Physiological and ultra-structural changes in Brassica napus seedlings induced by cadmium stress

The effects of cadmium on physiological and ultrastructural characteristics were evaluated in 6-d-old seedlings of two Brassica napus L. cultivars Zheda 619 and ZS 758. Results show that Cd at lower concentration (100 μM) stimulated the seedling growth but at higher concentration (500 μM) inhibited the growth of both cultivars, decreased content of photosynthetic pigments, activities of antioxidant enzymes, and increased the content of malondialdehyde and reactive oxygen species. Cd content in different parts of seedlings was higher in ZS 758 than in Zheda 619. Electron micrographs illustrated that 500 μM Cd severely damaged the leaf and root tip cells of both cultivars. Under Cd stress, the size and number of starch grains, plastoglobuli, and lipid bodies in the chloroplasts increased. In the root tip cells, enlarged vacuoles, diffused cell walls, and undeveloped mitochondria were detected.     

Leaf morphological and ultrastructural performance of eggplant (Solanum melongena L.) in response to water stress

The effects of water stress on leaf surface morphology (stomatal density, size, and trichome density of both adaxial and abaxial surfaces) and leaf ultrastructure (chloroplasts, mitochondria, and cell nuclei) of eggplant (Solanum melongena L.) were investigated in this study. Higher stomata and trichome densities were observed on abaxial surface compared with the adaxial surface. Compared with well watered (WW) plants, the stomata and trichome density of the abaxial surface increased by 20.39% and 26.23% under water-stress condition, respectively. The number of chloroplasts per cell profile was lesser, the chloroplasts became round in a shape with more damaged structure of membranes, the number of osmiophilic granules increased, and the number of starch grains decreased. The cristae in mitochondria were disintegrated. The cell nuclei were smaller and the agglomerated nucleoli were bigger than those of WW plants. Our results indicated that the morphological and anatomical responses enhanced the capability of plants to survive and grow during stress periods.     

Increasing seed mass and oil content in transgenic Arabidopsis by the overexpression of wri1-like gene from Brassica napus

Rapeseed (Brassica napus) is one of the most important edible oilseed crops in the world and is increasingly used globally to produce bio-diesel. Therefore, increasing oil content of oilseed corps is of importance economically in both food and oil industries. The wri1 genes are differentially expressed in B. napus lines with different oil content. To investigate the effects of B. napus WRI1 (BnWRI1) on oil content, two Bnwri1 genes with different lengths, Bnwri1-1 and Bnwri1-2, were identified and sequenced. Homology analysis shows 80% amino acids of Bnwri1s are homologous to Arabidopsis thaliana WRI1 (AtWRI1). Overexpression of Bnwri1 cDNAs driven by cauliflower mosaic virus 35S-promoter in 51 transgenic A. thaliana lines resulted in 10–40% increased seed oil content and enlarged seed size and mass. Detailed analysis on transgenic embryos indicates an increased cell size other than cell number. In addition, Bnwri1 sequence polymorphism is highly related to oil content (p < 0.001). Taking together, Bnwri1 has potential applications in food and oil industries and in rapeseed breeding.     

Effects of Hydrogen Sulfide on Growth,Antioxidative Capacity,and Ultrastructural Changes in Oilseed Rape Seedlings Under Aluminum Toxicity

The present study evaluates the beneficial effects of the hydrogen sulfide (H₂S) donor, sodium hydrosulfide (0 and 0.3 mM), on the growth of oilseed rape (Brassica napus L. cv. ZS 758) seedlings under aluminum (Al) stress (0, 0.1, and 0.3 mM). Results showed that Al stress decreased the seedling growth by reducing the shoot and root length, biomass, and antioxidant enzymes, which could be illustrated by increased levels of malondialdehyde (MDA), production of hydrogen peroxide (H₂O₂), and accumulation of Al in the shoots. Pretreatment with H₂S reduced MDA and H₂O₂ levels in the leaves and roots of B. napus seedlings. Moreover, activities of antioxidant enzymes (APX, CAT, APX, SOD, POD, and GR) were elevated significantly with the application of H₂S under Al stress. The microscopic examination confirmed that higher levels of Al completely impaired leaf mesophyll and root tip cells. Chloroplasts were spongy shaped with dissolved thylakoid membranes and more starch grains. Root tip cells showed visible symptoms under Al toxicity such as deposition of Al in vacuoles and disruption of whole cell organelles. Under pretreatment with exogenous H₂S, cell structures were improved and presented a clean mesophyll cell and chloroplast possessing well-developed thylakoid membranes as well as fewer starch grains. A number of modifications could be observed in root tip cells, that is, mature mitochondria, long endoplasmic reticulum as well as golgi bodies, under the combined application of H₂S and Al. On the basis of our results, we can conclude that H₂S has a promotive effect which could improve plant survival under Al stress.     

Characteristics of root boron nutrition confer high boron efficiency in Brassica napus cultivars

Background and aims

Brassica napus has high boron (B) demand, but significant genotype differences exist with respect to B deficiency. The aim of this research was to elucidate the relationship between the different sensitivities of Brassica napus cultivars to low B stress and the characteristics of B uptake and transport to characterise the regulation of B efficiency in Brassica napus.

Methods

B-efficient and B-inefficient Brassica napus cultivars were used to compare the uptake and transport of B using the stable isotope ¹⁰B tracer and grafting experiments, as well as expression of B transporters by RT-PCR.

Results

B-efficient cultivars have significant advantages with regard to B limitation. The B-efficient cultivar HZ showed less severe B deficiency symptoms and higher dry biomass than the B-inefficient cultivars LW and LB. Both the amount of total B and the ¹⁰B concentration and accumulation in the shoots and roots of B-efficient HZ were higher than those of B-inefficient cultivars. In B-inefficient LW, the amount of total B and the ¹⁰B that was transported into shoots was less than in the other three cultivars and the content and accumulation of total B and ¹⁰B in the roots of B-inefficient LB were the lowest among all of the cultivars. When the roots of B-efficient HZ were used as stocks, the grafted plants showed B-efficient characteristics, such as mild B deficiency symptoms, and higher dry biomass and B accumulation, regardless of whether they originated from B-efficient or B-inefficient cultivars. In contrast, the grafted plants with B-inefficient LW used as stocks were B-inefficient. The expressions of BnBOR1;1c, BnBOR1;2a and BnNIP5;1 were up-regulated in roots under low B stress compared with the normal B condition. However, there was no obvious difference in the expressions of the three genes or of four other BnBOR1s between B-efficient and B-inefficient cultivars in low or normal B environments.

Conclusions

These results indicate that the B efficiency of Brassica napus is controlled primarily by roots, which allow more uptake and accumulation of B in B-efficient cultivars than B-inefficient cultivars in a low B environment. However the molecular mechanism regulating B efficiency in Brassica napus remains to be determined.     

Differences in cell wall components and allocation of boron to cell walls confer variations in sensitivities of Brassica napus cultivars to boron deficiency

Aims

The cell wall is the main binding site of boron (B) in plants, and the differences in B requirements among different plant species are determined by pectic polysaccharide contents in the cell walls. The aim of this research was to illustrate the relationship between cell wall properties and allocation of B to cell wall and the differential sensitivity of Brassica napus cultivars to B deficiency.

Methods

Two cultivars with opposite B efficiency were used to analyse the relationship among cell wall pectin contents and glycosyl composition, B uptake and allocation, gene expression and cell wall ultrastructure.

Results

The Brassica napus B-efficient cultivar Qingyou 10 was more tolerant to B deficiency, exhibiting a higher biomass production, milder B deficiency symptoms and less cell wall thickening compared to the Brassica napus B-inefficient cultivar Westar 10. These differences were attributed to two factors; the first was that Qingyou 10 accumulated more B and distributed significantly higher proportion of it to the cell wall pectins than did Westar 10 under low B supply. Also, the cell walls of Qingyou 10 exhibited relatively less B-binding sites than those of Westar 10, which was indicated by the lower cell wall extraction rates, less pectin and glycosyl residue contents under the B-deficient and B-sufficient conditions. A comparison of the KDOPS gene expression levels in the two conditions suggests that Westar 10 had a higher potential for biosynthesizing B-binding substances than did Qingyou 10, regardless of B levels.

Conclusions

These results suggest that both higher cell wall pectin polysaccharide content, and limited accumulation and allocation of B to the cell walls contribute to the greater sensitivity of Westar 10 to B deficiency. These two physiological aspects may determine the differences in B deficiency tolerance between Brassica napus cultivars Qingyou 10 and Westar 10. Comparably, the difference in accumulation and allocation of B to cell wall plays a much more important role than cell wall components to sensitivity difference of Brassica napus cultivars to B deficiency.     

Physiological and ultrastructural responses of Catharanthus roseus cell suspension to salt stress

Catharanthus roseus (L.) cell response to salinity was investigated. Seven days after cell treatment with 100 mM NaCl, they showed a decrease in dry weight and an increase in sodium and chloride contents (about 12.4- and 1.5-fold, respectively, in comparison to the control). At the ultrastructural level, NaCl treatment reduced cell size and increased plastid density. In addition, it reduced the starch grain size and their number per plastid; however, starch content was 1.5-fold increased, which was due to the increase in the plastid density. At the ultrastructural level, the applied salinity had no obvious effects, such as swelling or disorganization of plastids except a slight decrease in the stroma electron density. Equally, no deleterious effect was observed on mitochondria except a small increase of their crista volume and matrix electron density. It was shown that, although the relative sensitivity of C. roseus cells to salt stress pointed by the reduction in the dry weight, a decrease in the cell size, and the high accumulation of toxic ions, they preserved the integrity of their plastids and mitochondria.     

Pronounced effects of slug herbivory on seedling recruitment of Brassica cultivars and accessions,especially those with low levels of aliphatic glucosinolates

In demographic studies on Brassica cultivars and accessions we observed large genotypic variation in the ability of seedlings to establish. Here we quantify the role of slugs during establishment by including and excluding slug herbivory.Slug exclusion during the first 10 days after germination led to 26.5 times more surviving seedlings in modern canola varieties of Brassica napus, 4.3 times more in feral B. napus and 1.9 times more in wild Brassica rapa. While seed and seedlings of canola were low in aliphatic glucosinolates (AGS), feral B. napus and wild B. rapa had much higher AGS levels. Consequently, we find a positive correlation between the survival of young seedlings and AGS concentration. Concentrations of indole glucosinolates (IGS) in seeds were much lower than those of AGS and did not correlate significantly with survival. Subsequently, we exposed 10-day-old seedlings to herbivory. In the 4 days following exposure, slug herbivory was negatively correlated to AGS concentration but not to IGS.In choice experiments in the lab, the preference of the slug Arion lusitanicus also correlated negatively with AGS content. A. lusitanicus preferred to feed on B. napus rather than on wild B. rapa, but had no significant preference when presented with plants similar in AGS content.Slugs can be a limiting factor for seedling recruitment in populations of B. napus, especially for modern canola cultivars with a low AGS content.     

Mitochondrial free fatty acid β-oxidation supports oxidative phosphorylation and proliferation in cancer cells

Oxidative phosphorylation (OxPhos) is functional and sustains tumor proliferation in several cancer cell types. To establish whether mitochondrial β-oxidation of free fatty acids (FFAs) contributes to cancer OxPhos functioning, its protein contents and enzyme activities, as well as respiratory rates and electrical membrane potential (ΔΨm) driven by FFA oxidation were assessed in rat AS-30D hepatoma and liver (RLM) mitochondria. Higher protein contents (1.4–3 times) of β-oxidation (CPT1, SCAD) as well as proteins and enzyme activities (1.7–13-times) of Krebs cycle (KC: ICD, 2OGDH, PDH, ME, GA), and respiratory chain (RC: COX) were determined in hepatoma mitochondria vs. RLM. Although increased cholesterol content (9-times vs. RLM) was determined in the hepatoma mitochondrial membranes, FFAs and other NAD-linked substrates were oxidized faster (1.6–6.6 times) by hepatoma mitochondria than RLM, maintaining similar ΔΨm values. The contents of β-oxidation, KC and RC enzymes were also assessed in cells. The mitochondrial enzyme levels in human cervix cancer HeLa and AS-30D cells were higher than those observed in rat hepatocytes whereas in human breast cancer biopsies, CPT1 and SCAD contents were lower than in human breast normal tissue. The presence of CPT1 and SCAD in AS-30D mitochondria and HeLa cells correlated with an active FFA utilization in HeLa cells. Furthermore, the β-oxidation inhibitor perhexiline blocked FFA utilization, OxPhos and proliferation in HeLa and other cancer cells. In conclusion, functional mitochondria supported by FFA β-oxidation are essential for the accelerated cancer cell proliferation and hence anti-β-oxidation therapeutics appears as an alternative promising approach to deter malignant tumor growth.     

Changes in Tomato Leaves Induced by NaCl Stress: Leaf Organization and Cell Ultrastructure

The alterations of organization of leaf tissues and cell ultrastructure as a consequence of salt stress (75 and 150 mM NaCl) were studied in two tomato (Lycopersicum esculentum Mill.) cultivars showing different salinity tolerance. The salinity brought changes in cell shape, volume of intercellular spaces and chloroplast number, shape and size. These characteristics were specific in each cultivar. The ultrastructural changes were also different in the two tomato cultivars studied and the most important ones were in the number and size of starch granules in chloroplasts, the number of electron-dense corpuscules in the cytoplasm, the structure of mitochondria, and number of plastoglobuli. This revised version was published online in July 2006 with corrections to the Cover Date.     

Use of 5´-anchored primers for the enhanced recovery of specific microsatellite markers in Brassica napus L.

Microsatellite markers have assumed great significance in biological research. The isolation and characterisation of microsatellites involves DNA library construction and screening, DNA sequencing, primer design and PCR optimisation. When a microsatellite is situated close to the beginning or end of a cloned fragment, specific primers cannot be designed for one of the flanking sequences, thus hindering the utilisation of such microsatellites as markers. The present approach was to use one 5′-anchored primer complementary to the microsatellite sequence in combination with one specific Cy5- labelled primer with a view to retrieving useful microsatellites, which would otherwise be lost. Six pairs of a 5′ anchored primer and a specific primer were used across a set of 31 Brassica napus winter cultivars and one accession each of five additional Brassica species. Using laser fluorometry a single labelled product was observed after amplification with each of four primer pairs, and one primer pair gave two labelled products. Three products corresponded in size with the products expected if 5′ anchoring was effective, indicating the amplification of locus-specific full-length products including all of the microsatellite repeats. All six primer pairs showed polymorphisms across the Brassica species examined, but only one primer pair showed polymorphisms within B. napus, making it useful for genetic analysis in rapeseed cultivars. The other primer pairs could be useful in studying gene introgression into B. napus or for investigating interspecific crosses involving different Brassica species. Received: 5 August 1999 / Accepted: 1 November 1999     

AN ELECTRON MICROSCOPE STUDY OF TOBACCO MOSAIC VIRUS LESIONS IN NICOTIANA GLUTINOSA L

Ultra-thin sections of Nicotiana glutinosa L. leaves inoculated with a concentrated solution of tobacco mosaic virus were made at short intervals from 0 to 78 hours after inoculation. Eight hours after inoculation, the size of starch grains increased. This was followed by rupture of cytoplasmic and chloroplast membranes. At about 24 hours there was a great increase in number of mitochondria, which persisted until about 60 hours, when some became electron opaque while others appeared to disintegrate. Finally, the cell contents were compressed into one area of the cell, where they became electron opaque. This was accompanied by collapse of the rest of the cell and tearing away of the cell walls from adjacent cells. The nucleus remained stable and intact for as long as observations could be made. No identifiable virus particles were seen.     

The immunolocalization of plasma membrane H+-ATPase in the transfer cell region of Brassica napus (Brassicaceae) ovules

Unfertilized mature ovules of Brassica L. contain an abundance of starch in the integument cells from the micropyle to a plane approximately at the level of the central cell polar nuclei. Inside the embryo sac central cell, in the coinciding region, there are transfer cell-like wall projections with plasma membranes appressed to their inner surfaces. H⁺-ATPase is present along the inner surfaces of the wall projections as indicated by reactivity with antibody raised against plasma membrane H⁺-ATPase. A number of mitochondria are in close association with wall projections in the region of the egg apparatus. Antibody raised against corn plasma membrane H⁺-ATPase cross reacts with a protein of the same size in extracts of Brassica napus indicating that the two species contain a similar plasma membrane H⁺-ATPase.     

Subcellular distribution of chromium in accumulating plant Leersia hexandra Swartz

The subcellular distribution of chromium in Leersia hexandra Swartz, a Cr-accumulating plant found in China, was studied by differential centrifugation, transmission electron microscope and energy dispersive analysis of X-ray. Subcellular fractionation of Cr-containing tissues showed that most of the accumulated Cr was isolated to the cell walls in roots and the vacuoles in leaves. When the plant was grown in a nutrient solution containing 60 mg L^?1 Cr, 83.2% of the root Cr was localized in the cell wall fraction, while 57.5% of leaf Cr was localized in the vacuole and cytoplasm fraction. Transmission electron microscopic analysis revealed that those cell compartments contained especially electron dense areas. Energy dispersive X-ray spectra showed the electron dense areas contained high Cr. However, the dark electron precipitates were never observed in the plant cells without Cr treatment. In all treatment groups (5, 30 and 60 mg L^?1), the fraction containing the lowest level of Cr was the organelle fraction in roots as well as leaves. These results indicated that Cr accumulated in the L. hexandra was preferentially stored in the cell walls of roots and the vacuoles of leaves. This phenomenon diverted Cr ions from metabolically active compartment (chloroplast, mitochondria), resulting in a reduction of Cr toxicity in the plant cell.     

Interspecific transfer of isolated plant mitochondria by microinjection

Summary Data on isolation, purification and transfer of mitochondria from a cytoplasmic male sterile line of the Ogura type of Raphanus sativus to a male fertile line of Brassica napus are reported. Microinjection has been used for the transfer of the donor mitochondria to the recipient protoplasts. The injected protoplasts were identified and followed individually throughout their development using a computerized microscope stage which greatly enhanced the number of injections (five-fold). The transferred donor mitochondria were stably maintained during several successive cell divisions, revealing that they were viable and functional. Several calluses were obtained from injected protoplasts without using any selection pressure. Restriction fragment length analysis of seventeen calluses, using mitochondrial DNA probes, indicated that three contained the donor Ogura type mitochondria. No recombinant types of mitochondria have been observed. Flow cytometric and karyotype analyses of the calluses revealed the presence of similar amount of DNA and chromosome number as those of the recipient plants of B.napus. The application of microinjection for the manipulation of cytoplasmic composition is discussed.Abbreviations ATPase adenosine triphosphate phosphohydrolase - BSA bovine serum albumin - CMS cytoplasmic male sterility - DIOC6(3) 3,3-dihexyloxacarbocyanine-iodide - mt mitochondria(I) - PEG polyethylene glycol - RFLP restriction fragment length polymorphism - UV ultraviolet     

Effect of Temperature on Respiration of Mitochondria and Shoot Segments from Cold hardened and Nonhardened Wheat and Rye Seedlings

The effect of temperature on respiration of mitochondria and tissue segments from three wheat (Triticum aestivum L.) and one rye (Secale cereale L.) cultivar grown at 2 and 24 C has been examined. Discontinuities in Arrhenius plots of respiratory activity against temperature were observed for mitochondria and tissue segments from seedlings grown at both temperatures. The rates of respiration decreased abruptly below the transition temperatures, resulting in increased energy of activation values for respiration. Transition temperatures were observed from 6 to 10 C during tissue segment respiration, and from 10 to 14 C during respiration by isolated mitochondria. Respiratory control and efficiency of phosphorylation were not affected markedly by either reaction temperature or growth temperature of the seedlings. No correlation was observed between the cold hardiness of the cultivars and the temperature at which structural transitions occurred in the mitochondria. Dry matter content of the seedlings increased markedly during growth at 2 C, but no appreciable changes in the levels of mitochondrial protein were observed. The results support the view that changes other than fatty acid unsaturation are involved in the abrupt change in mitochondrial membrane properties at low temperature.     

In vitro Propagation and Isozyme Polymorphism of the Medicinal Plant Hypericum brasiliense

Responses of 20 d-old plants of two Brassica napus L. cultivars Dunkeld and Cyclon to NaCl salinized soil [electrical conductivity 2.4 (control), 4.0, 8.0 or 12.0 dS m⁻¹] were examined. The salt tolerant line Dunkeld had significantly higher fresh and dry masses of shoots, and seed yield than salt sensitive line Cyclon in all salinities. The effect of salt stress on reduction in total leaf soluble sugars was markedly greater in Dunkeld as compared to that in Cyclon. No effect of salt stress was observed on leaf soluble proteins but there was a slight increase in total free amino acids of both cultivars. Leaf proline content increased markedly in both cultivars and Dunkeld had greater proline content than Cyclon at all salinities. Salt stress had no significant effect on seed oil content and erucic acid content of seed oil, however, content of glucosinolates in the seed meal increased and Cyclon had greater content of glucosinolates than Dunkeld. This revised version was published online in July 2006 with corrections to the Cover Date.     

Uptake of Exogenous DNA by Brassica napus Dry Seed Embryos via Cellular Permeabilization and Transient Expression of NPTII Activity

A simple method of exogenous DNA uptake by dry seed embryos has been examined which is evaluated. by transient gene expression. The uptake and expression of foreign DNA was tested employing a plasmid containing nptil gene and assaying, later, the enzyme activity. To facilitate the uptake of foreign DNA by dry seed embryos of Brassica napus ISN706, various permeabilizing agents, like dimethylsulphoxlde, toluene, Triton X-100 and saponin, were tested for their effectiveness in transiently permeabilizing the plasma membranes in the concerned tissues. The treatment of dry seed embryos with 1.0 mg/ml saponin for 45 min was found to be the most effective for uptake of plasmid DNA without adversely affecting embryo viability. The method was tested also with several other cultivars and also species, namely B. napus ISN 129, B. napus ISN 106, B. napus ISN 128, B. napus ISN 706, B. napus 8054, B. carinata, B. juncea, and B. oleracea. Transient gene expression was seen in all cases, indicating that the method may be relatively free of genotypic constraints for its wider application.