Saturday, May 27, 2006

Artemisia annua and Proteomics


Abiotic stresses such as temperature, drought and salinity puts the major hurdles in yield and production of the crops. As a matter of fact, A. annua L. has to face a definite phase of a season of very low temperature (below 10 0C) in the month of December/January mainly in northern and associated parts of India. Further, much (32-84%) of groundwater surveyed in different Indian states is rated either saline or alkali and found to substantially decrease the yield of crops including Artemisia annua L. (Qureshi et al., 2005), affecting plant growth and production of artemisinin. Crop yields start declining when pH of the soil solution exceeds 8.5 or ECe value goes above 4 dS m-1. At higher ECe values the crop yields are reduced so drastically that crop cultivation is not economical without soil amendments. Addition of salts to water lowers its osmotic potential, resulting in decreased availability of water to root cells. Salt stress thus exposes the plant to secondary osmotic stress, which implies that all the physiological responses, which are invoked by drought stress, can also be observed in salt stress. Fogle and Munns (1973) reported that osmotic restriction of root growth decreased the ability of wheat seedling to obtain nutrients from root medium. In addition to its injurious osmotic effects, a salt may injure plant by way of specific toxic effect. Salt stress causes inhibition of growth and development, reduction in photosynthesis, respiration, and protein synthesis and disturbs nucleic acid metabolism (Kaiser, 1987). Decrease in uptake of K+, Mg2+, Ca2+, and thereby decrease in growth at higher sodium concentration have also been reported.

Thus, the productivity and therapeutic properties of medicinal plants is subjected to the environmental and nutrient factors and hence, the ratio of demand and supply too.

Plants exposed to abiotic stress invariably showed marked alteration in the electron transport in both chloroplast and mitochondria. This results in the production of toxic oxygen species such as 1O2, OH and H2O2 (Foyer et al., 1997). Due to the multiplicity of the processes that produce excessive reactive oxygen species (ROS) during metabolism, including photosynthesis and photorespiration (Nocter and Foyer, 1998), fatty acid oxidation, response to pathogen attack and senescence; plants have developed a series of detoxification reaction mechanisms (Vitoria et al., 2001).

The impressive achievements in genome and expressed sequence tag (EST) sequencing have yielded a wealth of information for many model organisms, including the plants Arabidopsis thaliana and Medicago truncatula. Unfortunately, sequence information alone is insufficient to answer questions concerning gene function, developmental/regulatory biology, and the biochemical kinetics of life. To address these questions, more comprehensive approaches that include quantitative and qualitative analyses of gene expression products are necessary at the transcriptome, proteome, and metabolome levels. Transcriptome, approaches using microarray and serial analysis of gene expression technologies are powerful tools; however, mRNA and protein levels (Futcher et al., 1999; Gygi et al., 1999) cannot be correlated due inability of translation of total mRNA into protein. In contrast, proteomics provides a more direct assessment of biochemical processes of monitoring the actual proteins performing the enzymatic, regulatory, structural functions encoded by the genome and transcriptome. Recent improvement in high resolution two dimensional PAGE (2-DE; Klose and Kobalz, 1995; Gorge et al., 1999), increased content of protein and nucleotide databases, and increased capabilities for protein identification utilizing modern mass spectrometry methods such as matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS; Pappin et al., 1993; Yates, 1998a, 1998b; Corthals et al., 2000) have made the large-scale profiling and identification of proteins a dynamic new area of research in plant biology.

Although there is a substantial amount of work in literature on bacterial (Guerreiro et al., 1999; Morris and Djordevic, 2001), there is relatively less information on plant proteomes (Wan Wijk, 2001). Costa and co-workers have identified proteins from xylem and needles of maritime pine (Pinus pinaster; Costa et al., 1998, 1999), and Tsugita and co-workers have worked on the rice (Oriza sativa) proteome with some success (Tsugita et al., 1994). Both of these groups have relied heavily on Edman sequencing, which suffers due to the inability to sequence proteins blocked at the N terminus. More recently, researchers have reported on subcellular proteomes such as the chloroplast membrane (Peltier et al., 2000; 2002) whereas other have focused on single tissues including Arabidopsis seeds (Gallardo, et al., 2001), Arabidopsis mitochondria (Kruft et al., 2001; Millar et al., 2001), maize (Zea mays) root tips (Cheng et al., 2000), and barrel medic roots (Mathesius et al., 2001, 2002). To date, there has been no large-scale project to identify proteins from multiple tissues of the same plant species.

Being an important medicinal plant, Artemisia annua L. has been used as an experimental material in these studies.


Taking into consideration the above facts, this project has been planned with the following objectives:

*To grow cultures of Artemisia annua L. under in vitro conditions.

*To study the enzymatic and non-enzymatic antioxidant in Artemisia annua L. under salinity and heavy metal stress.

*To analyze the magnitude of expression of different proteins in shoot and their possible role in imparting the abiotic stress tolerance to Artemisia annua L.

*To sequence the amino acids of the stress-induced proteins.

Identify the stress-inducible genes under different abiotic stresses imparting tolerance against abiotic stresses.


Plant material and treatments

In vitro raised Artemisia annua L. plants will be exposed to NaCl and heavy metal with MS media in separate sets. Moderate salinity will be created by the application of NaCl in the nutrient media; drought by removal of the nutrient media for a standard set time; chilling and heat stress will be given by reducing and increasing the temperature of the growth chamber, respectively.

Protein extraction

Total protein of the tissue will be extracted according to the method of Tsugata et al., 1994. In brief, plant tissues will be homogenized in liquid N2 and proteins precipitated at –20 0C with 10% (w/v) TCA in acetone containing 0.07%(w/v) 2-mercaptoethanol for at least 45 min. The mixture will be centrifuged at 35,000g at 4 0Cfor 15 min, and the precipitates will be washed with acetone containing 0.07% (w/v) 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 2 mM EDTA. Pellets will be dried by vacuum centrifugation and solubilized in 8 M urea, 4% (w/v) CHAPS, 20 mM DTT, 0.1% (v/v) Biolytes (pH 3-10; Bio-Rad laboratories, Hercules, CA; Molloy et al., 1998).

Protein quantification and electrophoresis

Protein concentration of all tissue extracts will be quantified using the Bradford method (Bradford, 1976) with bovine serum albumin as a standard isoelectric focusing and acrylamide gel electrophoresis of will be done according to the method of Asirvatham et al., 2002. Gels will be stained over-night with Coomassie Brilliant Blue R-250 and destained the next day. Gel images will be digitized with UVI-tech gel documentation system equipped with image camera. Experimental molecular mass and Rf/pI will be calculated from digitized 2-DE images using standard molecular mass marker proteins.

Digestion and MALDI-TOFMS

Protein spots will be excised from the gel, washed twice with water for 15 min, and destained with a 1:1 (v/v) solution of acetonitrile and 50mM ammonium bicarbonate while changing solutions every 30 min until the blue colour of Coomassie was removed. SDS-PAGE/2-DE gel bands/spots will be then dehydrated by washing with 100% acetonitrile and dried by vacuum centrifugation. Further processing for sequencing of amino acids of the novel proteins will be done according the method of Watson et al., 2003.

Database queries and protein identifications

The peptide mass fingerprints will be compared with sequences in different databases such as NCBInr, SwissPort and/or dbESTothers, and queried using MS-Fit (


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