No abstract

HPLC coupled to a mass spectrometer (MS) was used for the analysis of galanthamine and lycorine in natural extracts of Leucojum aestivum and in their in vitro cultures grown with a precursor (ACC), inhibitors (AgNO(3), STS), or an absorber (KMnO(4)) of ethylene. The maximum galanthamine (0.002%) and lycorine (0.02%) concentrations in tissue cultures were obtained in the presence of KMnO(4). GCMS was used to investigate underivatized alkaloid mixtures from L. aestivum. Seven alkaloids were identified in in vivo bulbs. KMnO(4) led to the highest diversity of alkaloids in tissue culture extracts.

Vigor and selected physiological parameters (content of phenolic compounds, soluble sugars, chlorophyll a and b, and carotenoids) of eight naked and two husked oat cultivars harvested at 15% moisture content were determined. Oat seeds were threshed using two rotational speeds of the threshing drum: 1.6 ms −1 (LS) and 2.4 ms −1 (HS). They were then inoculated with a medium pathogenicity strain of Fusarium culmorum, strain IPO 348-01. In naked cultivars, the use of HS resulted in more severe mechanical damage; in consequence, seedling vigor decreased by 16%. In naked cultivars chlorophyll a and b and carotenoids content were significantly reduced-by more than 64%-when the HS was used. The inoculation caused over a 100% increase of carbohydrates in roots at LS but only a slight increase at HS. Phenolic compound content was twice as high in roots than in leaves after inoculation for both LS and HS. Area of microdamage and reduction of root fresh weight (f.wt.) are significantly correlated with biochemical parameters. Smaller microdamage area and root f.wt. reduction are connected with higher physiological parameters, which confirms lower seedling susceptibility to pathogen infection.

Previously, our in silico analyses identified four candidate genes that might be involved in uptake and/or accumulation of arsenics in plants: arsenate reductase 2 (ACR2), phytochelatin synthase 1 (PCS1) and two multi-drug resistant proteins (MRP1 and MRP2) [Lund et al. (2010) J Biol Syst 18:223-224]. We also postulated that one of these four genes, ACR2, seems to play a central role in this process. To investigate further, we have constructed a 3D structure of the Arabidopsis thaliana ACR2 protein using the iterative implementation of the threading assembly refinement (I-TASSER) server. These analyses revealed that, for catalytic metabolism of arsenate, the arsenate binding-loop (AB-loop) and residues Phe-53, Phe-54, Cys-134, Cys-136, Cys-141, Cys-145, and Lys-135 are essential for reducing arsenate to arsenic intermediates (arsenylated enzyme-substrate intermediates) and arsenite in plants. Thus, functional predictions suggest that the ACR2 protein is involved in the conversion of arsenate to arsenite in plant cells. To validate the in silico results, we exposed a transfer-DNA (T-DNA)-tagged mutant of A. thaliana (mutation in the ACR2 gene) to various amounts of arsenic. Reverse transcriptase PCR revealed that the mutant exhibits significantly reduced expression of the ACR2 gene. Spectrophotometric analyses revealed that the amount of accumulated arsenic compounds in this mutant was approximately six times higher than that observed in control plants. The results obtained from in silico analyses are in complete agreement with those obtained in laboratory experiments.

This paper reports a continuation of our previous research on the phytochelatin synthase1 (PCS1) gene involved in binding and sequestration of heavy metals or metalloids in plant cells. Construction of a 3D structure of the Arabidopsis thaliana PCS1 protein and prediction of gene function by employing iterative implementation of the threading assembly refinement (I-TASSER) revealed that PC ligands (3GC-gamma-glutamylcysteine) and Gln50, Pro53, Ala54, Tyr55, Cys56, Ile102, Gly161, His162, Phe163, Asp204 and Arg211 residues are essential for formation of chelating complex with cadmium (Cd²⁺) or arsenite (AsIII). This finding suggests that the PCS1 protein might be involved in the production of the enzyme phytochelatin synthase, which might in turn bind, localize, store or sequester heavy metals in plant cells. For validation of the in silico results, we included a T-DNA tagged mutant of Arabidopsis thaliana, SAIL_650_C12, (mutation in AtPCS1 gene) in our investigation. Furthermore, using reverse transcriptase PCR we confirmed that the mutant does not express the AtPCS1 gene. Mutant plants of SAIL_650_C12 were exposed to various amounts of cadmium (Cd²⁺) and arsenite (AsIII) and the accumulation of these toxic metals in the plant cells was quantified spectrophotometrically. The levels of Cd²⁺ and AsIII accumulation in the mutant were approximately 2.8 and 1.6 times higher, respectively, than that observed in the wild-type controlled plants. We confirmed that the results obtained in in silico analyses complement those obtained in in vivo experiments.