pH, abscisic acid and the integration of metabolism in plants under stressed and non-stressed conditions. II. Modifications in modes of metabolism induced by variation in the tension on the water column and by stress.

The hydrolysis of ATP(4-) by the plasmalemma and tonoplast H(+)/ATPases and by the tonoplast pyrophosphatase results in the export of a proton to the apoplast or vacuole with remaining in the cytoplasm. As the enzymes that synthesize ATP(4-) require as a substrate it is proposed that protons are an essential substrate for ATP(4-) synthesis. Thus, the entry of protons to the cytoplasm by sym- and antiports will control the rate of ATP(4-) synthesis. Evidence is adduced that plants control the tension on the water column by removing water to or from the 'cellular reservoir' and guard cells by generating osmotic gradients. Schemes are presented that propose a series of metabolic changes that result in a seamless transition through the following states: (1) the import of K(+), Cl(-) and water from the apoplast to the vacuole, the K(+) being admitted to the cytoplasm via a Ca(2+)-activated K(+)-H(+) symport and the water via a Ca(2+)-activated aquaporin; (2) the continued import of K(+) and water from the apoplast to the vacuole with the concomitant export of protons and the synthesis of malate from glucose in the cytoplasm for importation into the vacuole; (3) when the tension on the water column is optimal, respiration and photosynthesis is maximal resulting in biosynthetic reactions and growth; (4) when tension on the water column increases, K(+), Cl(-) and water are exported from the vacuole to the apoplast; (5) the continued export of K(+) and water from the vacuole to the apoplast with malate for export being synthesized in the cytoplasm; the export of K(+) resulting in the acidification of the vacuole; and (6) a further increase in tension results in the deactivation of the plasmalemma H(+)/ATPase by a further increase in cytoplasmic Ca(2+) which also indirectly activates the alternative oxidase. It is suggested that mitochondrial pyruvate is partly oxidized by the TCA cycle and is partly exported to the cytoplasm where it is carboxylated to form malate(1-) for continued export to the apoplast. K(+) is transferred from the vacuole to the apoplast, the K(+) being replaced by protons from the export of mitochondrial pyruvate. The maintenance of the tonoplast electrochemical gradient is thought to result in an increase in the pH of the apoplast which may cause the hydrolysis of abscisic acid precursors with the resulting abscisic acid opening Ca(2+) channels so that the above events are reinforced. (7) This mode is proposed to continue by the metabolism of glucose to four phosphoenolpyruvate, three of which are carboxylated to malate(1-) for continued export to the apoplast with K(+) from the vacuole, the 'stress-tolerant quiescent state'.

Methods for the quantitation of abscisic acid and its precursors from plant tissues.

Methods are given for the quantitation of the plant stress hormone, abscisic acid (ABA), and its two metabolic precursors, ketone and enolate, that are applicable to all species tested so far. The plant extract is homogenized at neutral pH, hexane-soluble neutrals are extracted and discarded, and then the free ABA and other organic acids are extracted as ion pairs. The remaining aqueous phase is acidified, allowed to stand, neutralized, and extracted to give the ABA ex ketone fraction and then the aqueous phase is treated with base and again extracted to give the ABA ex enolate fraction. Each of these three fractions, free ABA, ABA ex ketone, and ABA ex enolate, along with a deuteriated internal standard, [side-chain-(2)H(4)]ABA, is then derivatized with pentafluorobenzyl bromide and purified on an automated sample preparation system. The resulting pentafluorobenzyl abscisate samples are then quantified using electron capture negative ionization mass spectrometry with methane as the reagent gas. Using these procedures free ABA, and ABA from its precursors, can be quantified at the level of 100 fg on column. If a large volume injector is used so that the total sample is injected it should be possible to quantify ABA and its precursors in the parts per billion range on a few milligrams of plant tissue.

pH, abscisic acid and the integration of metabolism in plants under stressed and non-stressed conditions: cellular responses to stress and their implication for plant water relations.

A paradigm for the response of plants to stress is presented which suggests that plants move towards a state of minimal metabolic activity as a stress intensifies and remain in that state until that stress is relieved. The paradigm is based on the proposition that cells that interface with the transpiration stream employ variations on the following theme to move towards that state. Tension on the apoplastic water opens a mechanosensitive Ca2+ channel, a response that is augmented by apoplastic ABA. The resulting elevated cytoplasmic Ca2+ deactivates a plasmalemma H+/ATPase and also activates a K(+)-H+ symport. The inflow of K+ and H+ depolarizes the membrane and renders the apoplast less acidic, the protons being removed to the vacuole and the K+ ions being re-exported via the K+ outward rectifying channel. The onset of darkness in guard and mesophyll cells deactivates the plasmalemma H+/ATPase and then the events outlined above ensue except that these cells do not appear to utilize either Ca2+ or ABA during these changes. In stressed cells it is proposed that elevated cytoplasmic Ca2+ activates the release of an ABA precursor from a stored form. ABA is then released in the apoplast after export of the precursor if the activity of the K(+)-H+ symport has brought the apoplastic pH close to 7.0. It is proposed that aquaporins in the xylem parenchyma and mesophyll cells are opened by elevated cytoplasmic Ca2+ when the water potential of the transpiration stream is high so that water can be stored in the 'xylem parenchyma reservoir'. The water in this reservoir is then used to increase the water potential in the transpiration stream when the water column is under tension and to help repair embolisms by a mechanism that resembles stomatal closure.

Use of digoxigenin-labelled ampicillin in the identification of penicillin-binding proteins in Helicobacter pylori.

Amoxycillin is used in current therapeutic regimens to treat the infection caused by the human gastric pathogen, Helicobacter pylori. The penicillin-binding proteins (PBPs) are the primary targets for the beta-lactam antibiotics, such as amoxycillin, and are involved in the terminal stages of peptidoglycan synthesis. They also play active roles in the determination and maintenance of cellular morphology. It was believed that an organism with a complex morphology, such as H. pylori, would have more than the three PBPs previously suggested. Using digoxigenin-labelled ampicillin (DIG-ampicillin), we report the identification of eight PBPs in H. pylori with masses of 72, 62, 54, 50, 44, 33.5, 30.5 and 28 kDa. A smaller (21 kDa) ninth band was also detected, which may represent another PBP. However, the relatively small size of this apparent PBP raises questions as to whether this is a true PBP. In an attempt to identify the PBPs to which amoxycillin preferentially binds, amoxycillin was used in competition assays with DIG-ampicillin. It appeared that amoxycillin inhibited the binding of DIG-ampicillin to only the 72 kDa PBP. The experimental data were also compared with the seven putative PBPs identified in the two published H. pylori genomes, most of which correlate with the experimental data. To investigate further the properties of these PBPs, the seven putative PBP genes identified in the H. pylori genomes were examined. The derived amino acid sequences of the putative PBPs were examined for the three characteristic motifs found in all conventional PBPs, SXXK, SXN and KTG. We were able to determine that all of the putative PBPs had at least one of these motifs, but none possessed all three motifs with the characteristics of conventional PBPs. These findings suggest that the PBPs of H. pylori are unique.

Effects of singlet oxygen on membrane sterols in the yeast Saccharomyces cerevisiae.

Photodynamic treatment of the yeast Saccharomyces cerevisiae with the singlet oxygen sensitizer toluidine blue and visible light leads to rapid oxidation of ergosterol and accumulation of oxidized ergosterol derivatives in the plasma membrane. The predominant oxidation product accumulated was identified as 5alpha, 6alpha-epoxy-(22E)-ergosta-8,22-dien-3beta,7a lpha-diol (8-DED). 9(11)-dehydroergosterol (DHE) was identified as a minor oxidation product. In heat inactivated cells ergosterol is photooxidized to ergosterol epidioxide (EEP) and DHE. Disrupted cell preparations of S. cerevisiae convert EEP to 8-DED, and this activity is abolished in a boiled control indicating the presence of a membrane associated enzyme with an EEP isomerase activity. Yeast selectively mobilizes ergosterol from the intracellular sterol ester pool to replenish the level of free ergosterol in the plasma membrane during singlet oxygen oxidation. The following reaction pathway is proposed: singlet oxygen-mediated oxidation of ergosterol leads to mainly the formation of EEP, which is enzymatically rearranged to 8-DED. Ergosterol 7-hydroperoxide, a known minor product of the reaction of singlet oxygen with ergosterol, is formed at a much lower rate and decomposes to give DHE. Changes of physical properties of the plasma membrane are induced by depletion of ergosterol and accumulation of polar derivatives. Subsequent permeation of photosensitizer through the plasma membrane into the cell leads to events including impairment of mitochondrial function and cell inactivation.

L-nucleoside analogues as potential antimalarials that selectively target Plasmodium falciparum adenosine deaminase.

The L-stereoisomer analogues of D-coformycin selectively inhibited P. falciparum adenosine deaminase (ADA) in the picomolar range (L-isocoformycin, Ki 7 pM; L-coformycin, Ki 250 pM). While the L-nucleoside analogues, L-adenosine, 2,6-diamino-9-(L-ribofuranosyl)purine and 4-amino-1-(L-ribofuranosyl)pyrazolo[3,4-d]-pyrimidine were selectively deaminated by P. falciparum ADA, L-thioinosine and L-thioguanosine were not. This is the first example of 'non-physiological' L-nucleosides that serve as either substrates or inhibitors of malarial ADA and are not utilised by mammalian ADA.

Normal-phase high-performance liquid chromatography of triacylglycerols.

Triacylglycerols have been separated by normal-phase high-performance liquid chromatography (HPLC) on silica utilising a solvent system consisting of dry acetonitrile-half water saturated hexane (0.7:99.3). This solvent system is UV transparent allowing detection at 200 nm and affords a separation in which retention is primarily dependent on the number of constituent double bonds. There is also a slight separation on chainlength, the longer chainlengths being eluted first. The system is therefore complementary to currently used reversed-phase HPLC systems. Chromatograms for some polyunsaturated fats and oils are given, and the most polyunsaturated triacylglycerols from linseed oil are analysed in more detail. Data are given for the separation and quantitation of the pentafluorobenzyl esters of constituent fatty acids from these triacylglycerols by a similar normal-phase HPLC system.

The physico-chemical basis of leaf wettability in wheat.

Wild type wheat (Triticum aestivum L.) and three mutant lines that have reduced glaucousness on the flag leaf sheath have been examined for variations in glaucousness, contact angles, wax chemistry and wax morphology. On the sheath and culm, organs that are glaucous in the wild type, increasing glaucousness is correlated with increasing contact angles, an increasing proportion of ß-diketones plus hydroxy-ß-diketones in the was and an increasing proportion of wax tubes. Organs that were non-glaucous in all four lines, namely both surfaces of the vegetative leaves and the adaxial surface of the flag leaf, had high contact angles, a dense covering of wax plates and waxes rich in primary alcohols but devoid of ß-diketones and hydroxy-ß-diketones. The abaxial surface of the flag leaf was the most complex of the organ surfaces studied. In the wild type the glaucousness of the sheath continued onto this surface for 1-2 cm and this was correlated with the other characters studied as it was on the sheath. In the mutants, however, the tubes were absent. Flat ribbons of varying widths, a new wax structure in wheat, as well as various types of plates were found instead. These structures continued to the flag leaf tip and were also present on the abaxial surface of the wild type flag leaf. Changes in contact angle at the tip could not be correlated with the other measured parameters.