Rubén H. Vallejos

Hygromycin resistance as an efficient selectable marker for wheat stable transformation.

SummaryA highly efficient method for stable wheat transformation using hygromycin resistance as a selectable marker is described. Young embryogenic calli growing from immature wheat embryos were transformed using a gunpowder-driven microparticle accelerator. Transgenic wheat plants were determined by PCR amplification of transgene fragments and confirmed by Southern hybridization, activity of the transgene expression and by analysis of the progeny. The hpt gene was as good as or a better selectable marker than the bar gene with an average efficiency (number of transgenic plants relative to the number of bombarded calli) of 5.5% compared with 2.6% for the bar gene.

A Simple Method for Isolating DNA of High Yield and Quality from Cotton (shape Gossypium hirsutum L.) Leaves

An easy, reproducible and fast procedure to isolate DNA from cotton leaves is described. The addition of 0.5 M glucose in the extraction buffer avoids browning by polyphenolic compounds and improves the quality of DNA for molecular analysis. The DNA yield ranged between 150–400 mg per gram of fresh tissue. The DNA was suitable for digestion by restriction enzymes and amplificatiion by Taq DNA polymerase.

On the conformation of reconstituted ferredoxin:NADP+ oxidoreductase in the thylakoid membrane. Studies via triplet lifetime and rotational diffusion with eosin isothiocyanate as label

Eosin isothiocyanate was covalently bound to isolated ferredoxin-NADP+ reductase under protection of the NADP-binding domain. The bound label did not impair the functional reconstitution of the enzyme into depleted thylakoid membranes. Laser spectrophotometric experiments were carried out on thylakoids which were reconstituted with labeled ferredoxin-NADP+ reductase. Bound eosin isothiocyanate was used as a spectroscopic probe for conformational changes of ferredoxin-NADP+ reductase in either of two ways: We studied the rotational diffusion of labeled ferredoxin-NADP+ reductase in the membrane by the photoselection technique, and we studied the triplet lifetime of bound eosin, which measures polypeptide chain flexibility (via access of oxygen) around the binding site. The latter technique was complemented by measurements of the librational motion of bound dye. We observed: (1) When ferredoxin is absent, ferredoxin-NADP+ reductase undergoes very rapid rotational diffusion in the thylakoid membrane (correlation time less than 1 μs at 10°C). This is drastically slowed down (40 μs) upon addition of water-soluble ferredoxin. We propose that ferredoxin mediates the formation of a ternary complex with ferredoxin-NADP+ reductase and the Photosystem I complex. According to our data, this complex would live longer than required for the photoreduction of ferredoxin-NADP+ reductase by Photosystem I via ferredoxin. (2) Under the given incubation conditions, the binding sites for eosin isothiocyanate were located in the FAD domain of ferredoxin-NADP+ reductase. We found increased chain flexibility in this domain upon addition of NADP. This suggests induced fit for the binding of NADP and allosteric control of the FAD domain by the remote NADP domain. (3) Acidification of the internal phase of thylakoids decreased the chain flexibility in the FAD domain. This is of particular interest, since ferredoxin-NADP+ reductase is a peripheral external membrane protein. It suggests the existence of a binding protein for the oxidoreductase which spans the membrane and senses the internal pH

Multiple shoot induction and plant regeneration from embryonic axes of cotton

Cytokinins are involved in shoot development of plants. Events of multiple bud formation and shoot development in apical embryonic axes of cotton treated for 2 or 20 days with the cytokinin benzyladenine (BA), were compared with the development of untreated control axes. Meristematic regions (supernumerary vegetative buds) were observed in axes treated for 20 days with BA. An average of 3.4 shoots per embryonary axis was obtained when explants were cultured on medium supplemented with 3 mg l-1 BA. Higher and lower concentrations of the growth regulator yielded fewer shoots per explant. Results shown in this report suggest that BA is directly responsible for re-programming the embryonic apical meristem axes of cotton toward the production of multiple buds and subsequent shoot development.

Isolation and sequencing of an active-site peptide from spinach ferredoxin-NADP+ oxidoreductase after affinity labeling with periodate-oxidized NADP+

Spinach ferredoxin-NADP+ oxidoreductase was inactivated by treatment with 2,3-dialdehyde NADP+ (periodate-oxidized NADP+), which selectively modifies a lysine residue at the nucleotide-binding domain of the enzyme. The identity of the derivatized residue was ascertained by thin-layer chromatography of the protein hydrolysate. Reductase that had been labeled with periodate-oxidized NADP+ and NaB3H4 was treated with trypsin, and samples of the tryptic digest were subjected to reverse-phase high-performance liquid chromatography. The radioactivity profiles showed modification of one specific peptide. The primary structure of this peptide was found to be Gly-Glu-Lys*-Met-Tyr-Ile-Gln-Thr-Arg, where Lys* represents the derivatized lysine. The sequence obtained corresponds to residues 242-250 in the primary structure of spinach ferredoxin-NADP+ reductase recently reported [Karplus et al. (1984) Biochemistry 23, 6576-6583].

Stable wheat transformation obtained without selectable markers

Transgenic wheat plants without the selectable marker gene were obtained either in the presence or in the absence of selective pressure during the transformation protocol. When using hygromycin as selective agent in a co-transformation experiment involving a mixture of plasmids pGL2, containing the hpt gene, and pAI1Gus, containing the uidA gene, 3 out of 19 transgenic wheat plants had the uidA gene alone as shown by Southern blots. The gene was transmitted to the progeny following Mendelian rules. Segregation and loss of the selectable marker gene was also found in three out of six events from other experiments where high-molecular-weight glutenin genes were expressed or over-expressed. On the other hand, in 7 experiments where no selective pressure was applied and that involved 1016 bombarded explants, 23 transgenic wheat plants were obtained. The uidA gene was stably integrated as suggested by its transmission to the progeny.

Effect of chelerythrine on mitochondrial energy coupling

Chelerythrine is a 1:2-benzo-phenanthridine alkaloid found in Papaveraceae and Rutaceae [ 1,2] . It is a powerful fungicide [l] and might be responsible, together with related alkaloids, for endemic glaucoma [31. In a study of the effect of several alkaloids on yeast metabolism, chelerythrine was found to be one of the stronger inhibitors of yeast respiration [4] . This inhibition seemed to take place in yeast mite chondria. We report here a study of the effect of chelerythtine on mitochondrial energy coupling mechanisms. Low concentrations of chelerythrine inhibited energy transfer and coupled respiration in rat liver mitochondria. Inhibition of respiration was not reversed by uncouplers of oxidative phosphorylation. Chelerythrine did not inhibit electron transport in submitochondrial particles. It behaved as an uncoupler at high concentrations but caused only a transient stimulation of state “4” [5] respiration.

Protein phosphorylation in the photosynthetic bacterium Rhodospirillum rubrum

Endogenous protein phosphorylation in cellular fractions from Rhodospirillum rubrum was manifested after exposure to [γ‐32P]ATP. At least six phosphorylated protein bands of 90, 86, 64, 31, 13 and 11 kDa were found in the cell‐free extract. Treatment of the 64‐kDa band with V8 protease yielded smaller radioactive bands. Phosphoserine, phosphothreonine and phosphotyrosine were detected after acid hydrolysis of the phosphorylated fractions. Protein phosphorylation in all the fractions was insensitive to cAMP, did not recognize exogenous protein substrates and was rapidly reverted upon elimination of the excess of [γ‐32P]ATP. The chlorophyll‐anthena apoprotein from R. rubrum chromatophores overlapped the 13‐kDa phosphorylated band during gel filtration by high‐pressure liquid chromatography suggesting that it is one of the substrates of the protein kinase(s) of R. rubrum.

Conformational changes of the isolated ferredoxin-NADP-oxidoreductase upon nucleotide binding as revealed by the triplet lifetime of bound eosin-SCN

The existence in chloroplasts of a flavoprotein with NADPH-speciflc diaphorase activity was first reported by Avron and Jagendorf [ 1 ] and later it was discovered that this enzyme is essential for the photoreduction of NADP + [2]. It is active when isolated in monomeric form at M r 35 000-40 000 [3]. In the thylakoid membrane it is accessible to impermeant antibodies and probably located at the outer side of the nonstacked parts of the membrane [4,5]. Recently, regulation of the reductase activity by pH and by light, via a transmemb rane pH-difference, was demonstrated [6,7]. In vitro the reductase forms an equimolar complex with NADP ÷ which differs from the uncomplexed form in its visible absorption spectrum [8], fluorescence yield of the flavin moeity [9] and in sensitivity to SH-reagents [ 10]. These alterations have been ascribed to conformational changes of the protein, without further specification of their nature and their physiological role. It is known, however, that the complex formation requires unmodified lysyl [ 11], arginyl [12] and carboxyl groups [7]. We attempted to further characterize the conformational changes with the probe eosin-isothiocyanate. We have applied eosin-SCN to study conformational changes of the membrane-bound [13] as well as of the isolated coupling factor of photophosphorylation [19,22]. When excited with a short laser flash eosin is efficiently transferred into a relatively stable triplet state. When iosin-SCN is covalently bound to an enzyme its triplet lifetime depends on the access of oxygen to the respective binding site. This can vary under conformational changes [13], shorter lifetime is a qualitative indicator for closer proximity of a site to the surface of a protein. Besides via its triplet lifetime we also used eosin-SCN for photoselection studies on the rotational diffusion of the coupling factor (CFI) in the thylakoid membrane. The rotational correlation time was also dependent, e.g., on the existence of a protonmotive force across the membrane and on ATP, served as another indicator of conformational changes [13]. In this study we labelled the isolated and purified ferredoxin-NADP reductase with eosin-isothiocyanate and measured the activity of the enzyme and the triplet lifetime of the probe. We found at least two binding sites which differ in their effect on the activity and in their triplet lifetime. Binding of eosin-SCN to site A inhibits the NADPH-diaphorase activity. The triplet lifetime is rather short, and therefore site A will be close to the surface of the protein. In contrast to this, binding of eosin-SCN to site B does not affect the diaphorase activity but rather the interaction with ferredoxin. The triplet lifetime of eosin-SCN at site B is longer than at A; however, it is shortened upon binding of NADPH to the (unmodified) site A. This may indicate that the environment of the ferredoxin binding site of the reductase is modified by the presence of NADPH at the other catalytic site.

Growth of water hyacinth (Eichhornia crassipes (Mart.) Solms) in the middle Paraná River (Argentina)

The productivity of water hyacinth in the middle Paraná River was measured at three initial biomass levels by means of periodic harvesting of plants contained in 2 m2 floating baskets. The measuring period extended between August 1981 and July 1982. Initial biomass densities were 2, 5 and 10 kg (fw) m−2.Compared with inner island ponds, flowing waters connected to the main river showed better conditions for water hyacinth growth. The duration of the growth period was some 30% longer than in island ponds, probably due to the relatively warmer river waters. Productivity ranged between 108 and 164 g (fw) m −2 d−1 (annual average) for the lower and higher initial biomass values, respectively.