Viscoelastic drops moving on hydrophilic and superhydrophobic surfaces.

So-called "superhydrophobic" surfaces are strongly non-wetting such that fluid droplets very easily roll off when the surface is tilted. Our interest here is in understanding if this is also true, all else held equal, for viscoelastic fluid drops. We study the movement of Newtonian and well-characterised constant-viscosity elastic liquids when various surfaces, including hydrophilic (smooth glass), weakly hydrophobic (embossed polycarbonate) and superhydrophobic surfaces (embossed PTFE), are impulsively tilted. Digital imaging is used to record the motion and extract drop velocity. Optical and SEM imaging is used to probe the surfaces. In comparison with "equivalent" Newtonian fluids (same viscosity, density surface tension and contact angles), profound differences for the elastic fluids are only observed on the superhydrophobic surfaces: the elastic drops slide at a significantly reduced rate and complex branch-like patterns are left on the surface by the drop's wake including, on various scales, beads-on-a-string-like phenomena. The strong viscoelastic effect is caused by stretching filaments of fluid from isolated islands, residing at pinning sites on the surface pillars, of order ∼30 µm in size. On this scale, the local strain rates are sufficient to extend the polymer chains, locally increasing the extensional viscosity of the solution, retarding the drop.

Purely elastic flow asymmetries.

Using a numerical technique we demonstrate that the flow of the simplest differential viscoelastic fluid model (i.e., the upper-convected Maxwell model) goes through a bifurcation to a steady asymmetric state when flowing in a perfectly symmetric "cross-slot" geometry. We show that this asymmetry is purely elastic in nature and that the effect of inertia is a stabilizing one. Our results are in qualitative agreement with very recent experimental visualizations of a similar flow in the microfluidic apparatus of Arratia et al.

Vacuolar H(+)-translocating pyrophosphatase is induced by anoxia or chilling in seedlings of rice.

The present study was undertaken to determine whether vacuolar H(+)-pyrophosphatase (V-PPase) might replace vacuolar H(+)-ATPase under energy stress due to anoxia or chilling in anoxia-tolerant species such as rice (Oryza sativa L.) and corn (Zea mays L.). The relative transcript level of V-PPase in rice seedlings, like that of alcohol dehydrogenase 1, increased greatly under anoxia and declined again when the seedlings were returned to air. However, the distribution of transcripts in root, shoot, and seed differed somewhat from that of alcohol dehydrogenase 1. Immunoreactive V-PPase protein and V-PPase enzyme specific activity in a tonoplast fraction from rice seedlings increased progressively with time of anoxia or chilling at 10 degrees C, showing a 75-fold increase after 6 d of anoxia, compared with a 2-fold increase of vacuolar H(+)-ATPase activity. When the seedlings were returned to air, the specific activity returned to its initial level within 2 d. After 6 d of chilling at 10 degrees C, V-PPase specific activity reached a level 20-fold of that at 25 degrees C. In microsomes of corn roots, V-PPase specific activity did not respond to anoxia but was constitutively high. It is proposed that V-PPase can be an important element in the survival strategies of plants under hypoxic or chilling stress.

Potassium transport into plant vacuoles energized directly by a proton-pumping inorganic pyrophosphatase.

Potassium is accumulated in plant vacuoles against an inside-positive membrane potential. The mechanism facilitating energized K+ transport has remained obscure. However, electrogenic activity of the inorganic pyrophosphatase (H(+)-PPase) at the vacuolar membrane is dependent on cytoplasmic K+, raising the possibility that the enzyme translocates K+ into the vacuole. Membrane currents generated by the H(+)-PPase were measured (using a patch clamp technique) in intact vacuoles isolated from Beta vulgaris storage tissue. A significant orthophosphate-dependent outward current mediated by the enzyme in reverse mode is evoked only when potassium is present at the vacuolar face of the tonoplast, suggesting that potassium is a translocated ion. Furthermore, current-voltage analysis of the effects of extravacuolar potassium and pH on the reversal potential of the H(+)-PPase-generated current points to direct translocation of K+ and H+ by the enzyme. Thus the H(+)-PPase represents a distinct class of eukaryote translocase and could facilitate vacuolar K+ accumulation in vivo.

Vacuolar H(+)-translocating pyrophosphatases: a new category of ion translocase.

The membrane surrounding the central vacuole of plant cells contains an H(+)-translocating ATPase (H(+)-ATPase) and an H(+)-translocating inorganic pyrophosphatase (H(+)-PPase). Both enzymes are abundant and ubiquitous in plants but the H(+)-PPase is unusual in its exclusive use of inorganic pyrophosphate (PPi) as an energy source. The lack of sequence identity between the vacuolar H(+)-PPase and any other characterized ion pump implies a different evolutionary origin for this translocase. The existence of the vacuolar H(+)-PPase, in conjunction with increasing recognition of PPi as a key metabolite in plant systems, necessitates reconsideration of ATP as the primary energy source for membrane transport in plant cells.

Proton transport and phosphorylation of tonoplast polypeptides from zucchini are stimulated by the phospholipid platelet-activating factor.

The ether phospholipid platelet-activating factor and certain similar phospholipids, including lysophosphatidylcholine, are known to stimulate both H(+) transport and protein phosphorylation in plant microsomal membranes. In the present work, several polypeptides in highly purified tonoplast membranes from zucchini (Cucurbita pepo L.) showed platelet-activating factor-dependent phosphorylation. Comparison of protein phosphorylation in different membrane fractions separated by sucrose step density gradient centrifugation indicated that some of the phosphoproteins were contaminants or were common to several membrane fractions, but platelet-activating factor-dependent phosphorylation of peptides at 30, 53, and perhaps 100 kilodaltons was tonoplast specific. The phosphoprotein of 53 kilodaltons was shown by three different approaches (one- and two-dimensional polyacrylamide gel electrophoresis, western blots, and immunoprecipitation) to cross-react with antibody raised against the B subunit of the tonoplast ATPase from red beet (Beta vulgaris L.).

Radiation-inactivation analysis of vacuolar H(+)-ATPase and H(+)-pyrophosphatase from Beta vulgaris L. Functional sizes for substrate hydrolysis and for H+ transport.

The functional sizes of the vacuolar H(+)-ATPase (V-ATPase; EC 3.6.1.34) and H(+)-pyrophosphatase (PPase; EC 3.6.1.1) from vacuolar membranes of red beet (Beta vulgaris L.) were estimated by radiation inactivation, both for substrate hydrolysis and for H+ transport. For the V-ATPase, the radiation-inactivation size for H+ transport was 446 (403-497) kDa and that for ATP hydrolysis was 394 (359-435) kDa. The low values of both of these estimates suggest that not all subunits which may co-purify with V-ATPases are required for either hydrolysis or transport. For the PPase, the radiation-inactivation size for hydrolysis was 91 (82-103) kDa, suggesting that the minimum functional unit for hydrolysis is the 81 kDa monomer. In contrast to the V-ATPase, the PPase gave a radiation-inactivation size for transport which was 3-4-fold larger than that for hydrolysis (two estimates for transport gave 307 and 350 kDa), indicating that a single catalytic subunit is insufficient for transport activity.

Molecular cloning and sequence of cDNA encoding the pyrophosphate-energized vacuolar membrane proton pump of Arabidopsis thaliana.

The energy-dependent transport of solutes across the vacuolar membrane (tonoplast) of plant cells is driven by two H+ pumps: a vacuolar ("V-type") H(+)-ATPase (EC 3.6.1.3) and a H(+)-translocating (pyrophosphate-energized) inorganic pyrophosphatase (H(+)-PPase; EC 3.6.1.1). The H(+)-PPase, like the V-type H(+)-ATPase, is abundant and ubiquitous in the vacuolar membranes of plant cells, and both enzymes make a substantial contribution to the transtonoplast H(+)-electrochemical potential difference. Here, we report the cloning and sequence of cDNAs encoding the tonoplast H(+)-PPase of Arabidopsis thaliana. The protein predicted from the nucleotide sequence of the cDNAs is constituted of 770 amino acids and has a molecular weight of 80,800. It is a highly hydrophobic integral membrane protein, and the structure derived from hydrophilicity plots contains at least 13 transmembrane spans. Since the tonoplast H(+)-PPase appears to be constituted of one polypeptide species and genomic Southern analyses indicate that the gene encoding the Mr 80,800 polypeptide is present in only a single copy in the genome of Arabidopsis, it is suggested that the H(+)-PPase has been cloned in its entirety. The lack of sequence identities between the tonoplast H(+)-PPase and any other characterized H+ pump or PPi-dependent enzyme implies a different evolutionary origin for this translocase.

Cytosolic calcium regulates a potassium current in corn (Zea mays) protoplasts.

The voltage- and time-dependent K+ current, IK+ out, elicited by depolarization of corn protoplasts, was inhibited by the addition of calcium channel antagonists (nitrendipine, nifedipine, verapamil, methoxyverapamil, bepridil, but not La3+) to the extracellular medium. These results suggested that the influx of external Ca2+ was necessary for K+ current activation. The IC50, concentration of inhibitor that caused 50% reduction of the current, for nitrendipine was 1 microM at a test potential of +60 mV following a 20-min incubation period. In order to test whether intracellular Ca2+ actuated the K+ current, we altered either the Ca2+ buffering capacity or the free Ca2+ concentration of the intracellular medium (pipette filling solution). By these means, IK+out could be varied over a 10-fold range. Increasing the free Ca2+ concentration from 40 to 400 nM also shifted the activation of the K+ current toward more negative potentials. Maintaining cytoplasmic Ca2+ at 500 nM with 40 nM EGTA resulted in a more rapid activation of the K+ current. Thus the normal rate of activation of this current may reflect changes in cytoplasmic Ca2+ on depolarization. Increasing intracellular Ca2+ to 500 nM or 1 microM also led to inactivation of the K+ current within a few minutes. It is concluded that IK+out is regulated by cytosolic Ca2+, which is in turn controlled by Ca2+ influx through dihydropyridine-, and phenylalkylamine-sensitive channels.

Pharmacology of the Ca2(+)-dependent K+ channel in corn protoplasts.

We investigated the sensitivity of the Ca2(+)-dependent K+ current, IK(Ca), present in corn protoplasts, to different K+ channel blockers. IK(Ca) was inhibited by external Cs+ (10 mM), Ba2+ (10 mM), and quinine (0.5 mM): reagents which block many types of outward-rectifying K+ channels. In contrast 4-aminopyridine (5 mM), an inhibitor of delayed rectifier or inactivating K+ currents, had no effect. Neither of the peptide toxins, apamin or charybdotoxin, specific for Ca2(+)-dependent K+ channels in animal cells, inhibited currents when used in the nanomolar concentration range. However, higher levels of charybdotoxin (10 microM) caused marked reduction of IK(Ca).

Evolution of the vacuolar H+-ATPase: implications for the origin of eukaryotes.

Active transport across the vacuolar components of the eukaryotic endomembrane system is energized by a specific vacuolar H+-ATPase. The amino acid sequences of the 70- and 60-kDa subunits of the vacuolar H+-ATPase are approximately equal to 25% identical to the beta and alpha subunits, respectively, of the eubacterial-type F0F1-ATPases. We now report that the same vacuolar H+-ATPase subunits are approximately equal to 50% identical to the alpha and beta subunits, respectively, of the sulfur-metabolizing Sulfolobus acidocaldarius, an archaebacterium (Archaeobacterium). Moreover, the homologue of an 88-amino acid stretch near the amino-terminal end of the 70-kDa subunit is absent from the F0F1-ATPase beta subunit but is present in the alpha subunit of Sulfolobus. Since the two types of subunits (alpha and beta subunits; 60- and 70-kDa subunits) are homologous to each other, they must have arisen by a gene duplication that occurred prior to the last common ancestor of the eubacteria, eukaryotes, and Sulfolobus. Thus, the phylogenetic tree of the subunits can be rooted at the site where the gene duplication occurred. The inferred evolutionary tree contains two main branches: a eubacterial branch and an eocyte branch that gave rise to Sulfolobus and the eukaryotic host cell. The implication is that the vacuolar H+-ATPase of eukaryotes arose by the internalization of the plasma membrane H+-ATPase of an archaebacterial-like ancestral cell.

Purification of an h-translocating inorganic pyrophosphatase from vacuole membranes of red beet.

An H(+)-translocating inorganic pyrophosphatase (PPase) was isolated and purified from red beet (Beta vulgaris L.) tonoplast. One major polypeptide of molecular weight 67 kilodalton copurified with fluoride-inhibitable PPase activity when subjected to one-dimensional polyacrylamide gel electrophoresis. Overall, a 150-fold purification of the PPase was obtained, from the tonoplast fraction, through anion exchange chromatography of the detergent-solubilized membranes followed by ammonium sulfate precipitation and gel filtration chromatography. The purified polypeptide showed no cross-reactivity with antibodies raised against the 67 kilodalton subunit of the tonoplast ATPase.

Characterization of potassium-dependent currents in protoplasts of corn suspension cells.

Protoplasts obtained from corn (Zea mays) suspension cells were studied using the whole cell patch-clamp technique. One time-independent current, as well as two time-dependent currents were identified. All three currents were reduced by tetraethylammonium (9 millimolar), a K(+) channel blocker. The time-independent current had a nearly linear current-voltage relationship and its reversal potential, defined as the voltage at which there is zero current, was highly dependent on the extracellular potassium concentration. One of the two time-dependent currents was activated, with rapid kinetics, by membrane hyperpolarization to potentials more negative than -100 millivolts. The second time-dependent current was activated with a sigmoidal time course by membrane depolarization to potentials more positive than -60 millivolts. It exhibited no inactivation and was carried primarily by potassium ions. These characteristics suggest that this latter current is caused by the voltage-dependent opening of delayed-rectifier K(+) channels. These three currents, which are not generated by the plasmalemma H(+)-ATPase, are likely to assist in the regulation of the cellular K(+) fluxes and membrane potential.

Molecular cloning and relationship to freezing tolerance of cold-acclimation-specific genes of alfalfa.

Cold-acclimation-specific (CAS) gene expression has been examined by screening a cDNA library prepared from poly(A)(+) RNA of cold-acclimated seedlings of a freezing-tolerant variety of alfalfa (Medicago falcata cv Anik). Three CAS cDNA clones, pSM784, pSM2201, and pSM2358, representing different sequence species, have been used to investigate the relative abundance and time-course of accumulation of corresponding transcripts. Results obtained show that the expression of these CAS genes is regulated in a coordinated manner most likely at the level of transcription. The expression of genes, as measured by mRNA abundance corresponding to the three CAS cDNA clones, is not stimulated or induced by heat shock, water stress, abscisic acid, or wounding. A positive correlation is observed between the expression of these cloned sequences and the degree of freezing-tolerance in four alfalfa cultivars.

cDNA sequence and homologies of the "57-kDa" nucleotide-binding subunit of the vacuolar ATPase from Arabidopsis.

Functional and structural similarities among a wide variety of endomembrane H+-ATPases suggest that they form a distinct class with a common origin. Immunological studies (Manolson, M. F., Percy, J. M., Apps, D. K., Xie, X. S., Stone, D. K., and Poole, R. J. (1987) in Proceedings of the Membrane Protein Symposium (Goheen, S. C., ed) pp. 427-434, Bio-Rad, Richmond, CA, and M. F. Manolson, J. M. Percy, D. K. Apps, X. S. Xie, D. K. Stone, M. Harrison, D. J. Clarke, R. J. Poole, unpublished data) support this idea and suggest an evolutionary relationship between the endomembrane and F0F1 ATPases. Further examination of relationships necessitates comparison of protein/nucleic acid sequence data. To this end, we have cloned and sequenced the cDNA encoding the 57-kDa polypeptide of the Arabidopsis vacuolar membrane H+-ATPase. To our knowledge, this is the first report of the sequence of a "57-kDa" subunit for plant or animal endomembrane H+-ATPase. This cDNA encodes a hydrophilic polypeptide containing a putative ATP binding site. Lack of a secretion signal sequence suggests it is not processed through the endoplasmic reticulum but translated on cytosolic ribosomes. Comparison of protein sequences shows the 57-kDa subunit from Arabidopsis to be nearly identical with the corresponding subunit in Neurospora vacuolar membrane H+-ATPase, very similar to the beta subunit of the archaebacterium Sulfolobus, and slightly, but nevertheless significantly, homologous to the alpha and beta subunits of the F0F1-ATPases. These results suggest that these different classes of ATPases have evolved from a common ancestor.

Abscisic Acid-regulated gene expression in relation to freezing tolerance in alfalfa.

A comparison of abscisic acid (ABA)-induced and cold-acclimation-induced freezing tolerance in two alfalfa cultivars (Medicago falcata cv Anik and Medicago sativa v Trek) indicates that ABA alone can increase freezing tolerance to some extent, but for the development of maximum tolerance, cold acclimation is essential. Analysis of in vivo-labeled proteins of ABA-treated seedlings reveals that ABA causes several changes in the pattern of protein synthesis. While some of these changes appear to be similar to those induced by cold acclimation, others seem to be specific to ABA treatment. From a cDNA library constructed against poly(A(+)) RNA of a freezing-tolerant alfalfa cultivar, Anik, a cDNA clone, pSM1409, has been isolated. Expression of the gene corresponding to this clone, as determined by northern hybridization, is regulated most likely at the transcriptional level by cold acclimation and exogenously supplied ABA. However, the increase in the transcript level is much greater in the freezing-tolerant cultivar Anik than in the relatively freezing-sensitive cultivar, Trek. The role of ABA in the acquisition of freezing tolerance is discussed.

Alterations in Membrane Protein-Profile during Cold Treatment of Alfalfa.

Changes in pattern of membrane proteins during cold acclimation of alfalfa have been examined. Cold acclimation for 2 to 3 days increases membrane protein content. Labeling of membrane proteins in vivo with [(35)S]methionine indicates increases in the rate of incorporation as acclimation progresses. Cold acclimation induces the synthesis of about 10 new polypeptides as shown by SDS-PAGE and fluorography of membrane proteins labeled in vivo.

Inhibition of na/h antiport activity in sugar beet tonoplast by analogs of amiloride.

The effects of amiloride and a series of amiloride analogs have been tested on the Na(+)/H(+) antiport activity in intact vacuoles and tonoplast vesicles isolated from sugar beet cell suspension cultures. There is a competitive interaction between amiloride analogs and sodium. Substitution of one or both H-atoms of the 5-amino group of amiloride (apparent K(i) about 150 micromolar) resulted in a 3- to 200-fold increase in inhibitory potency of the antiport activity.

Changes in Protein Patterns and Translatable Messenger RNA Populations during Cold Acclimation of Alfalfa.

Changes in the rate and pattern of protein synthesis and in translatable mRNA population during cold acclimation of alfalfa (Medicago falcata cv Anik) seedlings have been examined. There appears to be a positive correlation between the increase in ability to synthesize proteins at 4 degrees C and the increase in freezing resistance (survival at -10 degrees C). Results obtained with three different approaches using sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern visualized by (a) staining, (b) immunoblotting and autoradiography, and (c) fluorography of in vivo labeled proteins, show that at least eight polypeptides are newly synthesized during cold acclimation. Results of analysis of in vitro translation products of mRNA from nonacclimated and acclimated seedlings show the appearance of new translatable mRNAs. It is concluded that changes in gene expression occur during cold acclimation, most probably at the transcriptional level.