Complex I impairment, respiratory compensations, and photosynthetic decrease in nuclear and mitochondrial male sterile mutants of Nicotiana sylvestris.

We have previously shown that in Nicotiana sylvestris cytoplasmic male-sterile (CMS) mutants where the mtDNA lacks the nad7 gene coding for a subunit of respiratory Complex I (NADH:ubiquinone oxidoreductase, EC 1.6.5.3), glycine (Gly) oxidation was lower than in the wild type and insensitive to rotenone, suggesting Complex I dysfunction. In contrast, the oxidation rate of exogenous NADH and the capacity of the cyanide-resistant respiration (AOX) were enhanced. Here we report that, in contrast to Gly, the rate of malate oxidation was not affected, but proceeded totally in a rotenone-insensitive pathway, strongly suggesting that survival of CMS plants depends on the activation of internal and external alternative NAD(P) H dehydrogenases and that Gly decarboxylase activity depends on Complex I functioning. A similar defect in Complex I activity and Gly oxidation was found in the NMS1 nuclear mutant, defective in the processing of the nad4 transcript, but alternative NAD(P) H dehydrogenases were less activated. In CMS and NMS1, the fraction of the AOX pathway was increased, as compared to wild type, associated with higher amounts of aox transcripts, AOX protein, and plant resistance to cyanide. Non-phosphorylating respiratory enzymes maintained normal in vivo respiration levels in both mutants, but photosynthesis was decreased, in correlation with lower leaf conductance, emphasizing mitochondrial control on photosynthesis.

Purification and characterization of a novel chitinase-lysozyme, of another chitinase, both hydrolysing Rhizobium meliloti Nod factors, and of a pathogenesis-related protein from Medicago sativa roots.

The symbiosis between Rhizobium meliloti and Medicago sativa (Leguminosae) involves the interaction of lipochito-oligosaccharides (Nod factors) excreted by bacteria with specific proteins of the host plant. The cleavage of Nod factors can be used as an enzymic assay to identify novel hydrolytic enzymes. Here a soluble extract of 3-day-old roots was fractionated by anion exchange, affinity chromatography, gel filtration and native electrophoresis. Two acidic chitinases (pI 4.6-5.4), CHIT24 and CHIT36, designated in accordance with their molecular mass in kDa, were separated. CHIT24 cleaves all tested Nod factors to produce lipotrisaccharides with the preference NodRm-V(S)>NodRm-IV >NodRm-IV(S)>=NodRm-IV(Ac,S); it also hydrolyses colloidal 3H-chitin and has lysozyme activity. The kinetics of Nod factor degradation by CHIT24 depends on substrate structural parameters, namely the length of the oligosaccharide chain and sulphation (S) at the reducing end, but not much on acetylation (Ac) at the non-reducing end. The 25-residue N-terminal sequence of CHIT24 has no similarity with known chitinases or lysozymes, indicating that it is a novel type of hydrolase. CHIT36 also hydrolyses NodRm-V(S) into NodRm-III, but it is inactive towards NodRm-IV(S) and NodRm-IV(Ac,S) formed by R. meliloti. Finally, a 17 kDa protein, P17, was co-purified with CHIT24. It neither degrades Nod factors nor exhibits lysozyme activity and shows complete identity, at the 15-residue N-terminal sequence, with a class 10 pathogenesis-related protein, PR-10.

In vivo interference of paromomycin with mitochondrial activity of Leishmania.

Paromomycin is an aminocyclitol aminoglycoside antibiotic used for the treatment of leishmaniasis. In view of the central role of mitochondria in cellular energetics and metabolism, its effect on in vivo mitochondrial activities of Leishmania donovani promastigotes-the parasite flagellate form-was investigated. The approach used flow cytometry, amperometric measure of O2 consumption, and, as a global estimate of mitochondrial dehydrogenases, thiazolyl blue reduction (MTT test); some in vitro controls were also made. When added to promastigote cultures for 24-72 h at 150-200 microM (= LC50), paromomycin doubled the generation time, inhibited respiration, and lowered its associated electric potential difference across mitochondrial membranes, as measured by rhodamine 123 fluorescence. The chemical analogue neomycin was ineffective. Furthermore, the in vivo mitochondrial dehydrogenase activities were lower, seemingly because of the shortage of respiratory substrates. Indeed, succinate addition to paromomycin-treated cultures partly restored mitochondrial membrane potential. However, no immediate effect of paromomycin on respiration was observed, neither inhibition of redox chain nor increase of membrane permeability (uncoupling). It is proposed that paromomycin acts at a metabolic level upstream of the respiratory chain itself. This would have the observed delayed consequence because the cell energy supply would progressively decline since it depends upon the proton gradient-viz., membrane potential-generated by respiration. In conclusion, paromomycin is an antibiotic affecting the cell's energetic metabolism; the respiratory dysfunction it induces may be a crucial aspect of its action against Leishmania and possibly other cells.

Lack of mitochondrial and nuclear-encoded subunits of complex I and alteration of the respiratory chain in Nicotiana sylvestris mitochondrial deletion mutants.

We previously have shown that Nicotiana sylvestris cytoplasmic male sterile (CMS) mutants I and II present large mtDNA deletions and that the NAD7 subunit of complex I (the main dehydrogenase of the mitochondrial respiratory chain) is absent in CMS I. Here, we show that, despite a large difference in size in the mtDNA deletion, CMS I and II display similar alterations. Both have an impaired development from germination to flowering, with partial male sterility that becomes complete under low light. Besides NAD7, two other complex I subunits are missing (NAD9 and the nucleus-encoded, 38-kDa subunit), identified on two-dimensional patterns of mitochondrial proteins. Mitochondria isolated from CMS leaves showed altered respiration. Although their succinate oxidation through complex II was close to that of the wild type, oxidation of glycine, a priority substrate of plant mitochondria, was significantly reduced. The remaining activity was much less sensitive to rotenone, indicating the breakdown of Complex I activity. Oxidation of exogenous NADH (coupled to proton gradient generation and partly sensitive to rotenone) was strongly increased. These results suggest respiratory compensation mechanisms involving additional NADH dehydrogenases to complex I. Finally, the capacity of the cyanide-resistant alternative oxidase pathway was enhanced in CMS, and higher amounts of enzyme were evidenced by immunodetection.

Flow-force relationships in lettuce thylakoids. 1. Strict control of electron flow by internal pH.

The regulation by the proton gradient of the electron flow from water to ferricyanide was investigated in thylakoids extracted from lettuce leaves. When the transmembrane proton current was varied by an uncoupler or by the ATP synthase activity, a unique relationship was found between the rate of ferricyanide reduction and the proton gradient, restricted here to its delta pH component. This behavior was conserved in CF1-depleted thylakoids where the passive proton flow was varied by the concentration of an Fo inhibitor or by the concentration of an uncoupler after 100% inhibition of Fo. This shows that under our experimental conditions no direct proton transfer exists in steady state between the site of regulation of the redox chain and the ATPase. Studies at two different pH's indicate that the internal pH, and not the transmembrane pH difference, controls the electron transfer between PS2 and PS1. Modeling the data suggests that a single deprotonation step is kinetically limiting.

Flow-force relationships in lettuce thylakoids. 2. Effect of the uncoupler FCCP on local proton resistances at the ATPase level.

The relationship between the steady-state proton gradient (delta pH) and the rate of phosphorylation was investigated in thylakoids under various conditions. Under partial uncoupling by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), the rate of ATP synthesis was reduced by less than expected from the decrease of delta pH. This was observed in the case of the pyocyanine-mediated cyclic electron flow around photosystem 1, but not with the H2O-->photosystem 2-->cytochrome b6f-->photosystem 1-->methyl viologen system. In state 4, a unique relation was found between delta pH and the "phosphate potential", delta Gp, regardless of whether the energy level was controlled by light input or FCCP. The anomalous effect of FCCP on the rate of ATP synthesis disappeared when the ATPase was partially blocked by the reversible inhibitor venturicidin, but not in the presence of tentoxin, an irreversible inhibitor. These results are consistent with the existence of a small kinetic barrier for protons, limiting their access to the ATPase. This resistance would be collapsed by FCCP.

Electron transport, Photosystem-2 reaction centers and chlorophyll-protein complexes of thylakoids of drought resistant and sensitive Lupin piants.

Two genotypes ofLupinus albus L., resistant and susceptible to drought, were subjected to water deficiency for up to two weeks. Such treatment progressively lowered the leaf water content from about 85% to about 60% (water potential from -0.8 to -4.3 MPa). Light-saturation curves of the uncoupled electron transport were analyzed according to a simple kinetic model of separated or connected reversible photoreactions. It gives an extrapolated maximum rate (Vmax) and the efficiency for capturing light (Im, which is the light intensity at Vmax/2). For Photosystem 2, Vmax and, less markedly, Im, declined with increasing severity of drought treatment; the artificial donor, diphenylcarbazide, could not restore the activity. One cause of this Photosystem 2 inhibition could be the loss of active Photosystem 2 centers. Indeed, their concentration relative to chlorophyll, estimated by flash-induced reduction of dimethylquinone, was halved by a medium stress. To the extent that it was still not restored by diphenylcarbazide, the site of Photosystem 2 inactivation must have been close to the photochemical trap, after water oxidation and before or at plastoquinone pool. By relating electron transport rate to active centers instead of chlorophyll, no inhibition by drought was detected. Therefore, water stress inactivates specifically Photosystem 2, without impairing a downhill thermal step of electron transport. On the other hand, the decrease of Im suggests that antennae connected to inactive centers may transfer their excitation energy to active neighbors, which implies that antenna network remains essentially intact. Gel electrophoresis confirmed that the apoproteins of the pigment complexes were well conserved. In conclusion, the inactivation of Photosystem 2 may not be a physical loss of its centers and core antennae but probably reflects protein alterations or conformational changes. These may result from the massive decrease of lipids induced by drought (Meyer et al. 1992, Photosynth. Res. 32: 95-107). Both lupin genotypes behaved similarly but, for a same deficiency, the resistant seemed unexpectedly more sensitive to drought.

ATPase state and activity in thylakoids from normal and water-stressed lupin.

To compare with the case of phosphorylation, ATP hydrolysis was investigated in thylakoids from plants submitted to drought. For medium stresses the relationship between hydrolysis rate and deactivation constant of the de-energized enzyme when delta pH varies is unchanged, hence the basic mechanisms are preserved; (PS1-driven) energization than is neither affected, at any light intensity. As drought intensifies, hydrolysis and monogalactolipids fall somewhat in parallel, whereas the deactivation constant changes with digalactolipids and phosphatidic acid content. This alteration of the F0 lipid environment must be transduced to F1, the subunits of which, however, are preserved, as shown by LDS-PAGE. ATP synthesis (thiol-oxidized enzyme) and hydrolysis (thiol-reduced enzyme) show an identical decrease with stress. Thus the latter impairs a common early step, probably the activation of the oxidized enzyme.

An attempt to discriminate catalytic and regulatory proton binding sites in membrane-bound, thiol-reduced chloroplast ATPase.

The question of the possible identity of catalytic and regulatory proton pathways in the chloroplast FoF1 ATPase has been studied using different energy-transfer inhibitors. Venturicidin, a reversible inhibitor of Fo, affects neither the delta mu H(+)-dependent thiol reduction of the membrane-bound chloroplast ATPase nor its ability to be activated by the proton gradient. It seems therefore to block only the proton flow required by the catalytic function of the enzymes. Venturicidin, however, also slows down the deactivation of the thiol-reduced ATPases during uncoupled ATP hydrolysis, following a delta mu H+ activation, but phloridzin, a reversible F1 inhibitor, has the same effect. Tentoxin, an irreversible F1 inhibitor, decreases the rate of ATP hydrolysis but does not affect the rate of deactivation. These findings suggest that catalytic and regulatory H(+)-binding sites are different. No distinction can be made, if any, between protons involved in unmasking the thiol-sensitive groups of F1 and in activating the enzyme. The effect of venturicidin and phloridzin on the deactivation is consistent with an inhibitory effect of newly formed--by ATP hydrolysis--ADP molecules, which might affect the enzyme without passing through the medium. Phosphate at millimolar concentration has an effect similar to low concentrations of phloridzin and venturicidin, probably by a simple back-reaction effect.

Energy coupling, membrane lipids and structure of thylakoids of Lupin plants submitted to water stress.

Bioenergetic properties of thylakoids from plants submitted to a water stress stress (watering stopped for 6-15 days) have been measured in two lupin genotypes characterized as resistant or susceptible to drought. This energy coupling was assessed by flow-force relationships relating the phosphorylation rate to the magnitude of the proton gradient % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqef0uAJj3BZ9Mz0bYu% H52CGmvzYLMzaerbd9wDYLwzYbItLDharqqr1ngBPrgifHhDYfgasa% acOqpw0xe9v8qqaqFD0xXdHaVhbbf9v8qqaqFr0xc9pk0xbba9q8Wq% Ffea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qqQ8frFve9Fve9Ff0dme% GabaqaaiGacaGaamqadaabaeaafiaakabbaaa6daaahjxzL5gapeqa% aiabgs5aenaaxacabaGaeqiVd0galeqabaGaaiOFaaaakmaaBaaale% aacaWGibWaaWbaaWqabeaacqGHRaWkaaaaleqaaaaa!4D55!\[\Delta \mathop \mu \limits^\~ _{H^ + } \]. The fluorescent probe 9-aminoacridine was used to express, as a ΔpH, the whole % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqef0uAJj3BZ9Mz0bYu% H52CGmvzYLMzaerbd9wDYLwzYbItLDharqqr1ngBPrgifHhDYfgasa% acOqpw0xe9v8qqaqFD0xXdHaVhbbf9v8qqaqFr0xc9pk0xbba9q8Wq% Ffea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qqQ8frFve9Fve9Ff0dme% GabaqaaiGacaGaamqadaabaeaafiaakabbaaa6daaahjxzL5gapeqa% aiabgs5aenaaxacabaGaeqiVd0galeqabaGaaiOFaaaakmaaBaaale% aacaWGibWaaWbaaWqabeaacqGHRaWkaaaaleqaaaaa!4D55!\[\Delta \mathop \mu \limits^\~ _{H^ + } \] by calibrating fluorescence quenching against the phosphate potential ΔGp in 'state 4', i.e., when ATP synthesis is strictly balanced by its hydrolysis. This calibration procedure was shown to be unaffected by treatments. At equal energization (iso-ΔpH), ATP synthesis was halved by a medium stress and disappeared for a more severe stress, whereas ΔpH at equal energy input (light) declined only under a severe drought. For an identical ΔpH, PS 1-driven phosphorylation is always more efficient than PS 2, both in control and stressed plants. Thus, uncoupling is not the cause of the phosphorylation decline; moreover, retention of a 'micro-chemiosmotic' type of coupling implies that the distribution of photosystems and ATPases is unchanged. Parallel to these functional alterations, the lipid content of thylakoids dramatically dropped. As galactolipids fell strongly, neutral lipids rose slightly. Fatty acids decreased then increased with stress, yet phosphorylation did not recover in the latter case and membrane permeability to protons remained unaffected. Overall, these observations suggest a preserved thylakoid structure and this was indeed observed on electron micrographs, even for a severe stress. Therefore, the membrane integrity is probably preserved more by the protein network than by the lipid matrix and the loss of the phosphorylating activity mainly reflects a loss of ATPases or at least their inactivation, possibly due to their altered lipid environment. Finally, from the bioenergetic point of view, the susceptible genotype was unexpectedly less affected by drought than the resistant.

Dependence of kinetic parameters of chloroplast ATP synthase on external pH, internal pH, and delta pH.

ATP synthesis by the membrane-bound chloroplast ATPase in the oxidized state of its gamma disulfide bridge was studied as a function of the ADP concentration, delta pH, and external pH values, under conditions where delta pH was clamped and delocalized. At a given pH, the rate of phosphorylation at saturating ADP concentration (Vmax) and the Michaelis constant Km (ADP) depend strictly on delta pH, irrespective of the way the delta pH is generated: there evidently is no specific interaction between the redox carriers and the ATPase. It was also shown that both Km (ADP) and Vmax depend on delta pH, not on the external or internal pH. This suggests that internal proton binding and external proton release are concerted, so that net proton translocation is an elementary step of the phosphorylation process. These results appear to be consistent with a modified "proton substrate" model, provided the delta G0 of the condensation reaction within the catalytic site is low. At least one additional assumption, such as a shift in the pK of bound phosphate or the existence of an additional group transferring protons from or to reactants, is nevertheless required to account for the strict delta pH dependence of the rate of ATP synthesis. A purely "conformational" model, chemically less explicit, only requires constraints on the pK's of the groups involved in proton translocation.

Measurement of chloroplast internal protons with 9-aminoacridine. Probe binding, dark proton gradient, and salt effects.

A defined ratio, gamma, of the total proton uptake to the concentration change of free internal H+ is observed for illuminated envelope-free chloroplasts (Haraux, F. and de Kouchkovsky, Y. (1979) Biochim. Biophys. Acta, 546, 455-471). Proton uptake is measured by the external pH shift, free internal H+ by 9-aminoacridine fluorescence quenching. Extension of this work leads to the following conclusions, which, in the case of 9-aminoacridine behaviour, should apply to any kind of diffusible protonizable delta pH probe: 1. The gamma constancy is preserved when the internal volume (Vi) is modulated by chlorophyll and osmolarity changes: thus, 9-aminoacridine behaves as expected from the delta pH distribution of an amine of high pK; previous doubts on this point are attributed to the lack of control of the external proton uptake. 2. With variable 9-aminoacridine concentration, however, some variation of gamma confirms the existence of slight light-induced probe-membrane interactions. 3. According to the diffuse layer theory, salts decrease the negative potential at the 'plane of closest approach' of the thylakoids, thereby releasing the excess 9-aminoacridine in this diffuse layer, which increases its fluorescence. Although of equal valency, NH4+ is more potent than K+, suggesting competition between amines for specific anionic binding sites. 4. Two categories of membrane modifications are induced by salts: in addition to the above-mentioned electrical effect, mono- and divalent cations at high concentration increase the chloroplast proton binding capacity. La3+ is only able to release the excess dye in the diffuse layer and leaves gamma unchanged. Therefore the probe-membrane interactions should have limited importance for steady-state delta pH measurement. 5. A Donnan-type dark pH difference, which could seriously bias these delta pH estimates, is found experimentally to be less than 2 (no significant gamma change when Vi varies) and even theoretically less than 1 (on the basis of the concentration of the non-diffusible internal protonizable groups). Similarly, the predictable errors of Vi and its possible light-induced variations must have a small effect on delta pH under present experimental conditions.

Quantitative estimation of the photosynthetic proton binding inside the thylakoids by correlating internal acidification to external alkalinisation and to oxygen evolution in chloroplasts.

The external alkalinisation delta pHe, or the rate of oxygen evolution vO2, of a suspension of envelope-free chlorplasts was correlated with their internal acidification, estimated from the transmembrane delta pHei. Knowing the external buffer value, the concentration of the total protons moved Hi was calculated from the delta pHe, measured with a glass electrode ([Hi] was also obtained from vO2), and the free proton concentration [Hi+] was determined from delta pHei, measured with 9-aminoacridine. This gives a ratio gamma i = theta [Hi]/theta [Hi+], which is independent of the thylakoids internal volume. Within a large pHi range, scanned by varying the light intensity, gamma i was kept reasonably constant; it was hardly sensitive to pHi. This apparent invariability implies a continuous change of the internal buffer value beta i with pHi, since beta i/gamma i = -2.3.....10pHi, a relationship which inlcudes neither the total concentration of protonizable groups [Ai] nor pKi. As gamma i approximately Ki[Ai]/(Ki + [Hi+i]2, to keep gamma i constant when pHi drops, pKi and [Ai] must increase. This may be achieved by a progressive unmasking of anionic functions, initially inaccessible in the membrane. The relative slowness of this process may explain why gamma i calculated from the initial kinetics was sometimes smaller in high than in low light, where it always equalled that measured from the steady-state amplitude at all intensities. A small deficit of [Hi+] deduced from what could have been expected from delta pHe may reflect a limited binding of protons in the membrane itself, about 1 H+ for 30--130 chlorophylls (gamma i could be between 70 and 240, more frequently around 100); these numbers varied depending on the samples, but were constant for a given preparation.

Study of the chlorophyll fluorescence in chloroplasts and algae with the plastoquinone antagonist dibromothymoquinone.

The effect of the plastoquinone antagonist dibromothymoquinone on chlorophyll fluorescence in vitro and in vivo was investigated. 1. With chlorophyll a in solution quenching is observed, more efficient than that of p-benzoquinone (the Stern-Volmer constant K equals to 200 M-1); ascorbate removes this effect. 2. With isolated chloroplasts, a dramatic enhancement of quenching occurs (also abolished by ascorbate) in the following order of importance: thermal step, photochemical step (of variable fluorescence), and constant fluorescence; K for the total variable fluorescence approximately equal to 480 000 M-1, for constant approximately equal to 190 000 M-1. Parallel to the quenching effect, an enlargement of the complementary area of the variable fluorescence is observed. Addition of ascorbate suppresses the quenching and this enlargement. If this area in the presence of reduced dibromothymoquinone is similar to that of the control, this means that A, the pool of oxidant next to Q, remains connected to it, a result contradicted by the O2-burst measurement. However, when the excess of unbound dibromothymoquinone is washed out, which removes the quenching effect, a significant decrease of the area is seen, in agreement then with the burst results. Therefore, the meaning of the complementary area and the nature of the controlling factors may be questioned. 3. With whole cells (Chlorella), no quenching is observed; instead, an enhancement of the thermal step is noticed, together with an even larger increase of the complementary area. Dibromothymoquinone being in an oxidized form at the thylakoid level, it is proposed that in intact membranes, the chlorophyll molecules are not readily accessible to it: thus, no quenching is observable, and the predominant effect is on the redox chain. The specific effect of dibromothymoquinone on the thermal step supports the hypothesis that is normally controlled by a secondary quencher, related to plastoquinone.