Silicon and Iron Differently Alleviate Copper Toxicity in Cucumber Leaves

Copper (Cu) toxicity in plants may lead to iron (Fe), zinc (Zn) and manganese (Mn) deficiencies. Here, we investigated the effect of Si and Fe supply on the concentrations of micronutrients and metal-chelating amino acids nicotianamine (NA) and histidine (His) in leaves of cucumber plants exposed to Cu in excess. Cucumber (Cucumis sativus L.) was treated with 10 mu M Cu, and additional 100 mu M Fe or/and 1.5 mM Si for five days. High Cu and decreased Zn, Fe and Mn concentrations were found in Cu treatment. Additional Fe supply had a more pronounced effect in decreasing Cu accumulation and improving the molar ratio between micronutrients as compared to the Si supply. However, the simultaneous supply of Fe and Si was the most effective treatment in alleviation of Cu-induced deficiency of Fe, Zn and Mn. Additional Fe supply increased the His but not NA concentration, while Si supply significantly increased both NA and His whereby the NA:Cu and His:Cu molar ratios exceeded the control values indicating that Si recruits Cu-chelation to achieve Cu tolerance. In conclusion, Si-mediated alleviation of Cu toxicity was directed toward Cu tolerance while Fe-alleviative effect was due to a dramatic decrease in Cu accumulation

Establishment and in-house validation of stem-loop rt pcr method for microrna398 expression analysis

MicroRNAs (miRNAs) belong to the class of small non-coding RNAs which have important roles throughout development as well as in plant response to diverse environmental stresses. Some of plant miRNAs are essential for regulation and maintenance of nutritive homeostasis when nutrients are in excess or shortage comparing to optimal concentration for certain plant species. Better understanding of miRNAs functions implies development of efficient technology for profiling their gene expression. We set out to establish validate the methodology for miRNA gene expression analysis in cucumber grown under suboptimal mineral nutrient regimes, including iron deficiency. Reverse transcription by "stem-loop" primers in combination with Real time PCR method is one of potential approaches for quantification of miRNA gene expression. In this paper we presented a method for "stem loop" primer design specific for miR398, as well as reaction optimization and determination of Real time PCR efficiency. Proving the accuracy of this method was imperative as "stem loop" RT which consider separate transcription of target and endogenous control. The method was verified by comparison of the obtained data with results of miR398 expression achieved using a commercial kit based on simultaneous conversion of all RNAs in cDNAs

Cell wall localization of the aspartic proteinase from buckwheat (FeAPL1) over-expressed in tobacco BY-2 cells

The recombinant aspartic proteinase-like protein (FeAPL1-His6) was overexpressed in the tobacco BY-2 cell line and the expected pepstatin A-sensitive enzymatic activity was confirmed at pH 3.0. Immunocytochemistry and protein gel blot analysis of the transformed BY-2 cells and their protoplasts showed extracellular localization of rFeAPL1-His6 in the cell wall. Based on the obtained results, potential functions of FeAPL1 are discussed

Silicon alleviates copper (Cu) toxicity in cucumber by increased Cu-binding capacity

Aims Although silicon (Si) is known to increase plant resistance to metal toxicity stress, the mechanisms responsible for alleviation of copper (Cu) toxicity are still insufficiently clear. We investigated the role of Si on Cu-binding processes involved in buffering excessive Cu in cucumber (Cucumis sativus L.) tissues. Methods Cucumber plants were subjected to moderate Cu toxicity stress (10 mu M Cu) without (-Si) or with (+Si) supply of 1.5 mM Si. We analyzed total and cell wall concentrations of Cu and Cu-binding compounds (organic acids and Cu-proteins) along with parameters of oxidative stress (e.g. lipid peroxidation and lignification). Results Supply of Si decreased total Cu concentration in both root and leaf tissues, but increased the root cell wall Cu fraction. Also, Si increased superoxide dismutase (SOD) activity in 10 mu M Cu-treated plants. Concomitantly, protein levels of Cu/Zn SOD isoforms (CSD1 and CSD2) in root tissues also increased in +Si plants. The leaf Cu-binding compounds, such as aconitate and plastocyanin (including the expression of CsPC gene) were higher in the +Si plants. Consequently, Si supply effectively lowered lipid peroxidation in both roots and leaves of Cu-stressed plants. Conclusions Supply of Si enhanced both the accumulation of Cu-binding molecules (Zn/Cu SOD in roots; aconitate and plastocyanin in leaves), and the Cu-binding capacity of the root cell wall

Silicon increases phosphorus (P) uptake by wheat under low P acid soil conditions

Although silicon (Si) is known to improve plant growth under low phosphorus (P) conditions, the in planta mechanisms responsible for this effect are still unknown. Here, we investigated the role of Si on P uptake along with the expression of Pi transporters in wheat (Triticum aestivum L.) grown in low P acid soil in comparison with P fertilization and liming. A combined approach was performed including analyses of rhizosphere soil, tissue P content, the expression of the root Pi transporter genes (TaPHT1.1 and TaPHT1.2), and the root exudation of citrate and malate. Supply of Si in a form of Na2SiO3 increased shoot P concentration to an adequate level in the range of P-fertilized plants. Silicon ameliorated low soil pH and high Al3+ comparable to the effect of liming. The in planta effect of Si on up-regulating the expression of TaPHT1.1 and TaPHT1.2 was several fold higher and consequently P uptake doubled compared to both P fertilization and liming. In addition, Si directly stimulated root Pi acquisition by prominently increasing both malate and citrate exudation rate. Application of Si increased root exudation of organic acids that mobilize Pi in the rhizosphere and up-regulated Pi transporters in wheat roots

Silicon Alleviates Iron Deficiency in Barley by Enhancing Expression of Strategy II Genes and Metal Redistribution

The beneficial effects of silicon (Si) have been shown on plants using reduction-based strategy for iron (Fe) acquisition. Here we investigated the influence of Si on Fe deficiency stress alleviation in barley (Hordeum vulgare), a crop plant which uses the chelation-based strategy for Fe acquisition. Analyses of chlorophyll content, ROS accumulation, antioxidative status, concentrations of Fe and other micronutrients, along with the expression of Strategy II genes were studied in response to Si supply. Si successfully ameliorated Fe deficiency in barley, diminishing chlorophyll and biomass loss, and improving the activity of antioxidative enzymes, resulting in lowered reactive oxidative species accumulation in the youngest leaves. Alleviation of Fe deficiency stress correlated well with the Si-induced increase of Fe content in the youngest leaves, while it was decreased in root. Moreover, Si nutrition lowered accumulation of other micronutrients in the youngest leaves of Fe deprived plants, by retaining them in the root. On the transcriptional level, Si led to an expedient increase in the expression of genes involved in Strategy II Fe acquisition in roots at the early stage of Fe deficiency stress, while decreasing their expression in a prolonged stress response. Expression of Strategy II genes was remarkably upregulated in the leaves of Si supplied plants. This study broadens the perspective of mechanisms of Si action, providing evidence for ameliorative effects of Si on Strategy II plants, including its influence on accumulation and distribution of microelements, as well as on the expression of the Strategy II genes

Transient expression in tobacco Bright Yellow 2 cells and pollen grains: A fast, efficient and reliable system for functional promoter analysis of plant genes

Gene expression is mediated by DNA sequences directly upstream from the coding sequences, recruited transcription factors and RNA polymerase in a spatially-defined manner. Understanding promoter strength and regulation would enhance our understanding of gene expression. The goal of this study was to develop a fast, efficient and reliable method for testing basal promoter activity and identifying core sequences within its pollen specific elements. In this paper we examined the functionality of buckwheat metallothionein promoter by a histochemical GUS assay in two transient expression systems: BY2 cells and pollen grains. Strong promoter activity was observed in both systems

Antioxidative enzymes in the response of buckwheat (Fagopyrum esculentum Moench) to complete submergence

Oxidative stress and antioxidative defense system activity were studied in buckwheat leaves after complete submergence and re-aeration. The levels of H2O2 and lipid peroxidation were found to be significantly higher in stressed than in untreated buckwheat leaves. Enzymes catalyzing the degradation of H2O2 and peroxides were shown to participate actively, whereas superoxide dismutase did not take part in the buckwheat leaf response to flooding stress. The most prominent increase in antioxidative enzyme activities was noticed upon return to air, when the strongest oxidative stress occurred and the need for antioxidative defense was the greatest

A FAST, EFFICIENT AND RELIABLE SYSTEM FOR FUNCTIONAL PROMOTER ANALYSIS OF PLANT GENES

Abstract – Gene expression is mediated by DNA sequences directly upstream from the coding sequences, recruited transcription factors and RNA polymerase in a spatially-defined manner. Understanding promoter strength and regulation would enhance our understanding of gene expression. The goal of this study was to develop a fast, efficient and reliable method for testing basal promoter activity and identifying core sequences within its pollen specific elements. In this paper we examined the functionality of buckwheat metallothionein promoter by a histochemical GUS assay in two transient expression systems: BY2 cells and pollen grains. Strong promoter activity was observed in both systems

TWO METALLOTHIONEIN GENE FAMILY MEMBERS IN BUCKWHEAT: EXPRESSION ANALYSIS IN FLOODING STRESS USING REAL TIME RT-PCR TECHNOLOGY

Abstract — Metallothioneins (MTs) are an extensive and diverse family of small cysteine-rich proteins with metal-binding ability that are involved in metal homeostasis and detoxification. Two cDNA clones of the MT3 type, differing in 3’ UTRs, were isolated from the developing buckwheat seed cDNA library. Following sequence analyses, expression profiles during flooding stress were monitored by Real Time RT PCR technology