In vitro shortening of generation time in Arabidopsis thaliana

Shortening of generations is very important for gene validation and a rapid exploitation of genetic novelties. Even if the life cycle of Arabidopsis thaliana is theoretically short, shortening it further would be useful. We developed a strategy where seeds germinated on medium with picloram (0.1 mg/l) and benzylaminopurine (0.5 mg/l) set seed about 40–45 days after sowing. Green seeds from immature siliques (second generation) are sown on half-strength, hormone-free MS medium where they flower and fruit much faster than the first generation (between 20 [C24] and 26 [Col] days/generation). A cold treatment was not needed for germination of immature seeds, and the duration of each cycle could thus be halved compared with the first generation. This strategy, allowing comfortably more than 10 generations per year, may be helpful for genetic studies using this model plant species.     

Carbonate extraction process for the metabolic, isozymic and proteomic profiling of rose-scented geranium (Pelargonium sp.), a hyper-acidic plant

Rose-scented geranium (Pelargonium sp.) is a valuable monoterpene-yielding plant. It has been well characterised phytochemically through the isolation of >270 secondary metabolites, however, there is hardly any biochemical or metabolic information concerning this plant. Initial attempts to investigate its metabolism failed to produce any enzyme activity in the tissue extracts prepared in routine extraction buffers owing to the intrinsic properties of the tissue matrix. It was recognised that cellular hyper-acidity (cell sap pH approximately 3.0) gave rise to very low protein levels in the extracts, thus prohibiting detection of activities of even primary metabolic enzymes that are usually abundantly present in plants. Tissue extraction in Tris solution without pH adjustment (as used for studies involving citrus and banana) led to little or no improvement. Therefore, a novel approach using sodium carbonate solution as an efficient extraction system for enzymes and proteins from the plant was studied. Functionality of the carbonate extraction has been demonstrated through its effectiveness, a several-fold superior performance, in yielding protein, monitoring primary metabolism and secondary metabolic enzymes, and isozymic and polypeptide profiling. The process may also be helpful in the reliable analysis of other acidic plant tissues.     

Vermiconversion of industrial sludge for recycling the nutrients

The aim of the present study was to investigate the transformation of sugar mill sludge (PM) amended with biogas plant slurry (BPS) into vermicompost employing an epigeic earthworm Eisenia fetida. To achieve the objectives experiments were conducted for 13 weeks under controlled environmental conditions. In all the waste mixtures, a decrease in pH, TOC, TK and C:N ratio, but increase in TKN and TP was recorded. Maximum worm biomass and growth rate was attained in 20% PM containing waste mixture. It was inferred from the study that addition of 30-50% of PM with BPS had no adverse effect on the fertilizer value of the vermicompost as well as growth of E. fetida. The results indicated that vermicomposting can be an alternate technology for the management and nutrient recovery from press mud if mixed with bulking agent in appropriate quantities.     

Withanolide A is inherently de novo biosynthesized in roots of the medicinal plant Ashwagandha (Withania somnifera)

Ashwagandha ( Withania somnifera Dunal., Solanaceae) is one of the most reputed medicinal plants of Ayurveda, the traditional medical system. Several of its traditionally proclaimed medicinal properties have been corroborated by recent molecular pharmacological investigations and have been shown to be associated with its specific secondary metabolites known as withanolides, the novel group of ergostane skeletal phytosteroids named after the plant. Withanolides are structurally distinct from tropane/nortropane alkaloids (usually found in Solanaceae plants) and are produced only by a few genera within Solanaceae. W. somnifera contains many structurally diverse withanolides in its leaves as well as roots. To date, there has been little biosynthetic or metabolism-related research on withanolides. It is thought that withanolides are synthesized in leaves and transported to roots like the tropane alkaloids, a group of bioactive secondary metabolites in Solanaceae members known to be synthesized in roots and transported to leaves for storage. To examine this, we have studied incorporation of ¹⁴C from [2-¹⁴C]-acetate and [U-¹⁴C]-glucose into withanolide A in the in vitro cultured normal roots as well as native/orphan roots of W. somnifera . Analysis of products by thin layer chromatography revealed that these primary metabolites were incorporated into withanolide A, demonstrating that root-contained withanolide A is de novo synthesized within roots from primary isoprenogenic precursors. Therefore, withanolides are synthesized in different parts of the plant (through operation of the complete metabolic pathway) rather than imported.     

Plastid ultrastructure and DNA related to albinism in androgenetic embryos of various barley (Hordeum vulgare L.) cultivars

In order to understand the occurrence of albinism during androgenesis in barley, a number of plastid parameters were analyzed in microspore-derived embryos and androgenetic plantlets, and the results were compared in albino and non-albino producing cultivars. In the winter cv. Igri, plastids in microspore-derived embryos are characterized by numerous divisions, differentiated thylakoids, low amount of starch and a high DNA content examined by immunoelectron microscopy. After regeneration, the androgenetic plantlets were mostly chlorophyllous. In contrast, in the spring lines tested, the plastids of microspore-derived embryos were rarely dividing amyloplasts in which thylakoids and DNA were scarce and albino plantlets were mainly regenerated. After 2 weeks on the regeneration medium, plastids of Igri chlorophyllous androgenetic plantlets were typical chloroplasts, whereas in spring lines plastids of albino androgenetic plantlets were proplastids with the same characteristics as those in the corresponding microspore-derived embryos. These results strongly suggest that the origin of androgenetic albinism differs in winter and spring cvs.: in the winter cv. Igri plastid alteration may take place during the regeneration step of androgenesis whereas in the tested spring lines plastids are already affected in the microspore-derived embryos meaning that albinism is not initiated during regeneration but originates earlier during the androgenetic process likely as early as the sampling stage.     

Integration and expression of Sorghum C4 phosphoenolpyruvate carboxylase and chloroplastic NADP-malate dehydrogenase separately or together in C3 potato plants

We have integrated two cDNAs expressing Sorghum photosynthetic phosphoenolpyruvate carboxylase (C₄-PEPC) and NADP-malate dehydrogenase (cpMDH), two key enzymes involved in the primary carbon fixation pathway of NADP-malic enzyme-type C₄ plants, separately or together into a C₃ plant (potato). Analysis of the transgenic plants showed a 1.5-fold increase in PEPC and cpMDH activities compared to untransformed plants. Immunolocalization confirmed an increase at the protein level of these two enzymes in the transgenic plants and indicated that the Sorghum cpMDH was specifically addressed to the chloroplasts of potato mesophyll cells. However, integration of either or both of the cDNAs into the potato genome did not appear to significantly modify either tuber starch grain content or the rate of photosynthetic O₂ production compared to control untransformed plants. The low level of transgene expression probably explains the lack of influence on carbon metabolism and photosynthetic rates. This general observation suggests that some complex mechanism may regulate the level of production of foreign C₄ metabolism enzymes in C₃ plants.     

Aerobic growth and respiration of a delta-aminolevulinic acid synthase (hemA) mutant of Bradyrhizobium japonicum.

Oxygen-dependent growth of the Bradyrhizobium japonicum hemA mutant MLG1 (M.L. Guerinot and B.K. Chelm, Proc. Natl. Acad. Sci. USA 83:1837-1841, 1986) was demonstrated in cultured cells in the absence of exogenous delta-aminolevulinic acid (ALA), but growth of analogous mutants of Rhizobium meliloti or of Escherichia coli was not observed unless ALA was added to the yeast extract-containing media. No heme could be detected in extracts of strain MLG1 cells as measured by the absorption or by the peroxidase activity of the heme moiety, but the rates of growth and endogenous respiration of the mutant were essentially identical to those found in the parent strain. A role for ALA in the viability of strain MLG1 could not be ruled out since the ALA analog levulinic acid inhibited growth, but neither ALA synthase nor glutamate-dependent ALA synthesis activity was found in the mutant. The data show that the cytochromes normally discerned in wild-type B. japonicum cultured cells by absorption spectroscopy are not essential for aerobic growth or respiration.     

Comprehensive metabolic fingerprinting of Withania somnifera leaf and root extracts

Profiling of metabolites is a rapidly expanding area of research for resolving metabolic pathways. Metabolic fingerprinting in medicinally important plants is critical to establishing the quality of herbal medicines. In the present study, metabolic profiling of crude extracts of leaf and root of Withania somnifera (Ashwagandha), an important medicinal plant of Indian system of medicine (ISM) was carried out using NMR and chromatographic (HPLC and GC-MS) techniques. A total of 62 major and minor primary and secondary metabolites from leaves and 48 from roots were unambiguously identified. Twenty-nine of these were common to the two tissues. These included fatty acids, organic acids, amino acids, sugars and sterol based compounds. Eleven bioactive sterol-lactone molecules were also identified. Twenty-seven of the identified metabolites were quantified. Highly significant qualitative and quantitative differences were noticed between the leaf and root tissues, particularly with respect to the secondary metabolites.