionation method. Whereas triose phosphates and fructose-l,6-bisphosphate are concentrated in the stroma, the concentrations of hexose monophosphates were highest in the cytosol. Since the subcellular metabolite distribution in potato leaves reported here is very similar to that previously described for spinach and barley leaves, we conclude that it may be characteristic for mesophyll cells in general.     

Metabolite profiling and assessment of metabolome compartmentation of soybean leaves using non-aqueous fractionation and GC-MS analysis

In the present study, non-aqueous fractionation (NAQF) and GC-MS were used to obtain a spatially resolved view of metabolism in mature leaves of soybean (Glycine max Merr.). NAQF of lyophilized soybean leaves was performed using CCl₄-n-heptane and ultracentrifugation that yielded a gradient comprised of six fractions. Chlorophyll content, and marker enzyme activities, phosphoenolpyruvate carboxylase (PEPC) and α-mannosidase, were utilized as stroma, cytosol and vacuole markers, respectively. GC-MS analyses of each fraction resulted in the identification of around 100 different metabolites. The distribution of these identified compounds showed a decreasing order from the vacuole to cytosol to chloroplast stroma. In other words, a greater number of identified compounds were found in the vacuole when compared to the cytosol or stroma. Levels of sugars, organic acids and fatty acids showed greater relative abundances in the vacuole with 50, 55, and 50% of the respective pools. A greater relative abundance of amino acids was observed in the cytosol where 45% of the total of amino acids content was recorded. The relatively large pool of sugars and phenolic acids in the vacuole compartment implies high levels of starch metabolism and phenylpropanoid biosynthesis. The low amino acids pool, on the other hand, suggests low nitrogen accumulation in the leaves of soybean. Hierarchical cluster analysis on the most abundant metabolites revealed three clusters containing 10, 20, and 2 of the 32 selected metabolites. The data were discussed in term of NAQF and GC-MS analysis of soybean mature leaves, and also in term of distribution and compartmentation of metabolites at subcellular levels.     

Accumulation of hexoses in leaf vacuoles: Studies with transgenic tobacco plants expressing yeast-derived invertase in the cytosol,vacuole or apoplasm

The subcellular distribution of hexoses, sucrose and amino acids among the stromal, cytosolic and vacuolar compartments was analysed by a nonaqueous fractionation technique in leaves of tobacco (Nicotiana tabaccum L.) wild-type and transgenic plants expressing a yeast-derived invertase in the cytosolic, vacuolar or apoplasmic compartment. In the wild-type plants the amino acids were found to be located in the stroma and in the cytosol, sucrose mainly in the cytosol and up to 98% of the hexoses in the vacuole. In the leaves of the various transformants, where the contents of hexoses were greater than in wild-type plants, again 97–98% of these hexoses were found in the vacuoles. It is concluded that leaf vacuoles contain transporters for the active uptake of glucose and fructose against a high concentration gradient. A comparison of estimated metabolite concentrations in the subcellular compartments of wild-type and transformant plants indicated that the decreased photosynthetic capacity of the transformants is not due to an osmotic effect on photosynthesis, as was shown earlier to be the case in transformed potato leaves, but is the result of a long-term dedifferentiation of tobacco leaf cells to heterotrophic cells.Abbreviations apo-inv tobacco plant with yeast invertase in the apoplasm - Chl chlorophyll - cy-inv tobacco plant with yeast invertase in the cytosol - vac-inv tobacco plant with yeast invertase in the vacuole - WT wild-type tobacco plant The authors thank A. Großpietsch for her able technical assistance. This work has been supported by the Bundesminister für Forschung und Technologie.     

Subcellular volumes and metabolite concentrations in spinach leaves

Cellular and subcellular volumes in mature leaves of spinach (Spinacia oleracea L. US Hybrid 424) were determined stereologically from light and electron micrographs. Forty-nine-day-old leaves of spinach with a total leaf volume of 1177 μL per mg chlorophyll (Chl) were found to be composed of 3% epidermis, 58% mesophyll, 1% vascular tissue, 5% apoplasm and 32% gas space. In the epidermal cells 89% of the volume was occupied by the vacuole. The mesophyll cells consisted, expressed in mg·Chl⁻¹, of 546 μL (79%) vacuole, 66 μL (9.5%) chloroplast stroma, 24 μL (34%) cytosol, 3.7 μL (0.5%) mitochondria and 2.1 μL (0.3%) nucleus. From previous measurements of the subcellular levels of sucrose, of phosphorylated intermediates of carbohydrate metabolism, of malate, oxoglutarate and various amino acids in illuminated leaves, and the above subcellular volumes, the corresponding subcellular metabolite concentrations have been determined. Of the substances measured, only with malate was the concentration higher in the vacuole than in the cytosol. The concentration of sucrose in the cytosol was 5 times, and that of amino acids even 30 times higher than in the vacuole.     

Subcellular compartmentation of proline in the leaves of the subantarctic Kerguelen cabbage Pringlea antiscorbutica R. Br. In vivo13C-NMR study

Proline is one of the major solutes accumulated upon salt stress in leaves, stem and roots of the subantarctic Brassicaceae Pringlea antiscorbutica R. Br. (Kerguelen cabbage). Using in vivo ¹³C-NMR techniques, it was possible for the first time to visualize the subcellular compartmentation of proline between cytoplasmic and vacuolar compartments in Pringlea leaves. We observed that this osmolyte accumulated at a 2–3 times higher concentration in the cytoplasm than in the vacuole.     

Acclimation of potato plants to polyethylene glycol-induced water deficit II. Contents and subcellular distribution of organic solutes

Potato plants (Solanum tuberosum cv. Désirée) were grown hydroponically and subjected to water deficit induced by addition of 10% (w/v) PEG 6000. The potato plants were able to grow under water deficit by accumulating organic solutes (osmoregulation). Osmoregulation occurred in two phases. During the initial 2d hexoses were accumulated, and after 7 d of PEG treatment osmotic adjustment was mostly due to the accumulation of amino acids, especially proline, which accumulated up to 150 times the control content. Sucrose contents remained unchanged in leaves of PEG-treated plants compared with controls, whereas the starch content decreased during PEG treatment.In control leaves, the hexoses and malate were compartmented in the vacuole and sucrose was found in the cytosol and vacuole. Amino acids were distributed between the cytosol and stroma, but only minor amounts of amino acids could be detected in the vacuole. Under water deficit the subcellular distribution of hexoses, malate and sucrose remained unchanged. Most amino acids showed a slight to moderate higher concentration in the vacuole under water deficit. Proline, the metabolite contributing mainly to osmoregulation, was concentrated mostly in the chloroplast and the cytosol. This underlines the important role of proline as the osmolyte under water deficit.     

Subcellular immunocytochemical analysis detects the highest concentrations of glutathione in mitochondria and not in plastids

The tripeptide glutathione is a major antioxidant and redox buffer with multiple roles in plant metabolism. Glutathione biosynthesis is restricted to the cytosol and the plastids and the product is distributed to the various organelles by unknown mechanisms. In the present study immunogold cytochemistry based on anti-glutathione antisera and transmission electron microscopy was used to determine the relative concentration of glutathione in different organelles of Arabidopsis thaliana leaf and root cells. Glutathione-specific labelling was detected in all cellular compartments except the apoplast and the vacuole. The highest glutathione content was surprisingly not found in plastids, which have been described before as a major site of glutathione accumulation, but in mitochondria which lack the capacity for glutathione biosynthesis. Mitochondria of both leaf and root cells contained 7-fold and 4-fold, respectively, higher glutathione levels than plastids while the density of glutathione labelling in the cytosol, nuclei, and peroxisomes was intermediate. The accuracy of the glutathione labelling is supported by two observations. First, pre-adsorption of the anti-glutathione antisera with glutathione reduced the density of the gold particles in all organelles to background levels. Second, the overall glutathione-labelling density was reduced by about 90% in leaves of the glutathione-deficient Arabidopsis mutant pad2-1 and increased in transgenic plants with enhanced glutathione accumulation. Hence, there was a strong correlation between immunocytochemical and biochemical data of glutathione accumulation. Interestingly, the glutathione labelling of mitochondria in pad2-1 remained very similar to wild-type plants thus suggesting that the high mitochondrial glutathione content is maintained in a situation of permanent glutathione-deficiency at the expense of other glutathione pools. High and constant levels of glutathione in mitochondria appear to be particularly important in cell survival strategies and it is predicted that mitochondria must have highly competitive mitochondrial glutathione uptake systems. The present results underline the suggestion that subcellular glutathione concentrations are not controlled by a global mechanism but are controlled on an individual basis and it is therefore not possible to conclude from global biochemical glutathione analysis on the status of the various organellar pools.     

Methyl-beta-D-glucopyranoside in higher plants: accumulation and intracellular localization in Geum montanum L. leaves and in model systems studied by 13C nuclear magnetic resonance

Using (13)C-NMR, methyl-beta-D-glucopyranoside (MeG) was characterized as a major compound in the leaves of the alpine herb Geum montanum L. MeG continuously accumulated during the life span of G. montanum leaves, and accounted for up to 20% of the soluble carbohydrates in aged overwintering leaves, without being reallocated during senescence. Incubating intact plant tissues, culture cells, and purified organelles with (13)C-labelled substrates showed that MeG was synthesized in the cytosol of cells, directly from glucose and methanol molecules. There was no contribution of the C-1 pathway. MeG was subsequently stored in the vacuole without being re-exported to the cytoplasm. All the dicots tested contained the enzymatic machinery permitting MeG synthesis from methanol and glucose, but the plants accumulating this compound at concentrations higher than 1 micromol g(-1) wet wt were mainly members of the Rosaceae family belonging to the Rosoideae subfamily. It is suggested that the synthesis of MeG may contribute to reduce the accumulation in the cytoplasm of methanol and its derived compounds.     

Analysis of the compartmentation of glycolytic intermediates, nucleotides, sugars, organic acids, amino acids, and sugar alcohols in potato tubers using a nonaqueous fractionation method

The compartmentation of metabolism in heterotrophic plant tissues is poorly understood due to the lack of data on metabolite distributions and fluxes between subcellular organelles. The main reason for this is the lack of suitable experimental methods with which intracellular metabolism can be measured. Here, we describe a nonaqueous fractionation method that allows the subcellular distributions of metabolites in developing potato (Solanum tuberosum L. cv Desiree) tubers to be calculated. In addition, we have coupled this fractionation method to a recently described gas chromatography-mass spectrometry procedure that allows the measurement of a wide range of small metabolites. To calculate the subcellular metabolite concentrations, we have analyzed organelle volumes in growing potato tubers using electron microscopy. The relative volume distributions in tubers are very similar to the ones for source leaves. More than 60% of most sugars, sugar alcohols, organic acids, and amino acids were found in the vacuole, although the concentrations of these metabolites is often higher in the cytosol. Significant amounts of the substrates for starch biosynthesis, hexose phosphates, and ATP were found in the plastid. However, pyrophosphate was located almost exclusively in the cytosol. Calculation of the mass action ratios of sucrose synthase, UDP-glucose pyrophosphorylase, phosphoglucosisomerase, and phosphoglucomutase indicate that these enzymes are close to equilibrium in developing potato tubers. However, due to the low plastidic pyrophosphate concentration, the reaction catalyzed by ADP-glucose pyrophosphorylase was estimated to be far removed from equilibrium.     

Generation of transgenic Arabidopsis plants expressing mcherry-fused organelle marker proteins

In eukaryotic cells, a major proportion of the cellular proteins localize to various subcellular organelles where they are involved in organelle-specific cellular processes. Thus, the localization of a particular protein in the cell is an important part of understanding the physiological role of the protein in the cell. Various approaches such as subcellular fractionation, immunolocalization and live imaging have been used to define the localization of organellar proteins. Of these various approaches, the most powerful one is the live imaging because it can show in vivo dynamics of protein localization depending on cellular and environmental conditions without disturbing cellular structures. However, the live imaging requires the ability to detect the organelles in live cells. In this study, we report generation of a new set of transgenic Arabidopsis plants using various organelle marker proteins fused to a fluorescence protein, monomeric Cherry (mCherry). All these markers representing different subcellular organelles such as chloroplasts, mitochondria, peroxisomes, endoplasmic reticulum (ER) and lytic vacuole showed clear and specific signals regardless of the cell types and tissues. These marker lines can be used to determine localization of organellar proteins by colocalization and also to study the dynamics of organelles under various developmental and environmental conditions.     

Interactions between the diamondback moth, Plutella xylostella L. and glasshouse and outdoor-grown cabbage cultivars

Interactions between Brassica cultivars (cvs) and adult and larval stages of Plutella xylostella L. were examined. In six-way choice oviposition experiments, glasshouse-grown Chinese cabbage cv. Tip Top was preferred to five outdoor-grown temperate cabbage cultivars but was similar to four out of five tropical cultivars. Bionomic studies with neonate larvae on temperate cabbage cultivars showed that mean larval duration was longer and percentage survival less on older compared with younger plants but that plant age or cultivar had little effect on pupal weight. The fecundity of P. xylostella reared on outdoor-grown cabbage cultivars varied ten-fold between the poorest and the best cultivar but plant age had little effect on P. xylostella reared on Chinese cabbage. In a glasshouse experiment, survival of P. xylostella on Chinese cabbage was significantly greater compared with field-grown glossy-leaved, normal bloom green cabbage and red cabbage. In bioassays with neonate larvae, when the leaves were placed vertically instead of horizontally a significant level of intrinsic resistance was revealed for glasshouse and field-grown glossy-leaved cabbage but not for Chinese cabbage. Observed differences in host status to P. xylostella are discussed in relation to the potential for partial plant resistance in control programmes.     

The Mechanism of Nitrate Accumulation in Pakchoi [Brassica Campestris L.ssp. Chinensis(L.)]

Decreased nitrate in vegetables can improve crop nitrogen utilization efficiency and lessen the human health risk caused by the reduction of nitrate to nitrite in vegetables. This paper studied the mechanisms of differences in nitrate accumulation and distribution within organs of two cultivars of pakchoi (Brassica campestris L.ssp. Chinensis (L.) previously screened in hydroponic experiments from 12 cultivars popularly grown in China at present. The two typical cultivars used in this experiment were Shanghaiqing with low nitrate accumulation and Liangbaiye 1 with high nitrate accumulation. There was no significant difference of total nitrate uptake but a significant difference in nitrate content existed between the two cultivars. Compared with Liangbaiye 1, Shanghaiqing showed a significantly higher photosynthetic rate and nitrate reductase activity. Determination of nitrate concentration (activity) in vacuoles with double-barrelled nitrate-selective microelectrodes showed that Shanghaiqing had lower vacuolar nitrate activity than Liangbaiye 1. Two putative nitrate reductase genes, nia1 and nia2, were amplified from the leaf blades of these two cultivars. Nia1 mRNA fragments (887 bp, accession numbers DQ082868 and DQ082869) were amplified using degenerate primer and nia2 mRNA fragment was amplified using one pair of generate primers designed according to DQ001901. Sequence analysis of DQ082868 and DQ082869 both showed 97% and 87% similarity with two nitrate reductase mRNA sequences of Brassica napus, accession numbers D38219 and D38220, respectively. The results of real time PCR to compare the relative expression of the putative nitrate reductase genes (nia1 and nia2) showed that Shanghaiqing had significantly higher expression level than Liangbaiye 1 and nia2 was significantly higher than nia1 in leaf blade and petiole. Both the nitrate reductase activity and the relative expression level of nia1 were in the order of leaf blade > root > petiole, while that of nia2 was leaf blade > petiole > root. There was no statistically significant difference of nitrate activity stored in vacuoles between the different organs of the two cultivars. It can be concluded that Shanghaiqing took up slightly less nitrate, but had significantly higher nitrate reductase activity in cytosol and had a higher relative expression of the putative nitrate reductase genes than Liangbaiye 1; this leads to the fact that Shanghaiqing has a lower nitrate content than Liangbaiye 1.     

Toxoplasma evacuoles: a two-step process of secretion and fusion forms the parasitophorous vacuole.

Rapid discharge of secretory organelles called rhoptries is tightly coupled with host cell entry by the protozoan parasite Toxoplasma gondii. Rhoptry contents were deposited in clusters of vesicles within the host cell cytosol and within the parasitophorous vacuole. To examine the fate of these rhoptry-derived secretory vesicles, we utilized cytochalasin D to prevent invasion, leading to accumulation of protein-rich vesicles in the host cell cytosol. These vesicles lack an internal parasite and are hence termed evacuoles. Like the mature parasite-containing vacuole, evacuoles became intimately associated with host cell mitochondria and endoplasmic reticulum, while remaining completely resistant to fusion with host cell endosomes and lysosomes. In contrast, evacuoles were recruited to pre-existing, parasite-containing vacuoles and were capable of fusing and delivering their contents to these compartments. Our findings indicate that a two-step process involving direct rhoptry secretion into the host cell cytoplasm followed by incorporation into the vacuole generates the parasitophorous vacuole occupied by TOXOPLASMA: The characteristic properties of the mature vacuole are likely to be determined by this early delivery of rhoptry components.     

低温胁迫对两种圆柏属植物亚细胞抗氧化酶活性的影响

以祁连圆柏和圆柏幼苗为材料,研究不同处理时间下低温胁迫对圆柏属植物叶片亚细胞抗氧化酶活性的影响,探讨其在圆柏属植物叶片中的亚细胞定位。结果表明：低温胁迫下,丙二醛(MDA)含量和抗氧化酶活性随时间变化均呈先升后降趋势,祁连圆柏中抗氧化酶的种类比圆柏的多且活性强,而 MDA 含量低于圆柏,表明祁连圆柏在低温胁迫下具有更广泛的适应性。此外,两种圆柏植物叶片超氧化物歧化酶(SOD)和抗坏血酸过氧化物酶(APX)定位为叶绿体＞细胞溶质＞线粒体,过氧化氢酶(CAT)定位为线粒体＞叶绿体＞细胞溶质,谷胱甘肽还原酶(GR)定位为线粒体＞细胞溶质＞叶绿体,祁连圆柏过氧化物酶(POD)定位为细胞溶质＞叶绿体＞线粒体,圆柏POD定位为细胞溶质＞线粒体＞叶绿体,且抗氧化酶SOD、APX和 GR在亚细胞中分布差异达到极显著,这说明抗氧化酶在其中一种亚细胞中发挥主要作用,为克隆亚细胞组分中的抗氧化酶基因提供了理论依据。     

Uptake of sodium in protoplasts of salt-sensitive and salt-tolerant cultivars of rice, Oryza sativa L. determined by the fluorescent dye SBFI

In this study, the uptake of Na+ into the cytosol of rice (Oryza sativa L. cvs Pokkali and BRRI Dhan29) protoplasts was measured using the acetoxy methyl ester of the fluorescent sodium-binding benzofuran isopthalate, SBFI-AM, and fluorescence microscopy. By means of inhibitor analyses the mechanisms for uptake and sequestration of Na+ in the salt-sensitive indica rice cv. BRRI Dhan29 and in the salt-tolerant indica rice cv. Pokkali were detected. Less Na+ was taken up into the cytosol of Pokkali than into BRRI Dhan29. The results indicate that K+-selective channels do not contribute to the Na+ uptake in Pokkali, whereas they are the major pathways for Na+ uptake in BRRI Dhan29 along with non-selective cation channels. However, non-selective cation channels seem to be the main pathways for Na+ uptake in Pokkali. Protoplasts from Pokkali leaves took up Na+ only transiently in the presence of extracellular Na+ at 5-100 mM. Therefore, it is likely that the protoplasts have a mechanism for fast extrusion of Na+ out of the cytoplasm. Experiments with protoplasts pretreated with NH4NO3 and NH4VO3 suggest that the salt-tolerant Pokkali extrudes Na+ mainly into the vacuole. After cultivation of both cultivars in the presence of 10 or 50 mM NaCl for 72 h, the isolated protoplasts from Pokkali took up less Na+ than the control protoplasts. The results suggest that the salt-tolerance in Pokkali depends on reduced uptake through K+-selective channels and a fast extrusion of Na+ into the vacuoles.     

Distribution of polycyclic aromatic hydrocarbons in subcellular root tissues of ryegrass (Lolium multiflorum Lam.)

Background

Because of the increasing quantity and high toxicity to humans of polycyclic aromatic hydrocarbons (PAHs) in the environment, several bioremediation mechanisms and protocols have been investigated to restore PAH-contaminated sites. The transport of organic contaminants among plant cells via tissues and their partition in roots, stalks, and leaves resulting from transpiration and lipid content have been extensively investigated. However, information about PAH distributions in intracellular tissues is lacking, thus limiting the further development of a mechanism-based phytoremediation strategy to improve treatment efficiency.

Results

Pyrene exhibited higher uptake and was more recalcitrant to metabolism in ryegrass roots than was phenanthrene. The kinetic processes of uptake from ryegrass culture medium revealed that these two PAHs were first adsorbed onto root cell walls, and they then penetrated cell membranes and were distributed in intracellular organelle fractions. At the beginning of uptake (< 50 h), adsorption to cell walls dominated the subcellular partitioning of the PAHs. After 96 h of uptake, the subcellular partition of PAHs approached a stable state in the plant water system, with the proportion of PAH distributed in subcellular fractions being controlled by the lipid contents of each component. Phenanthrene and pyrene primarily accumulated in plant root cell walls and organelles, with about 45% of PAHs in each of these two fractions, and the remainder was retained in the dissolved fraction of the cells. Because of its higher lipophilicity, pyrene displayed greater accumulation factors in subcellular walls and organelle fractions than did phenanthrene.

Conclusions

Transpiration and the lipid content of root cell fractions are the main drivers of the subcellular partition of PAHs in roots. Initially, PAHs adsorb to plant cell walls, and they then gradually diffuse into subcellular fractions of tissues. The lipid content of intracellular components determines the accumulation of lipophilic compounds, and the diffusion rate is related to the concentration gradient established between cell walls and cell organelles. Our results offer insights into the transport mechanisms of PAHs in ryegrass roots and their diffusion in root cells.     

镉在黑藻叶细胞中的亚显微定位分布及毒害效应分析

Aquatic plants are known to accumulate and bioconcentrate heavy metals. In this study, aquatic vascular plant Hydrilla verticillata (L.f.) Royle was cultivated in water containing elevated concentrations of cadmium (up to 10 mg/L) for 7 d, the accumulation, subcellular distribution, ultrastructural localization, chemical form, toxic effects on mineral nutrient absorption of cadmium, photosynthesis rate and respiration rate were studied. It was found that H. verticillata fronds was able to accumulate cadmium, the bioconcentration factor was 193-307; subcellular fraction analysis revealed that cadmium major accumulated in cell wall (61.66%-52.00%) with decrease trends, the content of Cd enhanced in soluble fraction and remained stable in organelles, with the augment of pollutant concentration. The Cd levels occurred in different parts of leaf cell with the following sequence: cell wall > soluble fraction > organelles. Ultrastructural localization of cadmium with sulfide-silver method showed that Cd appeared in cell wall, chloroplast, nucleus and vacuole. Sequential extraction indicated that the ratio of different cadmium chemical form was different markedly, of which NaCl extractable Cd was predominated as compared with the other 5 forms; which could be seen in the following order: F(NaCl) > F(HAc) >F(Water) > F(Ethanol) > F(HCl) > F(Residue). The mineral nutrient absorption was also affected by cadmium stress, it increased the absorption of Ca, Mn, Cu, and Fe; but reduced that of P and K. Cd had a strong inhibitive effect on photosynthesis rate and respiration rate. The results suggested that the toxic symptoms of plant showed an evident correlation between dose and effect; the ultrastructural damage was closely related to the distribution of Cd. The conclusion could be reached that the death of plant was resulted from destruction of structure foundation of physiological function, unbalance of ion equilibrium and disorder of physiological metabolism.     

Subcellular distribution and chemical forms of cadmium in Bechmeria nivea (L.) Gaud.

Bechmeria nivea (L.) Gaud. (Ramie) is a promising species for Cd phytoextraction with large biomass and fast growth rate. Nevertheless, little information is available on its tolerance mechanisms towards Cd. Determination of Cd distribution and chemical speciation in ramie is essential for understanding the mechanisms involved in Cd accumulation, transportation and detoxification. In the present study, ramie plants were grown in hydroponics with increasing Cd concentrations (0, 1, 3, 7 mg l^?1). The subcellular distribution and chemical forms of Cd in different tissues were determined after 20 days exposure to this metal. To assess the effect of Cd uptake on plant performance, nitrate reductase activity in leaves and root activity were analyzed during the entire experimental period. Increased Cd level in the medium caused a proportional increase in Cd uptake, and the highest Cd concentration occurred in roots, followed by stems and leaves. Subcellular fractionation of Cd-containing tissues indicated that about 48.2–61.9% of the element was localized in cell walls and 30.2–38.1% in soluble fraction, and the lowest in cellular organelles. Cd taken up by ramie rapidly equilibrated among different chemical forms. Results showed that the greatest amount of Cd was found in the extraction of 1 M NaCl and 2% HAC, and the least in residues in all test tissues. In roots, the subdominant amount of Cd was extracted by d-H₂O and 80% ethanol, followed by 0.6 M HCl. While in stems and leaves, the amount of 0.6 M HCl-extractable Cd was comparable with that extracted by 80% ethanol or d-H₂O. 1 mg l^?1 Cd stimulated nitrate reductase activity in leaves and root activity, while a concentration-dependent inhibitory effect was observed with increasing Cd concentration, particularly at 7 mg l^?1 Cd. It could be suggested that the protective mechanisms evolved by ramie play an important role in Cd detoxification at relatively low Cd concentrations (below 3 mg l^?1 Cd) but become restricted to maintain internal homeostasis with higher Cd stress.     

蒌蒿对镉的富集特征及亚细胞分布特点

以镉(Cd)富集植物蒌蒿(Artemisia selengensis)为试材, 采用超声波细胞破碎处理和超速离心的方法, 对蒌蒿根和叶中亚细胞水平的Cd分布状况进行研究, 同时测定Cd在蒌蒿不同器官中的富集效果。结果表明, 在30 mg·kg^–1Cd胁迫下, 蒌蒿叶片中Cd的富集浓度是根和茎中的2–3倍, 但因叶片所占植株的生物量比例较小, 其对Cd的积累量远小于茎和根; Cd在蒌蒿叶片细胞壁、胞液和细胞器中含量比为16:5:1。细胞壁固定是叶片对Cd的主要防御机制。随着Cd处理浓度的增加, 细胞壁和胞液中的Cd含量大幅上升, 但细胞器中Cd含量仍维持在较低水平。长时间和高浓度的Cd胁迫可使细胞壁解毒机制失活并诱导细胞器中的Cd含量增加, 导致植株死亡。根中液泡的Cd贮存量较大, 解毒效果显著。