Rashmi Rai

Proteomics combines morphological, physiological and biochemical attributes to unravel the survival strategy of Anabaena sp. PCC7120 under arsenic stress

Proteomics in conjunction with morphological, physiological and biochemical variables has been employed for the first time to unravel survival strategies of the diazotrophic cyanobacterium Anabaena sp. PCC7120 under Arsenic (As) stress. Significant reduction in growth, carbon fixation, nitrogenase activity and chlorophyll content after 1 day (1d) and recovery after 15 days (15d) of As exposure indicates the acclimation of the test organism against As stress. The formation of akinete like structures is a novel observation never reported before in Anabaena sp. PCC7120. Proteomic characterization using 2-DE showed average 537, 422 and 439 spots in control, 1 and 15d treatment respectively. MALDI-TOF and LC-MS of As-treated Anabaena revealed a total of 45 differentially expressed proteins, of which 13 were novel (hypothetical) ones. Down-regulation of phosphoglycerate kinase (PGK), fructose bisphosphate aldolase II (FBA II), fructose 1,6 bisphosphatase (FBPase), transketolase (TK), and ATP synthase on day 1 and their significant recovery on the 15th day presumably maintained the glycolysis, pentose phosphate pathway (PPP) and turnover rate of Calvin cycle, hence survival of the test organism. Up-regulation of catalase (CAT), peroxiredoxin (Prx), thioredoxin (Trx) and oxidoreductase appears to protect the cells from oxidative stress. Appreciable induction in phytochelatin content (2.4 fold), GST activity (2.3 fold), and transcripts of phytochelatin synthase (5.0 fold), arsenate reductase (8.5 fold) and arsenite efflux genes - asr1102 (5.0 fold), alr1097 (4.7 fold) reiterates their role in As sequestration and shielding of the organism from As toxicity. While up-regulated metabolic and antioxidative defense proteins, phytochelatin and GST work synchronously, the ars genes play a central role in detoxification and survival of Anabaena under As stress. The proposed hypothetical model explains the interaction of metabolic proteins associated with the survival of Anabaena sp. PCC7120 under As stress.

UV-B and UV-C pre-treatments induce physiological changes and artemisinin biosynthesis in Artemisia annua L. - an antimalarial plant.

Present study was undertaken to investigate if short-term UV-B (4.2 kJ m(-2) day(-1)) and UV-C (5.7 kJ m(-2) day(-1)), pre-treatments can induce artemisinin biosynthesis in Artemisia annua. Twenty-one day old Artemisia seedlings were subjected to short-term (14 days) UV pre-treatment in an environmentally controlled growth chamber and then transplanted to the field under natural conditions. Treatment of A. annua with artificial UV-B and UV-C radiation not only altered the growth responses, biomass, pigment content and antioxidant enzyme activity but enhanced the secondary metabolites (artemisinin and flavonoid) content at all developmental stages as compared to non-irradiated plants. The extent of oxidative damage was measured in terms of the activities of enzymes such as catalase, superoxide dismutase and ascorbate peroxidase. Reinforcement in the antioxidative defense system seems to be a positive response of plants in ameliorating the negative effects of UV-B and UV-C radiations. While the carotenoid content was elevated, the chlorophyll content decreased under UV-B and UV-C pre-treatments. The reverse transcription PCR analysis of the genes associated in artemisinin/isoprenoid biosynthesis like 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), cytochrome P450 oxidoreductase (CPR) and amorpha-4,11-diene synthase (ADS) genes at different growth stages revealed UV induced significant over-expression of the above protein genes. UV-B and UV-C pre-treatments, led to an increase in the concentrations of artemisinin at full bloom stage by 10.5% and 15.7% than that of the control respectively. Thus, the result of our study suggests that short term UV-B pre-treatment of seedlings in greenhouse prior to transplantation into the field enhances artemisinin production with lesser yield related damages as compared to UV-C radiation in A. annua.

Enhanced Photosynthesis and Carbon Metabolism Favor Arsenic Tolerance in Artemisia annua, a Medicinal Plant as Revealed by Homology-Based Proteomics

This paper provides the first proteomic evidence of arsenic (As) tolerance and interactive regulatory network between primary and secondary metabolism in the medicinal plant, Artemisia annua. While chlorophyll fluorescence and photosynthetic rate depicted mild inhibition, there was a significant enhancement in PSI activity, whole chain, ATP, and NADPH contents in 100 μM As treatments compared to the control plants. However, a decrease in the above variables was recorded under 150 μM treatments. Proteomic decoding of the survival strategy of A. annua under As stress using 2-DE followed by MALDI-MS/MS revealed a total of 46 differentially expressed protein spots. In contrast to other plants where As inhibits photosynthesis, A. annua showed appreciable photosynthetic CO2 assimilation and allocation of carbon resources at 100 μM As concentration. While an increased accumulation of ATP synthase, ferredoxin-NADP(H) oxidoreductase, and FeS-rieske proteins supported the operation of cyclic electron transport, mdr ABC transporter protein and pcs gene might be involved in As detoxification. The most interesting observation was an increased accumulation of LEAFY like novel protein conceivably responsible for an early onset of flowering in A. annua under As stress. This study not only affirmed the role of energy metabolism proteins but also identified potential candidates responsible for As tolerance in plants.

Validation of SSR markers associated with rust (Uromyces fabae) resistance in pea (Pisum sativum L.).

Pea rust is a devastating disease of peas especially in the sub-tropical regions of the world and greatly influenced by the environmental conditions during disease development. Molecular markers associated with pea rust resistance would be useful in marker assisted selection (MAS). Utility of molecular markers associated with the pea rust resistance were evaluated in 30 diverse pea genotypes using four SSR markers (AA446 and AA505 flanking the major QTL Qruf; AD146 and AA416 flanking the minor QTL, Qruf1). QTL, Qruf flanking markers were able to identify all the resistant genotypes when used together, except Pant P 31. While, SSR markers AD146 and AA416 flanking the minor QTL, Qruf1 were able to identify all the pea resistant genotypes used for validation, except for HUDP-11 by AD146 and Pant P 31 by AA416. Similarly, SSR markers AA446 and AA505 were able to identify all the susceptible pea genotypes, except IPFD 99–13, HFP 9415 and S- 143. SSR markers AD146 and AA416 were together able to identify all the pea susceptible genotypes used for validation, except KPMR 526, KPMR 632 and IPFD 99–13. On the basis of marker allele analysis it may be concluded that SSR markers (AA446, AA505, AD146 and AA416) can be used in MAS of pea rust resistance.

Lignification and early abortive fungal colonies as indicators of partial resistance to rust in pea

Ten recombinant inbred lines (RILs) of pea were selected, on the basis of rust (Uromyces fabae) reaction under screenhouse conditions and molecular makers associated with rust resistance, to study the association of lignification and early abortive fungal colonies in response to attack of U. fabae. The present investigation indicated that partial resistance to rust in pea, as measured by the area under disease progress curve (AUDPC), is negatively influenced by lignification (r = −0.48). Histological studies indicated a higher number of early abortive fungal colonies and delayed development of colonies in resistant RILs than susceptible ones. Furthermore, investigations into the association of phenylalanine ammonia lyase activity and total phenolics during the partial resistance reaction to U. fabae in pea revealed a very low correlation with AUDPC in the RILs. This study infers that lignification plays a major role and is the best indicator of partial resistance towards U. fabae in pea, by influencing colony size and the number of early abortive colonies.

Biochemical and Molecular Basis of Arsenic Toxicity and Tolerance in Microbes and Plants

Arsenic is a ubiquitous toxic metalloid abundant in Earth’s crust. It is of major concern with respect to its increased accumulation in soils, in the food chain, or in drinking water. This chapter will focus on recent progress on the mechanisms of its uptake, toxicity, and detoxification in microbes and in planta. Due to widespread occurrence in nature, both microbes and plants have evolved a wide range of tolerance and detoxification mechanisms such as reduced uptake, immobilization, chelation, reduction/oxidation, methylation, and efflux. Among microbes, the ars operon is a well-characterized genetic system for arsenic detoxification. The mechanisms proposed for metal detoxification and hyperaccumulation within the plant involve chelation of the metal cation by ligands and binding with thiol groups or sequestration of metals away from sites or metabolism in the cytoplasm, notably into the vacuole or cell wall. Finally, this chapter will also shed light on hyperaccumulators and mechanisms of hyperaccumulation.