Ricardo A. Ravizzini

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.

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.

Inhibition of spinach chloroplasts photophosphorylation by the antibiotics leucinostatin and efrapeptin.

The ATPases that catalyze the synthesis of ATP in oxidative and photosynthetic phosphorylation are rather similar with respect to molecular weight, subunit distribution, amino acid composition and coupling activity (for a review see [ 11). However, they are immunologically different and do not replace each other as coupling factors. They also differ in their sensitivity towards some inhibitors. For instance, I.ardy and coworkers have introduced several antibiotics as specific inhibitors of oxidative phosphorylation [2]. The best known and more widely used are oligomycin and aurovertin [3,4]. Although both antibiotics inhibit oxidative phosphorylation their binding sites are different and only aurovertin inhibits the ATPase activity of soluble F1 [4]. On the other hand, they do not affect photophosphorylation except for a weak uncoupling by high concentrations of oligomycin [5]. Recently, Lardy et al; [2] have postulated four mitochondrial binding sites for antibiotics that inhibit phosphoryl transfer. Two of them are in F, and correspond to aurovertin and to efrapeptin. The other two are located on the membrane component of the ATPase complex; one of them is the oligomycin site and the fourth corresponds to leucinostatin. Since photophosphorylation is not affected by oligomycin and aurovertin, we thought it would be interesting to test the effects of leucinostatin and efrapeptin on spinach chloroplasts.This paper shows that both antibiotics inhibited photophosphorylation although by different mechanisms: leucinostatin

Sulphydryl groups in photosynthetic energy conservation. V. Localization of the new disulfide bridges formed by o-iodosobenzoate in coupling factor of spinach chloroplasts.

1. Chemical modification by o-iodosobenzoate of soluble chloroplast coupling factor 1 (CF1) during heat activation resulted in inhibition of its Ca-ATPase activity and in the formation of two new intrapeptide disulfide bridges as suggested by: (a) the disappearance of three out of four accessible thiol groups, two from gamma and one from a beta subunit as a consequence of CF1 modification by o-iodosobenzoate; (b) the total free sulphydryl groups of CF1 were reduced from 8 to 4 after modification of CF1 by o-iodosobenzoate. Two groups disappeared from beta and two from gamma subunits; (c) a second heating step of CF1 in the presence of 10 mM dithioerythritol reversed the inhibition of the ATPase and reduced both the newly formed disulfide bridges and those present in native CF1. 2. Modification of chloroplasts in the light with o-iodosobenzoate resulted in the inhibition of photophosphorylation and ATPase. CF1 isolated and purified from these chloroplasts had its Ca-ATPase activity inhibited and two new disulfide bridges. The total number of free sulphydryl groups was reduced from 8 to 4 and three accessible groups disappeared from beta and gamma subunits.

Effect of aurovertin on energy transfer reactions in Rhodospirillum rubrum chromatophores.

Aurovertin like oligomycin, was introduced by Lardy et al. [l] as an inhibitor of the synthesis of ATP in mitochondria. However, both antibiotics differ in some of their effects on mitochondrial energy transfer reactions. Aurovertin is a better inhibitor of the reactions leading to ATP synthesis than of the ATP utilizing reactions [ 1,2] . Moreover, it inhibits the mitochondrial soluble ATPase while oligomycin does not [3]. Therefore, their sites of action are different: aurovertin acts on the coupling factor 1 (Fr)‘while oligomycin acts on the ATPase complex [3] When aurovertin binds to mitochondria or to soluble Fr its fluorescence is greatly enhanced [4]. This property has lead to the use of aurovertin as a conformational probe of the mitochondrial ATPase [4-61. Oligomycin inhibits energy transfer reactions in R. rubrum chromatophores including photophosphorylation, oxidative phosphorylation and partial reactions, while other reactions like photosynthetic pyrophosphate formation and the activity of the soluble ATPase are not affected [7-91. In this paper we report a study of the effects of aurovertin on the energy transfer reactions of R. rubrum chromatophores and on the membranebound and soluble ATPases. It is concluded that aurovertin is better than oligomycin as inhibitor of these reactions and that its site of action may be the R. ;pubrum coupling factor.

Divalent-cation ionophores and Ca2+ transport in spinach chloroplasts

Processes such as ion transport and energy coupling have been studied with the aid of ionophores in several biological systems including chloroplasts [ 1 lo]. One group of ionophores used is that of the monocarboxylic polyethers of which nigericin, monesins, X-206 and X-537A are examples [3]. They act as potent uncouplers of photophosphorylation depending on the presence of suitable alkali metal cations [4-71. Valinomycin is representative of another group of neutral antibiotics that includes gramicidin, enniatins and macrotetrolides [2,9,10]. Among this group, only gramacidin is a strong uncoupler of photophosphorylation (for a review see [lo]). The actions of valinomycin in chloroplasts are more complex [ 11 ,121. We have recently shown [ 131 that the divalentcation ionophore A23187 uncouples photophosphorylation specially in the presence of Ca2’. One of the carboxylic ionophores, X-537A has been demonstrated to be different from others because it can also transport divalent cations across an organic solvent [I] or biological membranes such as sarcoplasmic reticulum vesicles [8,14] . Beauvericin, a cyclic hexadepsipeptide ionophore of the enniatin family [ 151, has been found to induce transport of monovalent cations across the mitochondrial membrane [ 10,161. Recently Roeske et al. [ 171 found that beauvericin also has a high affinity for calcium and barium ions and Prince et al. [ 181 found that beauvericin but not anniatin transport calcium ions in liposomes and bacterial chromatophores. In this paper we report the effects of beauvericin and X-537A on proton and calcium transport and on

The deactivation process of the proton-ATPase in isolated chloroplasts and whole leaves

Abstract Using rapid micromethods for chloroplast isolation and ATPase solubilization from preilluminated leaves, the deactivation of the proton-ATPase after different treatments was compared. The rate of decay of the ‘in vivo’ light-activated membrane-bound Mg 2+ -ATPase was highly dependent on temperature. However, the soluble Ca 2+ -ATPase, extracted from the temperature-inactivated membrane-bound ATPase, was active. Coupling factor 1 with a manifest and stable Ca 2+ -ATPase activity was also solubilized from chloroplasts activated by light in whole leaves and deactivated after chloroplast isolation with gramicidin D. Deactivation of the proton-ATPase in isolated chloroplasts was only associated with the dissipation of the proton gradient. Reaction of the accessible sulfhydryl groups of the membrane-bound proton-ATPase with iodoacetamide prevent inactivation of the enzyme by oxidants. However, the iodoacetamide treatment had not effect on the temperature-dependent decay. The rate of deactivation of the proton-ATPase in whole leaves was similar for both membrane-bound and soluble ATPases. Thus, the oxidation process may play an important role in physiological conditions.

Light Activation in vivo of the Chloroplast Proton ATPase: Effect on the Pi-ATP Exchange Reaction

ATP synthesis and hydrolysis in chloroplasts are catalyzed by the proton -ATPase complex. The turnover capacity of the enzyme seems to be highly regulated. Photophosphorylating and ATPase activities were higher in chloroplasts rapidly prepared from preilluminated leaves (Morita “el al”, 1982; Vallejos “et al”, 1983).