Keishiro Wada

A Recessive Arabidopsis Mutant That Grows Photoautotrophically under Salt Stress Shows Enhanced Active Oxygen Detoxification

Mutagenized Arabidopsis seedlings (ecotype Columbia) were screened for the ability to grow photoautotrophically on solid medium containing 200 mM NaCl. A novel mutant line, designated pst1 (for photoautotrophic salt tolerance1), was obtained. There were no significant differences between pst1 and wild-type plants with regard to their ability to induce proline as an osmoregulatory solute. In addition, the content of monovalent cations in pst1 plants grown with or without salt stress was equal to that in the wild type. We observed that light, even at moderate intensities, increased the effects of salt stress on wild-type plants. The pst1 seedlings were nearly 10 times more tolerant to methyl viologen than were wild-type seedlings. We also found that the activities of the active oxygen scavengers superoxide dismutase and ascorbate peroxidase were enhanced significantly in pst1 plants. The pst1 plants also were tolerant to other stresses, such as high light intensity and toxic monovalent cations. The recessive nature of the pst1 mutation indicates that the potential for salt-stress tolerance is blocked in wild-type Arabidopsis.

Characterization of the gene for Δ1-pyrroline-5-carboxylate synthetase and correlation between the expression of the gene and salt tolerance in Oryza sativa L.

A cDNA for Δ1-pyrroline-5-carboxylate (P5C) synthetase (cOsP5CS), an enzyme involved in the biosynthesis of proline, was isolated and characterized from a cDNA library prepared from 14-day-old seedlings of Oryza sativa cv. Akibare. The deduced amino acid sequence of the P5CS protein (OsP5CS) from O. sativa exhibited 74.2% and 75.5% homology to that of the P5CS from Arabidopsis thaliana and Vigna aconitifolia, respectively. Northern blot analysis revealed that the gene for P5CS (OsP5CS) was induced by high salt, dehydration, treatment of ABA and cold treatment, while it was not induced by heat treatment. Simultaneously, accumulation of proline was observed as a result of high salt treatment in O. sativa. Moreover, the levels of expression of OsP5CS mRNA and content of proline under salt stress condition were compared between a salt-tolerant cultivar, Dee-gee-woo-gen (DGWG) and a salt-sensitive breeding line, IR28. It was observed that the expression of the P5CS gene and the accumulation of proline in DGWG steadily increased, whereas those in IR28 increased slightly.

Molecular cloning and functional characterization of two kinds of betaine-aldehyde dehydrogenase in betaine-accumulating mangrove Avicennia marina (Forsk.) Vierh.

Glycinebetaine is an important osmoprotectant in bacteria, plants, and animals, but only little information is available on the synthesis of glycinebetaine in tree plants. Among four mangrove species, glycinebetaine could be detected only in Avicennia marina. Pinitol was the main osmoprotectant in the other three species. The level of glycinebetaine in A. marina increased under high salinity. Betaine-aldehyde dehydrogenase (BADH) was detected in all four species, but choline monooxygenase could not be detected. A cDNA library was constructed from the leaves of A. marina. Two kinds of BADH cDNAs were isolated, one homologous to the spinach chloroplast BADH, and the other with unique residues SKL at the end of C-terminus. The BADH transcription levels of the former were higher than those of the latter. The levels of the former BADH increased at high salinity whereas those of the latter were independent of salinity. BADHs were expressed in Escherichia coli and purified. Two kinds of A. marina BADHs exhibited similar kinetic and stability properties, but were significantly different from those of spinach BADH. A. marina BADHs efficiently catalyzed the oxidation of betainealdehyde, but not the oxidation of ω-aminoaldehydes and were more stable at high temperature than the spinach BADH.

The 8 kDa polypeptide in photosystem I is a probable candidate of an iron-sulfur center protein coded by the chloroplast gene frxA

The N‐terminal sequence of the 8 kDa polypeptide isolated from spinach photosystem I (PS I) particles was determined by a gas‐phase sequencer. The sequence showed the characteristic distribution of cysteine residues in the bacterial‐type ferredoxins and was highly homologous to that deduced from the chloroplast gene frxA of liverwort, Marchantia polymorpha. It is strongly suggested that the 8 kDa polypeptide has to be an apoprotein of one of the iron‐sulfur center proteins in PS I particles.

Complete amino acid sequence of 33 kDa protein isolated from spinach photosystem II particles

The amino acid sequence of the 33 kDa protein isolated as an extrinsic protein from spinach photosystem II particles was determined by using solid‐phase sequencing and conventional procedures. The 33 kDa protein was found to be composed of 248 amino acid residues, to lack histidine and to have a molecular mass of 26 663 Da, which was considerably smaller than the value deduced from SDS‐polycrylamide gel electrophoresis. The sequence of the 33 kDa protein was compared with those of the bacterial Superoxide dismutases (SOD) with Mn atoms at an active site. A part of the sequence of the 33 kDa protein was similar to a region in Mn‐SODs from Bacillus stearothermophilus and Escherichia coli, which was expected to be the Mn‐binding site.

The status of cysteine residues in the extrinsic 33 kDa protein of spinach photosystem II complexes

Two cysteine residues of the extrinsic 33 kDa protein in the oxygen-evolving photosystemII (PS II) complexes were found to exist as cystine residues in situ. The 33 kDa protein, when reduced by 2-mercaptoethanol in either the presence or the absence of 6 M guanidine-HCl (Gdn-HCl), could not rebind with the CaCl2-treated PS II complexes, from which the 33 kDa protein was removed, and evolve any oxygen. Two sulfhydryl (SH) groups of the 33 kDa protein were easily reoxidized to a disulfide (S-S) bond by stirring under aerobic conditions with the concomitant regaining of both the binding ability to the CaCl2-treated PS II complexes and the oxygen-evolving activity.The molecular conformation of the 33 kDa protein was examined by circular dichroic (CD) spectrometry in the UV regions to reveal that the conformation in the reduced state was completely different from those of the untreated and reoxidized states. The disulfide (S-S) bond of the 33 kDa protein is thus essential to maintain the molecular conformation required to function.

Purothionin: a seed protein with thioredoxin activity

Purothionins are basic proteins of -SOOOM, [l-3] that have 8 half-cystine residues bonded to form disulfide bridges [4-71. Purothionins occur in the seed endosperm of cereals such as wheat, barley and rye [8,9], and show the curious property of being toxic to animals, when injected intraperitoneally or intravenously [lo], and to certain yeasts [ 1 l] and cultured mammalian cells [ 121 when present in their growth media. T’he physiological function of purothionins has not been established. Purothionins show a chemical resemblance to thioredoxins a group of widely distributed multifunctional proteins oflowM,that undergo oxidation/reduction through the interconconversion of S-S/SH groups. Thioredoxins have been shown to function as regulatory proteins that alter enzyme activity through thiol redox changes [ 131. Thioredoxins appear to play a particularly important regulatory role in photosynthesis through their ability to link light to the activity of several key chloroplast enzymes [ 141. Because of the chemical similarity to thioredoxins, we considered it worthwhile to examine purothionin for thioredoxin activity. We now report that purothionin obtained from wheat endosperm (flour) can substitute for a specific thioredoxin from spinach chloroplasts in the activation of a photosynthetic enzyme. These findings provide a new in vitro assay for conveniently measuring purothionin activity and, in addition, constitute evidence that purothionins inay function as regulatory proteins.

Purification of four pyruvate kinase isozymes of rats by affinity elution chromatography

1. Purification of four isozymes of pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) L, M1, M2 and R was much improved to give good yields by affinity elution chromatography. The enzyme was eluted from a phosphocellulose column with 0.5 mM phosphoenolpyruvate. Types L, M2 and R were stabilized with fructose 1,6-diphosphate throughout the purification procedures. 2. The isozymes were crystallized under various conditions: types L and R were readily crystallized from medium of low ionic strength, types L, M1, and M2 were crystallized from ammonium sulfate solution in different forms in the presence and absence of phosphoenolpyruvate. Type M1 was also crystallized in different forms in the presence and absence of fructose 1,6-diphosphate. 3. Amino acid analyses showed that the compositions of types L and R, and of types M1 and M2, respectively, were very similar.

Estimation of labile sulfide in iron-sulfur proteins

The most commonly used method for labile sulfide determination in iron-sulfur proteins is the colorimetric procedure of Fogo and Popowsky (1) as modified by Lovenberg et al. (2). The sample is directly treated with a mixture of zinc acetate and sodium hydroxide, and then coupled with N,N′-dimethyl-p-phenylenediamine in the presence of ferric chloride to give methylene blue, which is then determined spectrophotometrically. The application of this method to the sulfide analysis of many iron-sulfur proteins has indicated that the amount of detectable labile sulfide is equivalent to the amount of iron present. However, lower results of labile sulfide content have been reported for some iron-sulfur proteins (3,4). Recently, Siegel et al. (5) reported that the total amount of released labile sulfide of NADPH-sulfide reductase depended upon the incubation time with the alkaline zinc reagent. We found that the original methylene blue method was also unsatisfactory for bovine adrenal ferredoxin and the reaction of the protein-bound sulfide with the alkaline zinc reagent was time1 dependent (6,7). In this communication is described an extension of the incubation with the alkaline zinc reagent in original methylene blue method which leads to a quantitative estimation of labile sulfide content in some iron-sulfur proteins.

Novel forms of ferredoxin and ferredoxin-NADP reductase from spinach roots

Ferredoxin and the enzyme catalyzing its reduction by NADPH, ferredoxin-NADP reductase (ferredoxin-NADP+ oxidoreductase or FNR), were found to be present in roots of spinach (Spinacia oleracea). Localization experiments with endosperm of germinating castor beans (Ricinus communis), a classical nonphotosynthetic tissue for cell fractionation studies, confirmed that ferredoxin and FNR are localized in the plastid fraction. Both proteins were purified from spinach roots and found to resemble their leaf counterparts in activity, spectral properties, and complex formation, but to differ in amino acid composition and amino terminal sequence. The results indicate that the primary structures of the FNR and ferredoxin of spinach roots differ from that of the corresponding leaf proteins. Together with earlier findings, the present results provide evidence that nonphotosynthetic plastids, including those of roots, are capable of reducing ferredoxin with heterotrophically generated NADPH.