Study of genetic modifications of flower development and methylation status in phytoplasma infected Brassica (Brassica rapa L.).

BACKGROUND: The plants of B. rapa (syn. B. campestris) are the most important food crop of Pakistan for the production of cooking oil. Brassica plants infected by phytoplasma exhibit floral abnormalities including phyllody, virescence, hypertrophied sepal and aborted reproductive organs and affected flower developmental genes which reduces the yield manifold. METHODS AND RESULTS: The expression level of flower developmental genes in healthy and phytoplasma infected brassica were compared by using semi-quantitative reverse transcription polymerase chain reaction and DNA hybridization. In infected brassica, LEAFY (LFY) gene, controlling the development and maintenance of floral organ, and directly involved in controlling the homeotic gene expression was affected, while APETALA2, regulate the production of sepals and petals, were not altered. Whereas the genes WUSCHEL, APETALA3 and AGAMOUS, were significantly down-regulated, that were responsible for the identity of shoot and central meristem, petals and stamens production, and stamens and carpels development, respectively. The GLUB gene, controlling the production of ß-1,3-glucanases enzyme, was highly up-regulated. According to DNA hybridization results, AGAMOUS and APETALA3 were restricted to floral organs territories in healthy and phytoplasma infected brassica, indicating that their expression was tissue-specific. These outcomes indicated that flower abnormalities of phytoplasma infected B. rapa are linked with DNA methylation in the expression of homeotic genes regulating flower development. CONCLUSIONS: Azacitidine act as a DNA demethylating reagent. By applying the foliar spray of azacitidine during the flower development, cells of Phytoplasma infected plants exhibits demethylation of DNA when treated with azacitidine chemical that incorporated as analogue of cytosine during the cell division stage. B. rapa showed the up-regulation of gene expression level significantly that restore the normal production of flowers, ultimately increase the oil production throughout the world.

Characterization of Ocimum basilicum synthesized silver nanoparticles and its relative toxicity to some insecticides against tobacco cutworm, Spodoptera litura Feb. (Lepidoptera; Noctuidae).

Tobacco cutworm, Spodoptera litura Feb. (Lepidoptera; Noctuidae) is a notorious cosmopolitan pest of numerous crops. Frequent evolution of insecticide resistance and polyphagous nature favors the survival of this voracious pest. Nanotechnology offers an alternative technique to overcome the limitations of existing pest management strategies. In the present study, the silver nanoparticles were synthesized from Ocimum basilicum leaf extract and UV-spectra at 464.17 nm confirmed their synthesis while crystalline nature of nanoparticles was evaluated by X-rays diffraction (XRD) pattern and scanning electron microscopy (SEM) revealed their octagonal to spherical shape. Insecticidal potential of O. basilicum synthesized silver nanoparticles (ObAgNPs) was evaluated in comparison with some synthetic insecticides owning different mode of actions, including Coragen®, Proclaim®, Tracer® and Talstar® against S. litura. Probit analysis of 2nd, 3rd, 4th and 5th instars of S. litura showed that lethal concentrations (LCs) of tested nanoparticles and pesticides were positively correlated with older instars. All values of LC50, LC90 and LC95 were statistically different from each other with maximum on the 4th instar and minimum on 2nd instar of S. litura. Tukey's HSD test revealed that ObAgNPs caused maximum mortality (21.67-96.67%) in 2nd instar larvae at the rate of 100-1500 mg/L followed by Coragen® (18.33-91.67%), Proclaim® (13.33-78.33%) and Talstar® (13.33-68.33%), while Tracer indicated the lowest larval mortality (11.67-66.67%) at the rate of 60-120 mg/L and a similar trend was assessed for all the successive instars of S. litura. Higher activity of carboxyl esterase and glutathione-s-transferases confirmed the development of insecticide resistance against the synthetic chemicals. Conclusively, ObAgNPs were found to be eco-friendly but the most effective biogenic compounds for the suppression of S. litura population as compared to the selected synthetic chemicals which may be hazardous for the environment as well as human health. This study will assist the pesticide industries to re-standardize the dose rates against Spodoptera litura.

The Imaging of Guard Cells of thioglucosidase (tgg) Mutants of Arabidopsis Further Links Plant Chemical Defence Systems with Physical Defence Barriers.

The glucosinolate-myrosinase system is a well-known plant chemical defence system. Two functional myrosinase-encoding genes, THIOGLUCOSIDASE 1 (TGG1) and THIOGLUCOSIDASE 2 (TGG2), express in aerial tissues of Arabidopsis. TGG1 expresses in guard cells (GCs) and is also a highly abundant protein in GCs. Recently, by studying wild type (WT), tgg single, and double mutants, we showed a novel association between the glucosinolate-myrosinase system defence system, and a physical barrier, the cuticle. In the current study, using imaging techniques, we further analysed stomata and ultrastructure of GCs of WT, tgg1, tgg2 single, and tgg1 tgg2 double mutants. The tgg mutants showed distinctive features of GCs. The GCs of tgg1 and tgg1 tgg2 mutants showed vacuoles that had less electron-dense granular material. Both tgg single mutants had bigger stomata complexes. The WT and tgg mutants also showed variations for cell wall, chloroplasts, and starch grains of GCs. Abscisic acid (ABA)-treated stomata showed that the stomatal aperture was reduced in tgg1 single and tgg1 tgg2 double mutants. The data provides a basis to perform comprehensive further studies to find physiological and molecular mechanisms associated with ultrastructure differences in tgg mutants. We speculate that the absence of myrosinase alters the endogenous chemical composition, hence affecting the physical structure of plants and the plants' physical defence barriers.

Arabidopsis myrosinases link the glucosinolate-myrosinase system and the cuticle.

Both physical barriers and reactive phytochemicals represent two important components of a plant's defence system against environmental stress. However, these two defence systems have generally been studied independently. Here, we have taken an exclusive opportunity to investigate the connection between a chemical-based plant defence system, represented by the glucosinolate-myrosinase system, and a physical barrier, represented by the cuticle, using Arabidopsis myrosinase (thioglucosidase; TGG) mutants. The tgg1, single and tgg1 tgg2 double mutants showed morphological changes compared to wild-type plants visible as changes in pavement cells, stomatal cells and the ultrastructure of the cuticle. Extensive metabolite analyses of leaves from tgg mutants and wild-type Arabidopsis plants showed altered levels of cuticular fatty acids, fatty acid phytyl esters, glucosinolates, and indole compounds in tgg single and double mutants as compared to wild-type plants. These results point to a close and novel association between chemical defence systems and physical defence barriers.