Masaaki Takahashi

Identification and molecular characterization of mitochondrial ferredoxins and ferredoxin reductase from Arabidopsis

We have identified and characterized novel types of ferredoxin and ferredoxin reductase from Arabidopsis. Among a number of potential ferredoxin reductase genes in the Arabidopsis genome, AtMFDR was identified to encode a homologue of mitochondrial ferredoxin reductase, and AtMFDX1 and AtMFDX2 were predicted to code for proteins similar to mitochondrial ferredoxin. First, we isolated cDNAs for these proteins and expressed them in heterologous systems of insect cells and Escherichia coli, respectively. The recombinant AtMFDX1 and AtMFDR proteins exhibited spectral properties characteristic of ferredoxin and ferredoxin reductase, respectively, and a pair of recombinant AtMFDX1 and AtMFDR proteins was sufficient to transfer electrons from NAD(P)H to cytochrome c inxa0vitro. Subcellular fractionation analyses suggested membrane association of AtMFDR protein, and protein-gel blot analyses and transient expression studies of green fluorescence protein fusions indicated mitochondrial localization of AtMFDX1 and AtMFDR. RNA-gel blot analyses revealed that the accumulation levels of AtMFDXs and AtMFDR gene transcripts were specifically high in flowers, while protein-gel blot analysis demonstrated substantial accumulation of AtMFDR protein in leaf, stem, and flower. Possible physiological roles of these mitochondrial electron transfer components are discussed in relation to redox dependent metabolic pathways in plants.

Experimental evaluation of model predictive control of ball and beam systems

In this paper, we discuss the control of a ball and beam system subject to an input constraint. Model predictive control (MPC) approaches are employed to derive a nonlinear control law satisfying the constraint. The control law is given by solving the optimization problem at each sample time, where the primal-dual interior point algorithm is implemented and used as the optimization solver. An experimental comparison of three control methods, two different MPCs and saturated LQR, has been presented for the control of the ball and beam system.

PCR-assisted differential display of the expression of genes that are induced by manganese-deficiency in tomato roots

Induction of Mn uptake activity by the deficiency of Mn in tomato roots were examined. Almost all of the Mn in tomato roots disappeared due to transport to leaf tissues within 10 days after the plants were shifted to a Mn-depleted hydroponic medium. Leaves showed typical Mn-deficiency symptoms in parallel with the increased rate of Mn uptake by roots. We screened the genes corresponding to the induced Mn transport in the Mn-stressed root tissues by polymerase chain reaction (PCR)-assisted differential display. Reverse transcription (RT)-PCR was carried out using several pairs of random primers and oligo-dT primer with total RNAs from roots that were exposed Mn-deficient culture medium for 0 to 10 days. Several PCR fragments had been amplified only in Mn-deficient roots, and they were cloned and sequenced.

NO 2 − Uptake by a NO 2 − Transporter of Chloroplast Envelope

Conversion of NO 3 − to NH 4 + in plants is comprised of two consecutive reduction steps catalyzed by reductases for NO 3 − and NO 2 − . The reduction of NO 3 − occurs in the cytoplasm while its product, NO 2 − , is reduced inside chloroplasts by plastid nitrite reductase [1, 2]. The uptake of NO 2 − by intact chloroplasts is strictly light dependent [3, 4]. The dependence of NO 2 − uptake on light was also proposed based on NO 2 − -induced phenomena such as swelling of chloroplasts [5], stromal pH shift [6-8], and inhibition of enzymes in CO2 fixation [6–8]. Participation of a NO 2 − -specific transport system in the uptake of NO 2 − by such photo-energized chloroplasts has been proposed based on the fact that the kinetics of NO 2 − uptake was saturable with increasing NO 2 − [3, 4], and inhibited by a thiol modifier [9]. Shingles et al. [10], on the other hand, proposed that the diffusional influx of HNO2 is faster than the consumpsion of NO 2 − by the reductive assimilation in chloroplast stroma if NO 2 − is present at a submillimolar level with outside-acidic pH gradient.

Modification of the nitrogen utilization in transgenic tobacco plants that have enhanced or repressed expression of a putative nitrite transporter in chloroplast envelope

Reductive assimilation of NO3 - in plant cells depends on an energy-requiring import of NO2 - into chloroplast. A cDNA (Nitr1) that encodes a nitrite transporter of chloroplast envelope was cloned and used to transform tobacco to modify the activity of transport.

Hydroponic culture of Arabidopsis thaliana to screen mutants defective in the absorption of manganese

Plant leaves accumulate Mn from soil by transporters in root plasma membranes, xylem systems, and subcellular membranes in mesophyll cells. Questions remain concerning how Mn is transferred to its major functioning site, i.e. the chloroplast lumen, and how Mn uptake is controlled. Genetic analysis of Mn-deficient mutants would help answer most of these questions. In this study four mutants have been found in genes of Arabidopsis thaliana. These mutants were selected for by appearance of characteristic Mn-deficiency symptoms on a hydroponic culture that contained 14 μM Mn2+. Mn content of the mutants was lowered by 1/3 – 1/4 in rosette leaves, 1/4 in caules, and 1/2 – 1/3 in siliques as compared to the wild type indicating that the mutations impaired Mn accumulation in whole plant cells.