Naoko Ohkama-Ohtsu

A γ -Glutamyl Transpeptidase-Independent Pathway of Glutathione Catabolism to Glutamate via 5-Oxoproline in Arabidopsis

The degradation pathway of glutathione (GSH) in plants is not well understood. In mammals, GSH is predominantly metabolized through the γ-glutamyl cycle, where GSH is degraded by the sequential reaction of γ-glutamyl transpeptidase (GGT), γ-glutamyl cyclotransferase, and 5-oxoprolinase to yield glutamate (Glu) and dipeptides that are subject to peptidase action. In this study, we examined if GSH is degraded through the same pathway in Arabidopsis (Arabidopsis thaliana) as occurs in mammals. In Arabidopsis, the oxoprolinase knockout mutants (oxp1-1 and oxp1-2) accumulate more 5-oxoproline (5OP) and less Glu than wild-type plants, suggesting substantial metabolite flux though 5OP and that 5OP is a major contributor to Glu steady-state levels. In the ggt1-1/ggt4-1/oxp1-1 triple mutant with no GGT activity in any organs except young siliques, the 5OP concentration in leaves was not different from that in oxp1-1, suggesting that GGTs are not major contributors to 5OP production in Arabidopsis. 5OP formation strongly tracked the level of GSH in Arabidopsis plants, suggesting that GSH is the precursor of 5OP in a GGT-independent reaction. Kinetics analysis suggests that γ-glutamyl cyclotransferase is the major source of GSH degradation and 5OP formation in Arabidopsis. This discovery led us to propose a new pathway for GSH turnover in plants where GSH is converted to 5OP and then to Glu by the combined action of γ-glutamyl cyclotransferase and 5-oxoprolinase in the cytoplasm.

A protocol for rapid DNA extraction fromArabidopsis thaliana for PCR analysis

We present a method for instant DNA extraction fromArabidopsis thaliana based on a simple DNA extraction method (Edwards et al., 1991). A piece of rosette leaf (typically 3–5 mg) was ground in a centrifuge tube in extraction solution. Extracted DNA was suitable for PCR analysis, without centrifugation. The feasibility of this method was confirmed by testing 24 primer sets. This method requires less than 1 mg of plant tissue and is useful for genetic mapping, transgene detection, and other experiments.

Recent Progress in Plant Nutrition Research : Cross-Talk Between Nutrients, Plant Physiology and Soil Microorganisms

Mineral nutrients taken up from the soil become incorporated into a variety of important compounds with structural and physiological roles in plants. We summarize how plant nutrients are linked to many metabolic pathways, plant hormones and other biological processes. We also focus on nutrient uptake, describing plant-microbe interactions, plant exudates, root architecture, transporters and their applications. Plants need to survive in soils with mineral concentrations that vary widely. Describing the relationships between nutrients and biological processes will enable us to understand the molecular basis for signaling, physiological damage and responses to mineral stresses.

Isolation and Characterization of an Arabidopsis Mutant That Overaccumulates O-Acetyl-l-Ser

O-Acetyl-l-Ser (OAS) is a positive regulator for the expression of sulfur (S) deficiency-inducible genes. In this study, through the isolation and analysis of Arabidopsis mutants exhibiting altered expression of S-responsive genes, we identified a thiol reductase as a regulator of the OAS levels. Ethyl methanesulfonate-mutagenized M2 seeds of transgenic Arabidopsis NOB7 carrying a chimeric S-responsive promoter driving the green fluorescent protein gene were screened for mutants with altered levels of green fluorescence compared to parental NOB7 line. One of the lines exhibited elevated levels of green fluorescence and mRNA accumulation of several endogenous S-responsive genes and carried a single recessive mutation responsible for the phenotype. OAS concentration in the rosette leaves of the mutant was about five times higher than that of wild-type plants. Based upon the high OAS levels, the mutant was named osh1-1 (OAS high accumulation). The OSH1 locus was mapped to a 30-kb region in chromosome V. DNA sequence analysis revealed no base change in this region; however, a demethylated C residue was found in the first exon of At5g01580. At5g01580 mRNA accumulation was higher in osh1-1 than in wild type, while transcript levels of other genes in the mapped region were not significantly altered in osh1-1. A line of transgenic plants overexpressing At5g01580 had elevated levels of endogenous S-responsive genes. These results suggest that elevated expression of At5g01580 is the cause of osh1 phenotype. Based on sequence similarity to animal thiol reductases, At5g01580 was tested for and exhibited thiol reductase activity. Possible roles of a thiol reductase in OAS metabolism are discussed.

12-Oxo-Phytodienoic Acid-Glutathione Conjugate is Transported into the Vacuole in Arabidopsis

While exogenous toxic compounds such as herbicides are thought to be sequestered into vacuoles in the form of glutathione (GSH) conjugates, little is understood about natural plant products conjugated with GSH. To identify natural products conjugated with GSH in plants, metabolites in the Arabidopsis γ-glutamyl transpeptidase (ggt) 4 knockout mutants that are blocked in the degradation of GSH conjugates in the vacuole were compared with those in wild-type plants. Among the metabolites identified, one was confirmed to be the 12-oxo-phytodienoic acid (OPDA)-GSH conjugate, indicating that OPDA, a precursor of jasmonic acid (JA), is transported into the vacuole as a GSH conjugate.

Physiological and genetic characterization of rice nitrogen fixer PGPR isolated from rhizosphere soils of different crops

AimsWe aimed to identify plant growth-promoting rhizobacteria that could be used to develop a biofertilizer for rice.MethodsTo obtain plant growth-promoting rhizobacteria, rhizosphere soils from different crops (rice, wheat, oats, crabgrass, maize, ryegrass, and sweet potato) were inoculated to rice plants. In total, 166 different bacteria were isolated and their plant growth-promoting traits were evaluated in terms of colony morphology, indole-3-acetic acid production, acetylene reduction activity, and phosphate solubilization activity. Moreover, genetic analysis was carried out to evaluate their phylogenetic relationships based on 16S rRNA sequence data.ResultsStrains of Bacillus altitudinis, Pseudomonas monteilii, and Pseudomonas mandelii formed associations with rice plants and fixed nitrogen. A strain of Rhizobium daejeonense showed nitrogen fixation activity in an in vitro assay and in vivo. Strains of B. altitudinis and R. daejeonense derived from rice rhizosphere soil, strains of P. monteilii and Enterobacter cloacae derived from wheat rhizosphere soil, and a strain of Bacillus pumilus derived from maize rhizosphere soil significantly promoted rice plant growth.ConclusionsThese methods are effective to identify candidate species that could be developed as biofertilizers for target crops.

Genetic diversity of native soybean bradyrhizobia from different topographical regions along the southern slopes of the Himalayan Mountains in Nepal.

Soybean-nodulating bradyrhizobia are genetically diverse and are classified into different species. In this study, the genetic diversity of native soybean bradyrhizobia isolated from different topographical regions along the southern slopes of the Himalayan Mountains in Nepal was explored. Soil samples were collected from three different topographical regions with contrasting climates. A local soybean cultivar, Cobb, was used as a trap plant to isolate bradyrhizobia. A total of 24 isolates selected on the basis of their colony morphology were genetically characterized. For each isolate, the full nucleotide sequence of the 16S rRNA gene and ITS region, and partial sequences of the nifD and nodD1 genes were determined. Two lineages were evident in the conserved gene phylogeny; one representing Bradyrhizobium elkanii (71% of isolates), and the other representing Bradyrhizobium japonicum (21%) and Bradyrhizobium yuanmingense (8%). Phylogenetic analyses revealed three novel lineages in the Bradyrhizobium elkanii clade, indicating high levels of genetic diversity among Bradyrhizobium isolates in Nepal. B. japonicum and B. yuanmingense strains were distributed in areas from 2420 to 2660 m above sea level (asl), which were mountain regions with a temperate climate. The B. elkanii clade was distributed in two regions; hill regions ranging from 1512 to 1935 m asl, and mountain regions ranging from 2420 to 2660 m asl. Ten multi-locus genotypes were detected; seven among B. elkanii, two among B. japonicum, and one among B. yuanmingense-related isolates. The results indicated that there was higher species-level diversity of Bradyrhizobium in the temperate region than in the sub-tropical region along the southern slopes of the Himalayan Mountains in Nepal.

Arabidopsis SNRK2.3 protein kinase is involved in the regulation of sulfur-responsive gene expression and O-acetyl-l-serine accumulation under limited sulfur supply

Abstract The role of the Arabidopsis thaliana genes SNRK2s, which are similar to the sulfur-regulatory gene SAC3 of Chlamydomonas reinhardtii, in the response of plants to sulfur was studied. The Arabidopsis genome contains 10 genes (SNRK2.1 to SNRK2.10) similar to SAC3. Among these genes, transcript accumulation of several genes including SNRK2.3 was induced in response to sulfur starvation. Independently isolated transgenic A. thaliana lines carrying T-DNA insertions in SNRK2.3 exhibited reduced sulfur-starvation induction of the transcript of the sulfate transporter SULTR2;2 gene, whereas the accumulation of O-acetyl-l-serine under sulfur deficiency conditions increased. These results suggest that SNRK2.3 plays a beneficial role in the regulation of gene expression and metabolism in response to sulfur starvation.

Enhanced arsenic sensitivity with excess phytochelatin accumulation in shoots of a SULTR1;2 knockout mutant of Arabidopsis thaliana (L.) Heynh

ABSTRACT One mechanism of arsenic detoxification in plants is synthesis of phytochelatins from glutathione in a sulfur-dependent manner. This study examined the contribution of a sulfate transporter, SULTR1;2, in arsenic tolerance in terms of sulfur metabolism in Arabidopsis thaliana (L.) Heynh. Comparative analysis of SULTR mutants showed that defective mutations of SULTR1;2 resulted in an increased arsenic sensitivity, in both shoots and roots, establishing that SULTR1;2 is required for arsenic tolerance. We subsequently quantified total sulfur content and levels of sulfur compounds (phytochelatins, glutathione, cysteine) in a SULTR1;2 mutant, sel1-8. Arsenic treatments increased sulfur uptake of sel1-8, but the mutant was unable to maintain the proper basal level of sulfur. Despite drastic reduction of total sulfur content, the mutant accumulated substantial or rather excess phytochelatins in shoots under arsenic stress conditions. The levels of glutathione and cysteine in shoots were lower in sel1-8 than in the wild type, possibly representing partial disorder of sulfur nutrition under limited sulfur supply. Taken together, we propose that SULTR1;2 contributes to arsenic tolerance by maintaining proper sulfur nutrient status at high demand for sulfur by phytochelatins-synthetic pathway, and/or optimal level of phytochelatin synthesis in shoots.

Phytochelatin Synthase has Contrasting Effects on Cadmium and Arsenic Accumulation in Rice Grains

Abstract Phytochelatin (PC) synthesis has been well demonstrated as a major metal tolerance mechanism in Arabidopsis thaliana, whereas its contribution to long-distance element transport especially in monocots remains elusive. Using rice as a cereal model, we examined physiological roles of Oryza sativa phytochelatin synthase 1 (OsPCS1) in the distribution and detoxification of arsenic (As) and cadmium (Cd), two toxic elements associated with major food safety concerns. First, we isolated four different transcript variants of OsPCS1 as well as one from OsPCS2. Quantitative real-time reverse transcription–PCR (RT-PCR) of each OsPCS transcript in rice seedlings suggested that expression of OsPCS1full, the longest OsPCS1 variant, was most abundant, followed by OsPCS2. Heterologous expression of OsPCS variants in PCS-deficient mutants of Schizosaccharomyces pombe and A. thaliana suggested that OsPCS1full possessed PCS activity in response to As(III) and Cd while the activity of other PCS variants was very low. To address physiological functions in toxic element tolerance and accumulation, two independent OsPCS1 mutant rice lines (a T-DNA and a Tos17 insertion line) were identified. The OsPCS1 mutants exhibited increased sensitivity to As(III) and Cd in hydroponic experiments, showing the importance of OsPCS1-dependent PC synthesis for rice As(III) and Cd tolerance. Elemental analyses of rice plants grown in soil with environmentally relevant As and Cd concentrations showed increased As accumulation and decreased Cd accumulation in grains of the T-DNA line. The Tos17 mutant also exhibited the reduced Cd accumulation phenotype. These contrasting effects on As and Cd distribution to grains suggest the existence of at least partially distinct PC-dependent pathways for As and Cd.