Hiroyuki Koyama

STOP1 Regulates Multiple Genes That Protect Arabidopsis from Proton and Aluminum Toxicities

The Arabidopsis (Arabidopsis thaliana) mutant stop1 (for sensitive to proton rhizotoxicity1) carries a missense mutation at an essential domain of the histidine-2-cysteine-2 zinc finger protein STOP1. Transcriptome analyses revealed that various genes were down-regulated in the mutant, indicating that STOP1 is involved in signal transduction pathways regulating aluminum (Al)- and H+-responsive gene expression. The Al hypersensitivity of the mutant could be caused by down-regulation of AtALMT1 (for Arabidopsis ALUMINUM-ACTIVATED MALATE TRANSPORTER1) and ALS3 (ALUMINUM-SENSITIVE3). This hypothesis was supported by comparison of Al tolerance among T-DNA insertion lines and a transgenic stop mutant carrying cauliflower mosaic virus 35S∷AtALMT1. All T-DNA insertion lines of STOP1, AtALMT1, and ALS3 were sensitive to Al, but introduction of cauliflower mosaic virus 35S∷AtALMT1 did not completely restore the Al tolerance of the stop1 mutant. Down-regulation of various genes involved in ion homeostasis and pH-regulating metabolism in the mutant was also identified by microarray analyses. CBL-INTERACTING PROTEIN KINASE23, regulating a major K+ transporter, and a sulfate transporter, SULT3;5, were down-regulated in the mutant. In addition, integral profiling of the metabolites and transcripts revealed that pH-regulating metabolic pathways, such as the γ-aminobutyric acid shunt and biochemical pH stat pathways, are down-regulated in the mutant. These changes could explain the H+ hypersensitivity of the mutant and would make the mutant more susceptible in acid soil stress than other Al-hypersensitive T-DNA insertion lines. Finally, we showed that STOP1 is localized to the nucleus, suggesting that the protein regulates the expression of multiple genes that protect Arabidopsis from Al and H+ toxicities, possibly as a transcription factor.

RNA isolation from siliques, dry seeds, and other tissues of Arabidopsis thaliana

Arabidopsis RNA Isolation Plants often present a challenge to researchers wanting to isolate nucleic acids for subsequent analyses as a result of the high amounts of polysaccharides and other contaminating metabolites present in plant tissues. Suzuki et al. (p. 542) describe an isolation method that produces RNA free of contamination by protein or polysaccharides or degradation, suitable for reverse transcription PCR (RT-PCR). The authors extracted RNA from Arabidopsis siliques, seeds, flower buds, leaves, roots, and stems with no decrease in the quality or quantity of RNA. Their method was both time- and cost-effective over more conventional methods and is simple enough to be routinely applied to other species.

Comparative transcriptomic characterization of aluminum, sodium chloride, cadmium and copper rhizotoxicities in Arabidopsis thaliana

BackgroundRhizotoxic ions in problem soils inhibit nutrient and water acquisition by roots, which in turn leads to reduced crop yields. Previous studies on the effects of rhizotoxic ions on root growth and physiological functions suggested that some mechanisms were common to all rhizotoxins, while others were more specific. To understand this complex system, we performed comparative transcriptomic analysis with various rhizotoxic ions, followed by bioinformatics analysis, in the model plant Arabidopsis thaliana.ResultsRoots of Arabidopsis were treated with the major rhizotoxic stressors, aluminum (Al) ions, cadmium (Cd) ions, copper (Cu) ions and sodium (NaCl) chloride, and the gene expression responses were analyzed by DNA array technology. The top 2.5% of genes whose expression was most increased by each stressor were compared with identify common and specific gene expression responses induced by these stressors. A number of genes encoding glutathione-S-transferases, peroxidases, Ca-binding proteins and a trehalose-synthesizing enzyme were induced by all stressors. In contrast, gene ontological categorization identified sets of genes uniquely induced by each stressor, with distinct patterns of biological processes and molecular function. These contained known resistance genes for each stressor, such as AtALMT1 (encoding Al-activated malate transporter) in the Al-specific group and DREB (encoding dehydration responsive element binding protein) in the NaCl-specific group. These gene groups are likely to reflect the common and differential cellular responses and the induction of defense systems in response to each ion. We also identified co-expressed gene groups specific to rhizotoxic ions, which might aid further detailed investigation of the response mechanisms.ConclusionIn order to understand the complex responses of roots to rhizotoxic ions, we performed comparative transcriptomic analysis followed by bioinformatics characterization. Our analyses revealed that both general and specific genes were induced in Arabidopsis roots exposed to various rhizotoxic ions. Several defense systems, such as the production of reactive oxygen species and disturbance of Ca homeostasis, were triggered by all stressors, while specific defense genes were also induced by individual stressors. Similar studies in different plant species could help to clarify the resistance mechanisms at the molecular level to provide information that can be utilized for marker-assisted selection.

Simple identification of transgenic Arabidopsis plants carrying a single copy of the integrated gene

Transgenic Arabidopsis thaliana plants carrying a single copy of integrated DNA can be identified by single-step genomic polymerase chain reaction. The reaction employs two sets of primer pairs with the same melting temperature that amplify the amplicons derived from the integrated T-DNA together with those from an endogenous single-copy gene as a reference. When the band intensity ratio is one, this means that the transgenic plants are carrying a single copy of the integrated gene per haploid.

Effect of Enhanced Calcium Supply on Aluminum Toxicity in Relation to Cell Wall Properties in the Root Apex of Two Wheat Cultivars Differing in Aluminum Resistance

The present study was conducted to investigate the effects of enhanced Ca supply on Al toxicity in relation to cell wall properties in two wheat (Triticum aestivum L.) cultivars differing in Al resistance. Seedlings of Al-tolerant Inia66 and Al-sensitive Kalyansona cultivars were grown in complete nutrient solutions for 4xa0days then subjected to treatment solutions containing Al (0, 50xa0μM) and Ca (500, 2500xa0μM) at pH 4.5 for 24xa0h. Root elongation was affected greatly by Al treatment in the Al-sensitive cultivar and a significant improvement in root growth was observed with enhanced Ca supply during Al stress. Pectin and hemicellulose contents in the root cell walls increased with Al stress, and this increase was more conspicuous in the Al-sensitive cultivar. The molecular mass of hemicellulosic polysaccharides increased with Al treatment in the Al-sensitive cultivar and decreased with enhanced Ca supply. The increase in the molecular mass of hemicellulosic polysaccharides was attributed to increased content of glucose, arabinose and xylose in neutral sugars. Enhanced Ca supply slightly decreased the content of these components with Al stress. Aluminum treatment increased the contents of ferulic and p-coumaric acid, especially in the Al-sensitive cultivar, by increasing peroxidase (POD, EC 1.11.1.7) and phenylalanine ammonia lyase (PAL, EC 4.3.1.5) activity, whereas enhanced Ca supply during Al stress decreased the content of these components by decreasing POD and PAL activity. These results suggest that the increased molecular mass of hemicellulosic polysaccharides and phenolic compounds in the Al-sensitive cultivar with Al stress might have inhibited root elongation associated with cell wall stiffening related to cross-linking among cell-wall polymers and lignin. Enhanced Ca supply might maintain the normal synthesis of these materials even with Al stress.

Characterization of NADP-isocitrate dehydrogenase expression in a carrot mutant cell line with enhanced citrate excretion

Citrate in the cytosol is converted to isocitrate, by the action of aconitase, and then isocitrate is converted to 2-oxoglutarate by the action of NADP-specific isocitrate dehydrogenase (NADP-ICDH). This pathway is modified in a mutant carrot (Daucus carotaL.) cell line [designated as insoluble phosphate grower (IPG): Koyama et al., 1992 Plant Cell Physiol. 33, 173–177], which releases large amounts of the citrate and grows faster than the wild-type cells in Al-phosphate medium. In the current study, the activities of aconitase in mitochondria and in crude extracts were similar in the mutant cells and the wild-type cells, suggesting that the activity of the cytosolic aconitase was similar among the cell types. By contrast, the NADP-ICDH activity in the crude extracts of the mutant cells was almost half that of the wild-type cells. Western blot analysis revealed that the cytosolic NADP-ICDH was the most abundant isoenzyme in both cell types. However the NADP-ICDH amount was lower in the mutant cells than that in the wild-type cells. Transcripts level of DcICDH1, which appeared to be a unique isoform encoding the cytosolic enzyme in carrot, was lower in the mutant cells than that of the wild-type cells. Thus, we inferred that the lower transcript level of the cytosolic isoform caused the lower NADP-ICDH activities in the mutant cells, compared with the wild-type cells.

Combined effects of Mg and Ca supply on alleviation of Al toxicity in wheat plants

Abstract A Bangladeshi wheat (Triticum aestivum) cultivar, Kalyansona, was exposed to 100 J.µ Al with varying concentrations of Ca (250, 500, 2,500 J.µM) and Mg (103, 1,029, 4,115 J.µ) in the nutrient solution for 14 d. Exposure to Al along with low concentrations of both Ca and Mg caused severe damage to the root apex. Under AI-stressed conditions, an increased supply of Mg from 103 to 1,029 J.µ enhanced the root length by 44, 22, and 3% at 250, 500, and 2,500 J.µ Ca, respectively. Root growth at 2,500 J.µ Ca was not affected by additional Mg concentration. Again, an increased supply of Mg from 103 to 4,115 J.µ enhanced the root length by 56% at 500 J.µ Ca. Under the Al-stressed conditions, the increase in the root length was correlated with the decrease in the Al content in roots. Our results suggest that Mg alleviates Al toxicity efficiently in the presence of Ca at a low level in the culture solution by decreasing Al accumulation in roots.