The alternative oxidase mediated respiration contributes to growth, resistance to hyperosmotic media and accumulation of secondary metabolites in three species.

Plant respiration, similar to respiration in animal mitochondria, exhibits both osmosensitive and insensitive components with the clear distinction that the insensitive respiration in plants is quantitatively better described as 'less' sensitive rather than 'insensitive'. Salicylic hydroxamic acid (SHAM)-sensitive respiration was compared with the respiration sensitive to other inhibitors in rice, yeast and Dunaliella salina. The influence of SHAM was largely in the osmotically less sensitive component and enhanced with external osmotic pressure unlike other inhibitors that inhibited the osmotically sensitive component. SHAM inhibited germination and root growth but not shoot growth. Osmotic remediation of respiration that developed in due course of time with rice seedlings was abolished by SHAM and was not due to water and ionic uptake mechanisms. Yeast and Dunaliella also showed susceptibility of growth and respiration to SHAM. Glycerol retention was influenced by all inhibitors, while growth was inhibited demonstrably by SHAM in Dunaliella. Respiration in plants needs to be seen as a positive contribution to overall growth and not merely for burning away of the biomass.

Respiration hastens maturation and lowers yield in rice.

Role of respiration in plant growth remains an enigma. Growth of meristematic cells, which are not photosynthetic, is entirely driven by endogenous respiration. Does respiration determine growth and size or does it merely burn off the carbon depleting the biomass? We show here that respiration of the germinating rice seed, which is contributed largely by the meristematic cells of the embryo, quantitatively correlates with the dynamics of much of plant growth, starting with the time for germination to the time for flowering and yield. Seed respiration appears to define the quantitative phenotype that contributes to yield via growth dynamics that could be discerned even in commercial varieties, which are biased towards higher yield, despite considerable susceptibility of the dynamics to environmental perturbations. Intrinsic variation, irreducible despite stringent growth conditions, required independent validation of relevant physiological variables both by critical sampling design and by constructing dendrograms for the interrelationships between variables that yield high consensus. More importantly, seed respiration, by mediating the generation clock time via variable time for maturation as seen in rice, directly offers the plausible basis for the phenotypic variation, a major ecological stratagem in a variable environment with uncertain water availability. Faster respiring rice plants appear to complete growth dynamics sooner, mature faster, resulting in a smaller plant with lower yield. Counter to the common allometric views, respiration appears to determine size in the rice plant, and offers a valid physiological means, within the limits of intrinsic variation, to help parental selection in breeding.

Shape anisotropy of lipid molecules and voids.

Biological polymers, viz., proteins, membranes and micelles exhibit structural discontinuities in terms of spaces unfilled by the polymeric phase, termed voids. These voids exhibit dynamics and lead to interesting properties which are experimentally demonstrable. In the specific case of phospholipid membranes, numerical simulations on a two-dimensional model system showed that voids are induced primarily due to the shape anisotropy in binary mixtures of interacting disks. The results offer a minimal description required to explain the unusually large permeation seen in liposomes made up of specific lipid mixtures (Mathai & Sitaramam, 1994). The results are of wider interest, voids being ubiquitous in biopolymers.

Osmotic perturbations induce differential movements in the core and periphery of proteins, membranes and micelles.

Polymeric structures, namely, micelles, membranes and globular proteins share the property of two distinct regions: a hydrophobic core and a hydrophilic exterior. The dynamics of these regions of the polymeric structures were probed using selective fluorophores 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-anilinonaphthalene-8-sulfonate (ANS), respectively. Perturbation of the polymers by external osmotic pressure, ionic strength and temperature was monitored in the two regions using steady state measurements of fluorescence intensity and anisotropy. While the fluorescence lifetime of DPH and ANS did not change significantly, parallel change in steady state anisotropy values and the rotational correlation time indicated mobility in the probe/probe-domain. Osmotic perturbation of the polymers in electrolyte media led to decreased DPH mobility. Enhanced ellipticity at 222 nm in bovine serum albumin was observed in 1.5 M NaCl and sucrose media. ANS exhibited a decreased anisotropy with progressive dehydration in proteins in NaCl media, in dimyristoylphosphatidylcholine (DMPC) vesicles in sucrose media, and in neutral laurylmaltoside micelles in both NaCl and sucrose media. Thus, ANS showed responses opposite to that of DPH in these systems. A comparison with several domain selective probes indicated that DPH reported findings common to depth probes while ANS reported data common to interfacial probes used for voltage monitoring.

Large solutes induce structural perturbations in proteins and membranes.

Structural perturbations in biopolymers with hydrophobic interiors i.e. specific proteins and dimyristoylphosphatidylcholine (DMPC) vesicles were investigated as a function of solute concentrations in the medium. 1,6-diphenyl-1,3,5-hexatriene (DPH) was used as fluorescent probe. Response of DPH was comparable to that of intrinsic tryptophan in BSA in terms of steady state and time resolved fluorescence. The solutes induced a decrease in steady state anisotropy as well as rotational correlation time (computed from lifetime measurements) for DPH in both proteins and membranes. Enhanced access of the quencher potassium iodide to tryptophan in bovine serum albumin (BSA) and ovalbumin, and enhanced terbium leakage in DMPC vesicles induced by various solutes concomitant with decreased anisotropy/correlation time were consistent with structural perturbations of the nature of defects or voids in these polymers.

Solvent interconnectedness permits measurement of proximal as well as distant phase transitions in polymer mixtures by fluorescence.

We monitored the fluorescence intensity and anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) incorporated in bovine serum albumin (BSA) and dimyristoylphosphatidylcholine (DMPC) vesicle membranes, which in turn were embedded in optically clear gelatin solutions, as a function of temperature. DPH in BSA gave unanticipated large changes in fluorescence intensity and anisotropy at the instant of gelatin gel melting. Both steady state anisotropy and fluorescence intensity reported the gel-sol transition point in gelatin unambiguously, which was independently confirmed as physical-pour point of the gel. In the case of DMPC vesicles, fluorescence intensity indicated the gelatin transition, while the anisotropy indicated DMPC phase transition. This fluorescence methodology uniquely offered a common probe for two distinct transitions in two distinct domains interconnected by the solvent, water.

What does a common channel for electrolytes and non-electrolytes in the sperm mean?

Cell volume is central to osmoregulation in intact cells. Bovine spermatozoa, as also other mammalian spermatozoa, exhibit a very rapid regulatory volume decrease (RVD) when exposed to hypotonic saline media. This response, fastest known in animal cells, is mediated by a putative potassium channel which the pharmacological properties of a conventional channel and yet admits both electrolytes and non-electrolytes. The evolutionary basis and functional role of this conserved quinine-sensitive channel in mammalian sperm could offer hitherto unexplored facets of the link(s) between ecology and reproduction.

The positive role of voids in the plasma membrane in growth and energetics of Escherichia coli.

Bacterial respiration, endogenous as well as induced respiration by glucose, lactose and glycine betaine, was found to be sensitive to external solute concentration. Permeability of hydrogen peroxide, a non-electrolyte of molecular size between water and urea, through the bacterial membranes changed directly with the rate of respiration (an activity residing in the bacterial plasma membrane) in E. coli and the enhanced permeability and respiratory activity were highly correlated. Hydrogen peroxide permeability and induction of voids (spaces in the matrix of the bilayer into which hydrophobic fluorescent probes partition, which in turn were used to assess the modulation of these cavities) were shown to be a direct and excellent measure of leak conductance. Fluorescence intensity and anisotropy of the extrinsic fluorescent probes (incorporated by growing bacteria in their presence) decreased with increased respiration in bacteria, consistent with lowered molecular restriction and enhanced hydration in the membrane phase for these probes as seen in dimyristoylphosphatidylcholine bilayers due to phase transition. The physical basis of osmotic phenomena, as a relevant (thermodynamic) volume, could relate to water exchange or compression, depending on the osmotic domain. In the domain of compression in bacteria, i.e. well above the isotonic range, the computed activation volume was consistent with voids in the membrane. This study emphasises a major role of leak conductance in bacterial physiology and growth.

Biology of the inner space: voids in biopolymers

Dynamic states of biopolymers are associated with appearance and disappearance of spaces, or voids, to an extent larger than what is hitherto recognized. Current awareness of the existence of free space in biopolymers is by and large restricted in terms of (i) essentially small pores obeying Boltzman distribution of sizes and energy and (ii) rigid structures dictated by structural constraints, whose volume merely exhibits fluctuations within the thermal limits. Interconnectivity of these spaces within biopolymers such as membranes give rises to new possibilities in addressing some of the long standing problems in understanding catalysis, transport and the derived biological phenomena. Given the a priori recognition that other kinds of space can exist in biopolymers, which are dynamic, predominantly inducible and possibly larger, a new kind of experimentation becomes possible as also a new set of constraints for the acceptability of molecular models of interactions in the explanation of biological phenomena. The theory of adsorption of liquids and presence of structural cavities as exemplified by zeolites competently accounts for much of the current thinking in our understanding of cavities in biopolymers. Induction of (larger) voids requires approaches that are significantly different. We suggest that it is necessary to consider a reservoir of inner space as a specific contribution to the energetics of polymer dynamics. We outline a methodological approach that helps identify these voids as well as biological phenomena in which the notion of dynamics of voids would bring novel insights. Copyright 1998 Academic Press

Volume regulation of spermatozoa by quinine-sensitive channels.

Bovine spermatozoa were shown to exhibit rapid regulatory volume decrease (RVD) when exposed to hypotonic saline media. This quinine- and quinidine-sensitive regulatory volume decrease was coincident with K+ release due to stretch-activation of inhibitor-specific presumptive K+ channels. The regulatory volume decrease response was much faster than a similar phenomenon observed in human peripheral blood lymphocytes. Studies on volume changes in different electrolyte and nonelectrolyte media suggested that: (1) this inhibitor-specific channel could also be a nonspecific pore in the spermatozoal membrane for nonelectrolytes below 150 daltons; (2) subpopulations (of nearly equal size) of the spermatozoa differ in the expression of the pore; (3) capacitation abolishes this distinction between subpopulations of spermatozoa; and (4) the general case of RVD for other mammalian spermatozoa was also established.

The energetic basis of osmotolerance in plants: physical principles

The current impasse in breeding for osmotolerance in plants has deep-seated reasons. The theoretical nature of the problem is not understood by the experimental biologists and agriculturists. Conversely, the diversity of the classes of responses that beset the experimenter are not anticipated by the theoretician. The evidence is mounting in favor of a diffusive mechanism for control of growth by the osmolarity of the medium/soil for plants and microbes. We examined a series of models as a locus of interaction of osmolarity of the medium and the simulations revealed a vast spectrum of possible biological behaviors. Of these, the diffusive mechanism for control velocity of respiratory enzymology was most dominant. The osmotic phenomena were re-evaluated by systematizing the theory in terms of time dependent changes in the organism, as initial responses and subsequent regulatory responses. The theoretical work presented herein leads some novel experimental approaches to define the problem of limits to yield by methods not thus far employed in plant research.Copyright 1997 Academic Press Limited Copyright 1997 Academic Press Limited

Assessment of molecular sieving across bacterial outer membrane of Pseudomonas.

The role of the permeability barrier of the outer membrane of Pseudomonas was re-evaluated based on the physical theory of molecular sieving in view of its intrinsic antibiotic resistance. We developed a set of analytical procedures based on parametric and non-parametric statistical tests to evaluate, validate and adopt the better among a set of competing non-linear models of diffusion. The molecular mass dependence of uptake of non-electrolytes in bacteria yielded a quantitative measure to distinguish between sieving mechanisms and specific uptake/efflux mechanisms. The experimental data, supported by the physical model of DEAE-Sephadex and various analytical models and extensive simulation of the errors, both in measurement and models, yielded evidence consistent with the relaxation of the outer membrane matrix barrier in Pseudomonas.

Influence of osmolality of the medium on photosynthetic electron transport, proton fluxes and photophosphorylation in isolated thylakoids.

The high osmotic potential inhibition of photosynthetic electron transport was determined to be related to membrane compaction rather than to an effect of primary thylakoid volume changes. Osmotic inhibition of proton fluxes and phosphorylation were entirely due to osmotic inhibition of electron transport. The ATPase activity, the nature of coupling and the rate constant of proton efflux were not influenced by osmotic pressure, while the rate constant and the extent of proton influx were inhibited by osmotic pressure.

Stretch sensitivity of transmembrane mobility of hydrogen peroxide through voids in the bilayer. Role of cardiolipin.

Availability of voids for diffusion of quinone in the membrane was shown to be the rate-limiting step in electron transport in mitochondria and chloroplasts (Mathai, J. C., Sauna, Z. E., John, O., and Sitaramam, V (1993) J. Biol. Chem. 268, 15442-15454). The primary role of voids in these diffusion-controlled reactions required a more rigorous documentation of the role of diffusion in membranes by independent measurements. The transbilayer diffusion of hydrogen peroxide as monitored by occluded catalase activity was developed as a kinetically valid probe to specifically address this question. This in turn led to unique results on the mechanistic basis of stretch (= hypo-osmotic) activation of hydrogen peroxide permeation via such voids. The rate of peroxide permeation is shown to be markedly stretch sensitive in some cells/organelles (e.g. peroxisomes) and insensitive in others (e.g. erythrocytes); this was equally true of liposomes prepared from lipids extracted from the corresponding cells/organelles. The molecular basis of stretch sensitivity was uncovered using specific binary mixtures of lipids: while pure phosphatidyl choline liposomes were stretch insensitive, these became sensitive when doped only with specific lipids, viz. cardiolipin and cerebrosides. Cholesterol abolished this stretch sensitivity in ternary mixtures. Induction of stretch sensitivity by cardiolipin was marked by lowering of activation energy for peroxide diffusion, a negative temperature coefficient for glucose permeation while further addition of cholesterol reversed these phenomena. The steady state fluorescence polarization studies revealed intimate correlations between anisotropy, hydrogen peroxide diffusion, and stretch sensitivity consistent with presence of voids in these binary mixtures.

Rate-limiting step in electron transport. Osmotically sensitive diffusion of quinones through voids in the bilayer.

Respiration in mitochondria and photosynthesis in chloroplasts varied with the osmotic stretch of the membrane such that these processes were uniformly inhibited at higher osmolalities. A systematic evaluation of segmental electron transport in these intact particles showed that no individual complex exhibited osmotic sensitivity, whereas osmotic sensitivity appeared wherever the assay involved crossing over the corresponding quinone in the electron transport chain. The evidence was consistent with the rate-limiting step in electron transport being the availability of voids for quinone migration rather than any of the components of electron transport chain per se. Evidence based on quinone reconstitution in mitochondria depleted of quinone by acetone treatment clearly distinguished the kinetic control in the hypotonic domain and diffusive control via availability of voids in the hypertonic domain. Influence as well as the presence of voids was further confirmed in quinone-depleted mitochondria reconstituted with quinone as well as cholesterol. Decrease in lateral diffusion of the fluorescent probe, 12-(9-anthroyl)stearic acid, on osmotic compression of the bilayer is consistent with a change in void size distribution on osmotic compression of the bilayer. A direct correlation between succinate cytochrome c oxidoreductase activity and diffusivity of fluorescent probe 12-(9-anthroyl)stearic acid confirmed the availability of voids as the rate-limiting step in electron transport.

Statistical testing of equality of two break-points in experimental data.

Two examples in quantitative biology are examined to emphasize the need for two-phase regression models: the osmotic behaviour of cells and the non-linear temperature kinetics of membrane-bound enzyme systems. Existing statistical techniques are inadequate to test the equality of break-points of two data sets for specific reasons. We suggest here a pragmatic solution by way of a computer programme useful in applying two-phase regression models to such data sets wherein a decision needs to be made whether the critical transition differs or not.

Membrane instability in respiring mitochondria: role of phosphate.

Metabolically-induced (spontaneous) high amplitude swelling of mitochondria has been shown to be due to a serial disruption of the mitochondrial membranes [D. Sambasivarao & V. Sitaramam (1985), Biochim Biophys Acta, 806, 195-209]. Phosphate- and arsenate-induced swelling was investigated in mitochondria to evaluate the role of phosphate transport in the instability created in the mitochondrial membranes. Phosphate-induced swelling in respiring mitochondria was similar to spontaneous swelling. Both represent essentially colloidal swelling due to the variable porosity induced in the inner membrane to polyols by respiration. Swelling of non-respiring mitochondria at high ammonium phosphate concentrations was, on the other hand, primarily due to high permeability to phosphate. This membrane instability created by phosphate transport in the surrounding lipid involves neither the endogenous nor the exogenous Ca2+.

Charge anisotropy across biological membranes: evidence and implications.

Membrane proteins exhibit charge anisotropy across the bilayer with the vector positive inwards. The proton pumps, primary or secondary, which have been examined as a subset of these membrane proteins, also reveal charge anisotropy based on their sequence data. The direction of the anisotropy appears to satisfy the observed directional gradient of protons mediated by these proteins. A correct description of transport requires attention to local as well as field effects of the charge anisotropy of membrane proteins.

Oxidative phosphorylation in rat liver mitochondria: influence of physical parameters.

Effects of pH, temperature, ionic strength and osmotic pressure on various respiratory states and indices of oxidative phosphorylation in well coupled rat liver mitochondria have been studied. It appears that temperature and osmotic pressure are the most important physical variables, whereas ionic strength and pH were devoid of any significant influence on oxidative phosphorylation. Thus any model for oxidative phosphorylation must critically account for the differential osmotic sensitivity of respiration as well as the curious fact that ADP/O ratio increases as temperature decreases.

Characterization of mitochondrial membrane fragments resulting from spontaneous swelling: novel stimulation of NADH-dependent respiration by carboxylic acids.

Metabolically induced high amplitude swelling of rat liver mitochondria has been found to result in the formation of a heterogeneous population of mitochondrial membranes consisting of right side-out particles with occluded fumarase activity and inside-out particles/fragments capable of NADH-dependent respiration. This rotenone-sensitive, uncoupler-insensitive, NADH-dependent respiration was specifically and instantaneously stimulated by several ligands such as glutamate and malate (which can be metabolized) and, interestingly, even lactate (which could not be metabolized by the swollen mitochondria). These observations suggest that high amplitude swelling results in a novel type of control of respiration in these fragments.