Impact of Some Toxic Metals on Important ABC Transporters in Soybean (Glycine max L.).

In plants, ATP-binding cassette (ABC) transporters facilitate the movement of substrates across membranes using ATP for growth, development, and defense. Soils contaminated with toxic metals such as cadmium (Cd) and mercury (Hg) might adversely affect the metabolism of plants and humans. In this study, a phylogenetic relationship among soybeans' (Glycine max) ATP binding cassette (GmABCs) and other plant ABCs was analyzed using sequence information, gene structure, chromosomal distribution, and conserved motif-domain. The ontology of GmABCs indicated their active involvement in trans-membrane transport and ATPase activity. Thirty-day-old soybean plants were exposed to 100 µM CdCl2 and 100 µM HgCl2 for 10 days. Physiological and biochemical traits were altered under stress conditions. Compared to Control, GmABC transporter genes were differentially expressed in response to Cd and Hg. The qRT-PCR data showed upregulation of seven ABC transporter genes in response to Cd stress and three were downregulated. On the other hand, Hg stress upregulated four GmABC genes and downregulated six. It could be concluded that most of the ABCB and ABCG subfamily members were actively involved in heavy metal responses. Real-time expression studies suggest the function of specific ABC transporters in Cd and Hg stress response and are helpful in future research to develop stress-tolerant varieties of soybean.

Trigonella foenum-graecum (fenugreek) differentially regulates antioxidant potential, photosynthetic, and metabolic activities under arsenic stress.

Arsenic (As) contamination is continuously increasing in the groundwaters and soils around the world causing toxicity in the plants with a detrimental effect on physiology, growth, and yield. In a hydroponic system, thirty-day-old plants of Trigonella foenum-graecum were subjected to 0, 50, or 100 µM NaHAsO40.7 H2O for 10 days. The magnitude of oxidative stress increased, whereas growth indices and photosynthetic parameters decreased in a dose-dependent manner. The efficiency of photosystem II in terms of Hill reaction activity (HRA) or chlorophyll-a was adversely affected by As stress. The antioxidant potential of plants regarding ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays was enhanced, indicating the augmented resistance mechanism in plants to counter As stress. The metabolite analysis of leaf extracts revealed many As responsive metabolites including amino acids, organic acids, sugars/polyols, and others. Phenylalanine and citrulline were highly accumulated at 50 or 100 µM As, salicylic acid accumulated more at 50 µM of As while ascorbic acid notably increased at 100 µM of As. At 50 or 100 µM As, the glucose and fructose contents increased while the sucrose content decreased. At both As doses, tagatose and glucitol contents were 13 times higher than controls. Varied accumulation of metabolites could be associated with the different As doses that represent the range of tolerance in T. foenum-graecum towards As toxicity. Pathway analysis of metabolites revealed that amino acid and carbohydrate metabolism and the citrate cycle play important roles under As stress. This study helps in a better metabolomic understanding of the dose-dependent toxicity and response of As in T. foenum-graecum.

Antimicrobial and antioxidant activities of fennel oil.

It is of interest to estimate the effects of foliar spray (ISA) on essential oil yield, chemical constituents, antioxidant, and antimicrobial activities of fennel (Foeniculum vulgare Mill).Fennel was treated with ISA solutions at 40 and 80 mg L-1 doses. Application of ISA significantly augmented antioxidant and antimicrobial activities in addition to essential oil yield and its principal elements in fennel. 80 mg L-1 dose of ISA was found to be pre-eminent. Antioxidant properties of EOs were determined through DPPH assays, metal chelators and lipid peroxidation. While antimicrobial activities were evaluated using agar well diffusion and microdilution techniques of broth. Gram positive and Gram negative bacteria were used to gauge the oil's antibacterial effectiveness. Data shows that antioxidant and antimicrobial activities of fennel oil were found to be the highest. According to GC analysis, trans-anethole (78.38-86.08%), methyl chavicol (2.32-2.54%), and fenchone (6.65-8.95%) were the three main constituents of fennel essential oil.

Triacontanol attenuates drought-induced oxidative stress in Brassica juncea L. by regulating lignification genes, calcium metabolism and the antioxidant system.

Effect of triacontanol on drought-induced stress was studied in Brassica juncea L. Foliage of sixteen-days-old plants was sprayed with concentrations (0, 10, 20 and 30 µM) of triacontanol (TRIA) for 7 days. Subsequently, plants were subjected to drought stress (10% polyethylene glycol, PEG6000) for 7 days. Drought stress increased oxidative stress (TBARS, O2●- and H2O2), however, their contents were reduced by TRIA. Total soluble sugars, reduced glutathione, and proline content in stressed plants were increased by TRIA. Activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), catalase (CAT), and phenylalanine ammonia-lyase (PAL) activity were significantly increased in a dose-dependent manner with TRIA. Potassium (K+) level declined, while magnesium (Mg2+) and calcium (Ca2+) contents increased. The elevated level of lignin under drought with TRIA was significantly associated with MYB46 and PAL gene expression patterns. Altogether, our results suggest that foliar spray of 20 µM TRIA was more operative in reducing the negative impact of drought stress in B. juncea by regulating the antioxidant system, calcium, and lignification.

Role of sulfur in combating arsenic stress through upregulation of important proteins, and in-silico analysis to study the interaction between phosphate transporter (PHO1), arsenic and phosphate in spinach.

An adequate amount of Sulfur (S) is essential for proper plant growth and defence against abiotic stresses including metals and metalloids. Arsenic (As) contamination is increasing in agricultural soils rapidly due to anthropogenic activities. Sulfur deficiency and arsenic stress could be more harmful than these individual stresses alone. To understand the impact of S-deficiency and arsenic (31 ppm Na3AsO4 of soil) on ecophysiology, growth, inorganic phosphate level, and proteomic profile of spinach, the present study was conducted. Interaction of arsenic with phosphate transporters, phytochelatins, and glutathione was also analyzed in silico. Comparative 2D MS/MS proteomics helped in the identification of important proteins which might be the key players under S-deficiency and As stress. Upregulation and downregulation of 36 and 21 proteins under As stress; 19 and 36 proteins under S-deficiency; 38 and 31 proteins under combined stress, respectively was observed. A total, 87 proteins subjected to identification via MS/MS ion search were found to be associated with important plant functions. PHO1 abundance was highly influenced by As stress; hence an in-silico homology modeling based molecular docking was performed which indicated high interaction between PHO1 and As/phosphate. Varied proximity of arsenic with phosphate transporters, phytochelatin, and glutathione revealed these components as a potential target of As toxicity/detoxification in Spinach, reflecting sulfur as an important criterion for arsenic tolerance.

Parthenium hysterophorus steps up Ca-regulatory pathway in defence against highlight intensities.

Parthenium hysterophorus exhibits tolerance to a great extent against abiotic stresses including high light intensities. In this study, P. hysterophorus was subjected to three different light intensities viz. control (CL, 250 µmol photons m-2 s-1), moderately high (ML, 500 µmol photons m-2 s-1) and high (HL, 1000 µmol photons m-2 s-1) for assessment of biochemical and physiological responses at 3 and 5 days after treatment (DAT). Proteomic responses were also observed at 5 DAT. Level of oxidative stress marker, abundance of H2O2 and O2- was highest in leaves exposed to HL followed by ML treatment. Biomass accumulation, photosynthetic parameters, chloroplast and mitochondrial integrity were also affected by both ML and HL treatments. Differential protein expression data showed modulation of thirty-eight proteins in ML and HL intensities. P. hysterophorus exhibited good ability to survive in ML then HL treatment as demonstrated by enhancement of the antioxidant system and photosynthesis. Furthermore, P. hysterophorus mobilized some key proteins related to calcium signaling, which in turn coordinate physiological homeostasis under stress. Proline and total soluble sugar content were high under stress; however, results of simulated experiment of our study indicate such accumulation of osmolytes may inhibit photon-availability to chloroplast. These results clarify our understanding of the mechanisms underlying the light stress tolerance of P. hysterophorus.

Organ-Specific Phytochemical Profiling and Antioxidant Analysis of Parthenium hysterophorus L.

Parthenium hysterophorus is a weed of global concern with high threshold of tolerance against most of biotic and abiotic stresses. Phytochemical profile and in vitro antioxidant analysis may help in understanding its tolerance to stresses. Root, stem, leaf, phyllary, and receptacle (including disc and ray florets) were chemotyped employing GC tof-MS and assessed for antioxidant activity by DPPH, FRAP, HRSA, and TAC assays. Phytochemicals identified were terpenes, fatty acids, hydrocarbons, phytosterols, and compounds of miscellaneous chemical nature. Organ-specific maximum concentration of metabolite was ß-vatirenene (root), hexadecanoic acid methylester (stem), aristolene epoxide (leaf), hexadecanoic acid methylester (phyllary), and hexadecanoic acid methylester (receptacle). Identified metabolites could be associated with stress tolerance mechanisms, basic metabolism, and allelopathy, etc. Root extracts showed highest antioxidant potential followed by receptacle. It can be concluded that diverse and unique phytochemical profile and great antioxidant potential make P. hysterophorus stress-tolerant, hence a weed of global habitat.

Comparative assessment of four RNA extraction methods and modification to obtain high-quality RNA from Parthenium hysterophorus leaf.

Isolation of high-quality RNA from weed plants such as Parthenium hysterophorus is a difficult task due to the hindrance caused by numerous secondary metabolites. Such metabolites not only affect the quality and yield of RNA, but also limit the quality of downstream applications. Therefore, the present study was undertaken to design a protocol for yielding RNA with better quality and quantity from P. hysterophorus leaf which could be suitable for functional genomics. To achieve the objective, four different important RNA extraction protocols, viz. acid guanidinium thiocyanate-phenol-chloroform, phenol-LiCl precipitation, TRIzol®, and PVP-ethanol were tested. The PVP-ethanol method proved to be best among the tested protocols. This method was further modified for obtaining improved quality and yield of RNA. The modified method successfully enhanced the yield of RNA from 280 to 334 µg g-1 fresh weight. The absorbance ratio (A260/A280) was in the purity range of 1.9 that indicated the good quality of RNA. To prove the feasibility of the extracted RNA in PCR-based cDNA synthesis, actin transcripts were targeted and successfully amplified using suitable primers. The improved protocol thus not only improved the yield and quality of RNA, but also gave better results in reverse transcriptase PCR.

Drought and salinity induced changes in ecophysiology and proteomic profile of Parthenium hysterophorus.

Parthenium hysterophorus is a plant that tolerates drought and salinity to an extremely high degree. Higher expression of stress-responsive proteome contributes for greater defence against abiotic stresses. Thus, P. hysterophorus could be a rich source of genes that encode stress-imparting mechanisms and systems. The present study utilizes comparative physiological and proteomic approaches for identification of key proteins involved in stress-defence of P. hysterophorus. Thirty-days-old plants were exposed to drought (10% PEG 6000) and salinity (160 mM NaCl) for 10 days duration. Both stresses induced oxidative stress estimated in terms of TBARS and H2O2. Levels of both enzymatic and non-enzymatic antioxidants were elevated, more by drought than salinity. Particularly, SOD, GR, CAT and GST proved to be assisting as very commendable defence under drought, as well as salinity. Levels of ascorbate, glutathione and proline were also increased by both stresses, more in response to drought. Comparative proteomics analysis revealed a significant change in relative abundance of 72 proteins under drought and salinity. Drought and salinity increased abundance of 45 and 41 proteins and decreased abundance of 24 and 26 proteins, respectively. Drought and salinity increased and decreased abundance of 31 and 18 proteins, respectively. The functions of identified proteins included those related to defence response (26%), signal transduction (13%), transcription and translation (10%), growth and development (8.5%), photosynthesis (8.5%), metabolism (7%), terpenoid biosynthesis (5.5%), protein modification and transport (7%), oxido-reductase (4%) and Miscellaneous (11%). Among the defence related proteins, antioxidants and HSPs constituted 26% and 21%, respectively. Present study suggests a potential role of defence proteins. Proteins involved in molecular stabilization, formation of osmolytes and wax and contributing to stress-avoiding anatomical features emerged as key and complex mechanisms for imparting stress tolerance to P. hysterophorus.

Proliferating effect of radiolytically depolymerized carrageenan on physiological attributes, plant water relation parameters, essential oil production and active constituents of Cymbopogon flexuosus Steud. under drought stress.

Carrageenan has been proved as potent growth promoting substance in its depolymerized form. However, relatively little is known about its role in counteracting the adverse effects of drought stress on plants. In a pot experiment, lemongrass (Cymbopogon flexuosus Steud.), grown under different water stress regimes [(100% field capacity (FC), 80% FC and 60% FC)], was sprayed with 40, 80 and 120 mg L-1 of gamma irradiated carrageenan (ICA). Foliar application of ICA mitigated the harmful effects of drought stress to various extents and improved the biochemical characteristics, quality attributes and active constituents (citral and geraniol) of lemongrass significantly. Among the applied treatments, ICA-80 mg L-1 proved the best in alleviating detrimental effects of drought. However, drought stress (80 and 60% FC), irrespective of the growth stages, had an adverse impact on most of the studied attributes. Generally, 60% FC proved more deleterious than 80% FC. At 80% FC, application of ICA-80 mg L-1 elevated the essential oil (EO) content by 18.9 and 25%, citral content by 7.33 and 8.19% and geraniol content by 9.2 and 8.9% at 90 and 120 days after planting (DAP), respectively, as compared to the deionized-water (DW) spray treatment (80% FC+ DW). Whereas, at 60% FC, foliar application of 80 mg L-1 ICA significantly augmented the EO content by 15.4 and 17.8% and active constituents viz. citral and geraniol, by 5.01 and 5.62% and by 6.06 and 5.61% at 90 and 120 DAP, respectively, as compared to the control (water-spray treatment).

A novel multicopper oxidase (laccase) from cyanobacteria: Purification, characterization with potential in the decolorization of anthraquinonic dye.

A novel extracellular laccase enzyme produced from Spirulina platensis CFTRI was purified by ultrafiltration, cold acetone precipitation, anion exchange and size exclusion chromatography with 51.5% recovery and 5.8 purification fold. The purified laccase was a monomeric protein with molecular mass of ~66 kDa that was confirmed by zymogram analysis and peptide mass fingerprinting. The optimum pH and temperature of the enzyme activity was found at 3.0 and 30°C using ABTS as substrate but the enzyme was quite stable at high temperature and alkaline pH. The laccase activity was enhanced by Cu+2, Zn+2 and Mn+2. In addition, the dye decolorization potential of purified laccase was much higher in terms of extent as well as time. The purified laccase decolorized (96%) of anthraquinonic dye Reactive blue- 4 within 4 h and its biodegradation studies was monitored by UV visible spectra, FTIR and HPLC which concluded that cyanobacterial laccase can be efficiently used to decolorize synthetic dye and help in waste water treatment.

Changes in rubisco, cysteine-rich proteins and antioxidant system of spinach (Spinacia oleracea L.) due to sulphur deficiency, cadmium stress and their combination.

Sulphur (S) deficiency, cadmium (Cd) toxicity and their combinations are of wide occurrence throughout agricultural lands. We assessed the impact of short-term (2 days) and long-term (4 days) applications of cadmium (40 µg/g soil) on spinach plants grown on sulphur-sufficient (300 µM SO42-) and sulphur-deficient (30 µM SO42-) soils. Compared with the control (+S and -Cd), oxidative stress was increased by S deficiency (-S and -Cd), cadmium (+S and +Cd) and their combination stress (-S and +Cd) in the order of (S deficiency) < (Cd stress) < (S deficiency and +Cd stress). SDS-PAGE profile of leaf proteins showed a high vulnerability of rubisco large subunit (RbcL) to S deficiency. Rubisco small subunit (RbcS) was particularly sensitive to Cd as well as dual stress (+Cd and -S) but increased with Cd in the presence of S. Cysteine content in low molecular weight proteins/peptide was also affected, showing a significant increase under cadmium treatment. Components of ascorbate-glutathione antioxidant system altered their levels, showing the maximum decline in ascorbate (ASA), dehydroascorbate (DHA), total ascorbate (ASA + DHA, hereafter TA), glutathione (GSH) and total glutathione (GSH + GSSG, hereafter TG) under S deficiency. However, total ascorbate and total glutathione increased, besides a marginal increase in their reduced and oxidized forms, when Cd was applied in the presence of sufficient S. Sulphur supply also helped in increasing the activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR) and catalase (CAT) under Cd stress. However, their activity suffered by S deficiency and by Cd stress during S deficiency. Each stress declined the contents of soluble protein and photosynthetic pigments; the highest decline in contents of protein and pigments occurred under S deficiency and dual stress respectively. The fresh and dry weights, although affected adversely by every stress, declined most under dual stress. It may be concluded that an optimal level of S is required during Cd stress for better response of SOD, APX, GR and CAT activity, as well as synthesis of cysteine. RbcS is as highly sensitive to S deficiency as RbcL is to Cd stress.

Allium sativum Protease Inhibitor: A Novel Kunitz Trypsin Inhibitor from Garlic Is a New Comrade of the Serpin Family.

PURPOSE: This study was aimed to purify and characterize the Protease inhibitor (PI) from a plant Allium sativum (garlic) with strong medicinal properties and to explore its phytodrug potentials. METHODS: Allium sativum Protease Inhibitor (ASPI) was purified using ammonium sulphate fractionation and Fast Protein Liquid Chromatography on anion exchanger Hi-Trap DEAE column. The purified protein was analyzed for its purity and molecular weight by SDS PAGE. The confirmation of presence of trypsin inhibiting PI was performed by MALDI TOF-TOF and analyzed by MASCOT database. The ASPI was further investigated for its kinetic properties and stability under extreme conditions of pH, temperature and chemical denaturants. Secondary structure was determined by Circular Dichorism (CD) spectroscopy. RESULTS: ASPI of ~15 kDa inhibited trypsin and matched "truncated kunitz Trypsin Inhibitor (Glycine max)" in MASCOT database. The purified ASPI showed 30376.1371 U/mg specific activity with a fold purity of 159.92 and yield ~93%. ASPI was quite stable in the range of pH 2-12 showing a decline in the activity around pH 4-5 suggesting that the pI value of the protein as ASPI aggregates in this range. ASPI showed stability to a broad range of temperature (10-80°C) but declined beyond 80°C. Further, detergents, oxidizing agents and reducing agents demonstrated change in ASPI activity under varying concentrations. The kinetic analysis revealed sigmoidal relationship of velocity with substrate concentration with Vmax 240.8 (µM/min) and Km value of 0.12 µM. ASPI showed uncompetitive inhibition with a Ki of 0.08±0.01 nM). The Far UV CD depicted 2.0% α -helices and 51% ß -sheets at native pH. CONCLUSIONS: To conclude, purified ~15 kDa ASPI exhibited fair stability in wide range of pH and temperature Overall, there was an increase in purification fold with remarkable yield. Chemical modification studies suggested the presence of lysine and tryptophan residues as lead amino acids present in the reactive sites. Therefore, ASPI with trypsin inhibitory property has the potential to be used as a non-cytotoxic clinical agents.

Compartmentalized Metabolic Engineering for Artemisinin Biosynthesis and Effective Malaria Treatment by Oral Delivery of Plant Cells.

Artemisinin is highly effective against drug-resistant malarial parasites, which affects nearly half of the global population and kills >500 000 people each year. The primary cost of artemisinin is the very expensive process used to extract and purify the drug from Artemisia annua. Elimination of this apparently unnecessary step will make this potent antimalarial drug affordable to the global population living in endemic regions. Here we reported the oral delivery of a non-protein drug artemisinin biosynthesized (∼0.8 mg/g dry weight) at clinically meaningful levels in tobacco by engineering two metabolic pathways targeted to three different cellular compartments (chloroplast, nucleus, and mitochondria). The doubly transgenic lines showed a three-fold enhancement of isopentenyl pyrophosphate, and targeting AACPR, DBR2, and CYP71AV1 to chloroplasts resulted in higher expression and an efficient photo-oxidation of dihydroartemisinic acid to artemisinin. Partially purified extracts from the leaves of transgenic tobacco plants inhibited in vitro growth progression of Plasmodium falciparum-infected red blood cells. Oral feeding of whole intact plant cells bioencapsulating the artemisinin reduced the parasitemia levels in challenged mice in comparison with commercial drug. Such novel synergistic approaches should facilitate low-cost production and delivery of artemisinin and other drugs through metabolic engineering of edible plants.

Designing, construction and characterization of genetically encoded FRET-based nanosensor for real time monitoring of lysine flux in living cells.

BACKGROUND: Engineering microorganisms in order to improve the metabolite flux needs a detailed knowledge of the concentrations and flux rates of metabolites and metabolic intermediates in vivo. Fluorescence resonance energy transfer (FRET) based genetically encoded nanosensors represent a promising tool for measuring the metabolite levels and corresponding rate changes in live cells. Here, we report the development of a series of FRET based genetically encoded nanosensor for real time measurement of lysine at cellular level, as the improvement of microbial strains for the production of L-lysine is of major interest in industrial biotechnology. RESULTS: The lysine binding periplasmic protein (LAO) from Salmonella enterica serovar typhimurium LT2 strain was used as the reporter element for the sensor. The LAO was sandwiched between GFP variants i.e. cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). Affinity, pH stability, specificity and metal ions effects was scrutinized for the in vitro characterization of this nanosensor, named as FLIPK. The FLIPK is specific to lysine and found to be stable with the pH within the physiological range. The calculated affinity (K d ) of FLIPK was 97 µM. For physiological applications, mutants with different binding affinities were also generated and investigated in vitro. The developed nanosensor efficiently monitored the intracellular level of lysine in bacterial as well as yeast cell. CONCLUSION: The developed novel lysine fluorescence resonance energy transfer sensors can be used for in vivo monitoring of lysine levels in prokaryotes as well as eukaryotes. The potential of these sensors is that they can be used as reporter tools in the development of metabolically engineered microbial strains or for real-time monitoring of intracellular lysine during fermentation.

Oxaliplatin-induced Oxidative Stress Provokes Toxicity in Isolated Rat Liver Mitochondria.

BACKGROUND AND AIMS: Oxaliplatin is a widely employed platinum-derived chemotherapeutic agent commonly used for the treatment of colorectal cancer. Unfortunately, the benefit of this important drug is compromised by severe side effects such as neuropathy, ototoxicity, gastrointestinal toxicity, and hematological toxicity. Recently, few studies have also suggested the occurrence of hepatotoxicity in oxaliplatin-treated patients. Mitochondria have emerged as targets for anticancer drugs in various kinds of toxicity including hepatotoxicity that can lead to neoplastic disease. Oxidative stress is a well-established biomarker of mitochondrial toxicity. The purpose of this study was to investigate the dose-dependent damage caused by oxaliplatin on isolated liver mitochondria under in vitro conditions. METHODS: The study was conducted in mitochondria isolated from liver of Wistar rats. Oxaliplatin was incubated with mitochondria in a dose-dependent manner under in vitro conditions. Oxidative stress indexes, non-enzymatic and enzymatic antioxidants were evaluated, looking at the overall armamentarium against the toxicity induced by oxaliplatin. RESULTS: Oxaliplatin caused a significant rise in the mitochondrial oxidative stress indexes lipid peroxidation and protein carbonyl. Alterations in the levels of non-enzymatic antioxidants and activities of enzymatic antioxidants were also observed. CONCLUSION: Oxidative stress plays an important role in the mitochondrial toxicity of oxaliplatin. The integrity of the hepatic tissue is compromised by the reactive oxygen species-mediated lipid peroxidation and protein carbonyl formation.

Spinach (Spinacia oleracea L.) modulates its proteome differentially in response to salinity, cadmium and their combination stress.

Cadmium (Cd) contamination and salinity are common stressors in agricultural soils all over the globe. Sensitivity and modulation of plant proteome lead to proper signal execution and adaptation to abiotic stress via molecular responses, which strengthen plant defence system. A comparative proteomic study, employing 2DE-MALDI TOF/TOF MS, of Spinacia oleracea plants exposed to cadmium (50 µg CdCl2 g(-1) soil), salinity (10 mg NaCl g(-1) soil) and their combination (NaCl + Cd) was conducted to understand the minimum common adaptation to multiple stress. Analysis of 2D gel maps showed significant increase and decrease in relative abundance of 14 and 39 proteins by Cd; 11 and 46 by salinity and 22 and 37 by combined stress of Cd and salinity, respectively. Peptide mass fingerprinting (PMF) helped in the identification of maturase K and PPD4 with increased relative abundance under all stresses; whereas salinity stress and combination stress silenced the presence of one protein (polycomb protein EZ2) and two proteins (cellulose synthase-like protein and ubiquitin conjugation factor E4), respectively. The identified proteins were functionally associated with signal transduction (15%), protein synthesis (16%), stress response and defence (33%), photosynthesis (13%), plant growth/cell division (9%), energy generation (4%), transport (4%), secondary metabolism (3%), and cell death (3%); clearly indicating the importance and necessity of keeping a higher ratio of defence and disease-responsive proteins. The results suggest that plant may increase the abundance of defence proteins and may also lower the abundance of catabolic proteins. Proteins with altered ratios of abundance belonged to different functional categories, suggesting that plants have differential mechanisms to respond to Cd, salinity, and their combined stress, but with unique sets of proteins.

Influence of sulfur and cadmium on antioxidants, phytochelatins and growth in Indian mustard.

Soils in many parts of the world are contaminated with heavy metals, leading to multiple, deleterious effects on plants and threats to world food production efficiency. Cadmium (Cd) is one such metal, being toxic at relatively low concentrations as it is readily absorbed and translocated in plants. Sulfur-rich compounds are critical to the impact of Cd toxicity, enabling plants to increase their cellular defence and/or sequester Cd into vacuoles mediated by phytochelatins (PCs). The influence of sulfur on Cd-induced stress was studied in the hyperaccumulator plant Indian mustard (Brassica juncea) using two sulfur concentrations (+S, 300 µM [Formula: see text] and S-deficient -S, [Formula: see text]) with and without the addition of Cd (100 µM CdCl2) at two different time intervals (7 and 14 days after treatment). Compared with control plants (+S/-Cd), levels of oxidative stress were higher in S-deficient (-S/-Cd) plants, and greatest in S-deficient Cd-treated (-S/+Cd) plants. However, additional S (+S/+Cd) helped plants cope with oxidative stress. Superoxide dismutase emerged as a key player against Cd stress under both -S and +S conditions. The activity of ascorbate peroxidase, glutathione reductase and catalase declined in Cd-treated and S-deficient plants, but was up-regulated in the presence of sulfur. Sulfur deficiency mediated a decrease in ascorbate and glutathione (GSH) content but changes in ascorbate (reduced : oxidized) and GSH (reduced : oxidized) ratios were alleviated by sulfur. Our data clearly indicate that a sulfur pool is needed for synthesis of GSH, non-protein thiols and PCs and is also important for growth. Sulfur-based defence mechanisms and the cellular antioxidant pathway, which are critical for tolerance and growth, collapsed as a result of a decline in the sulfur pool.

Effect of long-term salinity on cellular antioxidants, compatible solute and fatty acid profile of Sweet Annie (Artemisia annua L.).

Impact of long-term salinity and subsequent oxidative stress was studied on cellular antioxidants, proline accumulation and lipid profile of Artemisia annua L. (Sweet Annie or Qinghao) which yields artemisinin (Qinghaosu), effective against cerebral malaria-causing strains of Plasmodium falciparum. Under salinity (0.0-160 mM NaCl), in A. annua, proline accumulation, contents of ascorbate and glutathione and activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR) and catalase (CAT) increased, but the contents of reduced forms of glutathione (GSH) and ascorbate declined. The fatty-acid profiling revealed a major salinity-induced shift towards long-chain and mono-saturated fatty acids. Myristic acid (14:0), palmitoleic acid (16:1), linoleic acid (18:2) and erucic acid (22:1) increased by 141%, 186%, 34% and 908%, respectively, in comparison with the control. Contents of oleic acid (18:1), linolenic acid (18:3), arachidonic acid (22:0) and lignoceric acid (24:0) decreased by 50%, 17%, 44% and 78%, respectively. Thus, in A. annua, salinity declines ascorbate and GSH contents. However, increased levels of proline and total glutathione (GSH+GSSG), and activities of antioxidant enzymes might provide a certain level of tolerance. Modification in fatty-acid composition might be a membrane adaptation to long-term salinity and oxidative stress.

Iron stabilizes thylakoid protein-pigment complexes in Indian mustard during Cd-phytoremediation as revealed by BN-SDS-PAGE and ESI-MS/MS.

Two-dimensional BN-SDS-PAGE, ESI-MS/MS and electron microscopy (EM) were used to study the role of iron (Fe) under cadmium (Cd) stress in retention of thylakoidal multiprotein complexes (MPCs) and chloroplast ultrastructure of Indian mustard, a moderate hyperaccumulator plant. Mustard was grown hydroponically with or without iron for 17 days and then exposed to CdCl2 for 3 days. Fe deficiency led to an increase in oxidative stress and damage to chloroplast/thylakoids accompanied by a decrease in chlorophyll content; exposure of plants to Cd further enhanced the oxidative stress and Cd accumulation (more in -Fe plants). However, the presence of iron aided plants in the suppression of oxidative stress and retention of chloroplasts and chlorophylls under Cd stress. Proteomic analyses by 2D BN-SDS-PAGE and mass spectrometry showed that Fe deficiency considerably decreased the amount of LHCII trimer, ATPase-F1 portion, cyt b6/f and RuBisCO. No or less reduction, was observed for PSI(RCI+LHCI), the PSII-core monomer, and the PSII subcomplex, while an increase in the LHCII monomer was noted. Under iron deficiency, Cd proved to be very deleterious to MPCs, except for the PSII subcomplex, the LHCII monomer and free proteins which were increased. Iron proved to be very protective in retaining almost all the complexes. MPCs showed greater susceptibility to Cd than Fe deficiency, mainly at the level of RuBisCO and cyt b6/f; an increase in the amount of the PSII subcomplex, LHCII monomer and free proteins indicates differences in the mechanisms affected by Fe deficiency and Cd stress when compared to Fe-fed plants. This study furthers our understanding of the sites actually damaged in MPCs under Fe deficiency and Cd stress. A role emerges for iron in the protection of MPCs and, hence, of the chloroplast. The present study also indicates the importance of iron for efficient phytoextraction/phytoremediation.