Exposure of biosynthesized nanoscale ZnO to Brassica juncea crop plant: morphological, biochemical and molecular aspects.

The present work describes the in vitro synthesis and characterization of Zinc oxide nanoparticles (ZnO NPs) using an enzyme alpha amylase, the synthesized nanoparticles were used to study their beneficial effect in the growth and development of Brassica juncea. Transmission Electron Microscope (TEM) image reveals the average size of ZnO NPs was 11 nm and X-ray powder diffraction (XRD) suggests nanoparticles were crystalline in nature. In-silico study confirmed lysine, glutamine and tyrosine present in alpha amylase enzyme, plays a crucial role in the reduction of Zinc acetate dihydrate to ZnO NPs. The biochemical parameters and oxidative enzymes of Brassica juncea were compared with ZnO NPs treated plants. The effect of ZnO NPs on the cellular expression of metal tolerant protein (BjMTP) and cation efflux transporter gene (BjCET2) was also studied. The results indicate that nanoparticles can be used as a replacement for traditional harmful chemical fertilizers.

A quartet pollen phenotype identified in a population of Brassica interspecific hybrids shows incomplete penetrance and variable response to temperature.

Quartet pollen, where pollen grains remain attached to each other post-meiosis, is useful for tetrad analysis, crossover assessment and centromere mapping. We observed the quartet pollen phenotype for the first time in the agriculturally significant Brassica genus, in an experimental population of allohexaploid Brassica hybrids derived from the cross (Brassica napus × B. carinata) × B. juncea followed by two self-pollination generations. Quartet pollen production was assessed in 144 genotypes under glasshouse conditions, following which a set of 16 genotypes were selected to further investigate the effect of environment (warm: 25 °C and cold: 10 °C temperatures) on quartet pollen production in growth cabinets. Under glasshouse phenotyping conditions, only 92 out of 144 genotypes produced enough pollen to score: of these, 30 did not produce any observable quartet pollen, while 62 genotypes produced quartet pollen at varying frequencies. Quartet pollen production appeared quantitative and did not clearly fall into phenotypic or qualitative categories indicative of major gene expression. No consistent effect of temperature on quartet pollen production was identified, with some genotypes producing more and some producing less quartet pollen under different temperature treatments. The genetic heterogeneity and frequent pollen infertility of this population prevents strong conclusions being made. However, it is clear that the quartet phenotype in this Brassica population does not show complete penetrance and shows variable (likely genotype-specific) response to temperature stress. In future, identification of quartet phenotypes in Brassica would perhaps best be carried out via screening of diploid (e.g. B. rapa) TILLING populations.

Subcellular distribution and chemical forms of thorium in Brassica juncea var. foliosa.

Brassica juncea var. foliosa (B. juncea var. foliosa) is a promising species for thorium (Th) phytoextraction due to its large biomass, fast growth rate and high tolerance toward Th. To further understand the mechanisms of Th tolerance, the present study investigated the subcellular distribution and chemical forms of Th found in B. juncea var. foliosa Our results indicated that in both roots and leaves, Th contents in different parts of the cells follow the order of cell wall > membranes and soluble fraction > organelles. In particular, Transmission Electron Microscope (TEM) analysis showed that Th was abundantly located in cell walls of the roots. Additionally, when plants were exposed to different concentrations of Th, we have found that Th existed in B. juncea var. foliosa with different chemical forms. Much of the Th extracted by 2% acetic acid (HAc), 1 M NaCl and HCl in roots with the percentage distribution varied from 47.2% to 62.5%, while in leaves, most of the Th was in the form of residue and the subdominant amount of Th was extracted by HCl, followed by 2% HAc. This suggested that Th compartmentation in cytosol and integration with phosphate or proteins in cell wall might be responsible for the tolerance of B. juncea var. foliosa to the stress of Th.

Silver and gold nanoparticles in plants: sites for the reduction to metal.

Induced formation of metal nanoparticles in living plants is poorly understood. The sites for the reduction of Ag(+) and Au(3+) to Ag(0) and Au(0) metal nanoparticles in vivo in plants were investigated in order to better understand the mechanism of the reduction processes. Brassica juncea was grown hydroponically, followed by growth in solutions of AgNO(3), [Ag(NH(3))(2)]NO(3) or HAuCl(4). Harvested plants were sectioned and studied by transmission electron microscopy. Total metal content was analysed by atomic absorption spectroscopy. The chemical state of the metals was determined by X-ray absorption spectroscopy. Nanoparticles of Ag(0) and Au(0) were found in leaves, stem, roots and cell walls of the plants at a concentration of 0.40% Ag and 0.44% Au in the leaves. Particles which were approximately spherical were formed with sizes of 2-100 nm. The sites of the most abundant reduction of metal salts to nanoparticles were the chloroplasts, regions of high reducing sugar (glucose and fructose) content. We propose that these sugars are responsible for the reduction of these metals and other metal salts with reduction potentials over +0.16 V and that the amount of reducing sugar present or produced determines the quantity of metal nanoparticles that may be formed.

Optimization of ultrasonic-stimulated solvent extraction of sinigrin from Indian mustard seed (Brassica Juncea L.) using response surface methodology.

INTRODUCTION: Sinigrin, a major glucosinolate present in Indian mustard (Brassica juncea L.) seeds as the precursor of the anticancer compound allyl isothiocyanate, shows a wide range of biological activities. It's necessary to optimize the extraction methods and conditions, in order to improve the extraction productivity and save raw material. OBJECTIVE: To systemically investigate and optimize the most important factors affected the productivity of sinigrin in the process of extraction using response surface methodology. METHODOLOGY: The ranges of three main factors including the ethanol concentration, extraction time and extraction temperature were selected by the one-factor-at-a-time method. The conditions of ultrasonic-stimulated extraction of sinigrin from defatted Indian mustard seed powder were optimized by Box-Behnken design to obtain the maximum productivity. RESULT: The predicted productivity (3.81%) was obtained using 57% ethanol concentration at 81 °C for 60 min, with the coefficient of the model R² > 0.96 (n = 17). The actual productivity (3.84 ± 0.02%) of sinigrin under the optimized condition was increased by 70.67% compared with the result of conventional extraction. Meanwhile, HPLC, UV and IR were applied to examine if there is a difference between the ultrasonic-stimulated solvent extraction and conventional extraction, and the improvement of productivity of sinigrin depended on the destruction of cell wall caused by the elimination of outer pectinous material was explained by SEM and composition content analysis. CONCLUSION: The ultrasonic-stimulated solvent extraction was suggested to be a promising method to improve the productivity of sinigrin. And the results demonstrated that sinigrin productivity may be related to pectinous materials existed in the seeds.

Phytotoxicity of mercury in Indian mustard (Brassica juncea L.).

This study investigated the phytotoxicity of mercury to Indian mustard (Brassica juncea L.). Two common cultivars (Florida Broad Leaf and Long-standing) were grown hydroponically in a mercury-spiked solution. Mercury exhibited a significant phytotoxicity in these two cultivars of Indian mustard at elevated concentrations (>or=2 mg L(-1)). Mercury uptake induced a significant reduction in both biomass and leaf relative water content. Microscopy studies indicated that elevated mercury concentrations in plants significantly changed leaf cellular structure: thickly stained areas surrounding the vascular bundles; decreases in the number of palisade and spongy parenchyma cells; and reduced cell size and clotted depositions. The palisade chloroplasts exhibited decreases in their amounts and starch grains as well as a loss of spindle shape. However, due to high accumulation of mercury in plants, especially in the roots, Indian mustard might be a potential candidate plant for phytofiltration of contaminated water and phytostabilization of mercury-contaminated soils.

Cadmium-induced plant stress investigated by scanning electrochemical microscopy.

In vivo oxygen evolution above single stomata in Brassica juncea has been used to investigate, for the first time, the effect of Cd-induced stress as imaged by scanning electrochemical microscopy (SECM). SECM images showed a clear stomatal structure-a pore, whose aperture is modulated by two guard cells, serving as the conduit for the oxygen produced. Lower stomatal density and larger stoma size were found in plants treated with 0.2 mM CdCl2 compared with control plants. Either the introduction of Cd caused a slower cell replication in the plane of the epidermis, hence fewer stomata, and/or the number of open stomata was reduced when plants were under Cd-stress. Oxygen evolution above individual stomatal complexes in Cd-treated plants was lower than that from control plants, as determined from the electrochemical current above the middle of each stoma. All guard cells under illumination were swollen, indicating that the stomata were open in both control and treated plants. Thus, decreased oxygen evolution in response to Cd cannot be attributed to simple closing of the stomata, but to a lower photosynthetic yield. SECM provides an excellent tool for monitoring the effects of Cd on photosynthetic activity at the scale of individual stomata.

Cellular evidence of allelopathic interference of benzoic acid to mustard (Brassica juncea L.) seedling growth.

Cellular changes in the roots of mustard (Brassica juncea L.) grown in soil treated with 1.09, 1.46 and 1.83 mg benzoic acid per g soil, a known allelochemical, were analyzed after 7 days. The recoverable concentration of 1.09, 1.46 and 1.8 mg benzoic acid per g soil (measured by high performance liquid chromatography) was 68, 150 and 250 microg benzoic acid per g soil, respectively. The benzoic acid treatments suppressed root growth by 30.5%, 58.8% and 81.1% with increasing concentrations. Transmission electron microscopy studies of roots showed irregular shaped cells arranged in disorganized manner and disruption of cell organelles at cellular level. Root cells showed dissolution of middle lamella (at 68 and 150 microg benzoic acid per g soil) but intact middle lamella with increased wall deposits was observed with 250 microg benzoic acid per g soil. Damage to the mustard root at cellular level was evidenced by changes in cell morphology and internal organization.

[The ultrastructure of Brassica junceae chloroplast as a criterion of salt resistance]. / Ul'trastruktura khloroplastov gorchitsy Brassica junceae kak pokazatel' solerezistentnosti.

Two salt resistant forms of the mustard Brassica junceae were obtained. These forms demonstrated no chlorophyll defects. SR2 and SR3 salt resistant forms have constant parameters of productivity in condition of NaCl salting, in comparison with the initial plants of sort Donskaya-5. Results of a comparative ultrastructural analysis of plastids show that the salt influences the plastid ultrastructure of SR2 and SR3 lines of mustard in a lesser degree in comparison with the sort plants. The plastids of SR2 and SR3 salt resistant lines of mustard are functionally quite active under stress conditions.

The frequency of plasmodesmata increases early in the whole shoot apical meristem of Sinapis alba L. during floral transition.

The frequency of plasmodesmata increases in the shoot apical meristem of plants of Sinapis alba L. induced to flower by exposure to a single long day. This increase is observed within all cell layers (L1, L2, L3) as well as at the interfaces between these layers, and it occurs in both the central and peripheral zones of the shoot apical meristem. The extra plasmodesmata are formed only transiently, from 28 to 48 h after the start of the long day, and acropetally since they are detectable in L3 4 h before they are seen in L1 and L2. These observations indicate that (i) in the Sinapis shoot apical meristem at floral transition, there is an unfolding of a single field with increased plasmodesmatal connectivity, and (ii) this event is an early effect of the arrival at this meristem of the floral stimulus of leaf origin. Since (i) the wave of increased frequency of plasmodesmata is 12 h later than the wave of increased mitotic frequency (A. Jacqmard et al. 1998, Plant cell proliferation and its regulation in growth and development, pp. 67 78; Wiley), and (ii) the increase in frequency of plasmodesmata is observed in all cell walls, including in walls not deriving from recent divisions (periclinal walls separating the cell layers), it is concluded that the extra plasmodesmata seen at floral transition do not arise in the forming cell plate during mitosis and are thus of secondary origin.

Separation of two classes of plastid DNA-dependent RNA polymerases that are differentially expressed in mustard (Sinapis alba L.) seedlings.

Chloroplast and etioplast in vitro transcription systems from mustard have different functional properties, which is reflected in differences in phosphorylation status. Here we report another transcription control mechanism, which involves two plastid DNA-dependent RNA polymerases designated as peak A and peak B enzymes. Both are large multi-subunit complexes, but differ in their native molecular mass (> 700 kDa for peak A and ca. 420 kDa for peak B) and in their polypeptide composition. The A enzyme is composed of at least 13 polypeptides, while the B enzyme contains only four putative subunits. Peak B activity is inhibited by rifampicin, whereas that of peak A is resistant. RNA polymerase activity was compared for plastids from cotyledons of 4-day-old seedlings that were grown either under continuous light (chloroplasts) or in darkness (etioplasts), or were first dark-grown and then transferred to light for 16 h ('intermediate-type' plastids). While the total activity was approximately the same in all three cases, enzyme B was the predominant activity obtained from etioplasts and enzyme A that obtained from chloroplasts. Both had equal activity in preparations from the 'intermediate-type' plastid form. Both activation/inactivation and differential gene expression seem to play a role in the regulation of the plastid transcription machinery.

Nucleolar activation and vacuolation in embryo radicle cells during early germination.

The activation of the nucleolus of primary root cells of Sinapis alba embryos during the first 72 h of germination was monitored by autoradiographic, ultrastructural and microstereological methods. Autoradiographs showed that within 48 h, the nucleolus progressively resumed the capacity to synthesize pre-rRNA molecules at a high rate. In quiescent embryos the nucleolus was small, compact and composed of mixed granular and fibrillar components. Within the first 6 h of germination a strong nucleolar vacuolation occurred, accompanied by a decrease in the volume of the nucleolus and a concomitant high loss of its ribonucleoproteins (RNPs). From 6 to 24 h, nucleolar vacuolation decreased to reach a stable level. During this last period the volume of the nucleolus increased by the accumulation of the fibrillar component resulting from a slow pre-rRNA processing. At 24 h the nucleolus presented a predominantly fibrillar texture. After 24 h, nucleolus growth continued but was due to the accumulation of the granular component, indicating that pre-rRNA processing occurred at a higher rate than during the first day of germination. From 48 h the nucleolus was composed of well-delineated granular and fibrillar areas. Dense nucleolus-associated chromatin as well as fibrillar centres were always observed during the whole period of observation. In addition, previous studies on the nucleolus of radicle cells of Zea mays embryo during early germination were completed by studying changes in the nucleolar volume and in the density of pre-ribosomal subunits of the granular component. On the basis of the data obtained with both species we suggest that a possible function for the nucleolar vacuoles is the increase in the nucleolus-nucleoplasm exchange interface in response to a rapid increase in the output of nucleolar RNPs. The nucleolar growth pattern during early germination is also discussed.

Transitory development of rough endoplasmic reticulum aggregates during embryo maturation in seeds of mustard (Sinapis alba L.).

During the final period of maturation of mustard (Sinapis alba L.) seeds conspicuous aggregates of rough endoplasmic reticulum are found specifically in some tissues of the differentiation zone of the radicle. The appearance of these structures is temporally correlated with the disappearance of single-stranded reticulum and the onset of seed dehydration. These aggregates can be demonstrated also in the dry, mature seed and during the first few hours after imbibition with water; they disappear however during germination. In germinated root tips reformation of the aggregates can be induced by severe water stress. It is concluded that the observed membrane aggregates represent a storage form of rough endoplasmic reticulum during periods of low protoplasmic hydration.