JOURNAL AND PROCEEDINGS OF THE mayrAt SOCIETY O F NEW SOUTH WALES Volume 135 Parts 3 and 4 (Nos 405-406) 2003 ISSN 0035-9173 PopbInne BY THE SOCIETY PO BOX 1525, MACQUARIE CENTRE, NSW 2113 Issued April 2003 THE ROYAL SOCIETY OF NEW SOUTH WALES OFFICE BEARERS FOR 2002-2003 Patrons His Excellency the Right Reverend Dr Peter Hollingworth AC, OBE, Governor General of the Commonwealth of Australia. Her Excellency Professor Marie Bashir, AC, Governor of New South Wales. President Mr D.A. Craddock, BSc(Eng) NSW, Grad.Cert. Management UWS. Vice Presidents Prof. P.A. Williams, BA (Hons), PhD Macq. Dr W.E. Smith, MSc Syd, MSc Oxon, PhD NSW, MInstP, MAIP. Mr C.F. Wilmot Hon. Secretary (Gen.) vacant (acting Hon. Sec. Prof. P.A. Williams) Hon. Secretary (Ed.) Mrs M. Krysko von Tryst, BSc, Grad.Dip.Min.Tech NSW, MAusIMM. Hon. ‘Treasurer Prof R.A. Creelman, BA, MSc, PhD Hon. Librarian Dr E.V. Lassak, MSc, PhD NSW, ASTC, FRACI Councillors Mr J.R. Hardie, BSc Syd, FGS, MACE. Prof. J. Kelly, BSc Syd, PhD Reading, DSc NSW Ms K. F. Kelly, BSc(Hons) Mr M.F. Wilmot, BSc Prof M.A. Wilson, PhD, DSc. Auck, FRACI, C.Chem. Southern Highlands Rep. Mr C.M. Wilmot The Society originated in the year 1821 as the Philosophical Society of Australasia. Its main function is the promotion of Science by: publishing results of scientific investigations in its Journal and Proceedings; conducting monthly meetings; organising summer science schools for senior secondary school students; awarding prizes and medals; and by liason with other scientific societies. Special meetings are held for: the Pollock Memorial Lecture in Physics and Mathematics, the Liversidge Research Lecture in Chemistry, the Clarke Memorial Lecture in Geology, Zoology and Botany, and the Poggendorf Lecture in Agricultural Science. Membership, as an Ordinary, Associate or Absentee Member, is open to any person whose application is acceptable to the Society. An application must be supported by two members of the Society. 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This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Responsibility for interpretations, opinions, reproductions and data published on behalf of authors rests with the relevant authors, not with the Royal Society of New South Wales. Journal € Proceedings of the Royal Society of New South Wales, Vol. 135, p. 57-71, 2003 ISSN 0035-9173/03/020057-15 $4.00/1 The 33rd Liversidge Lecture for the Royal Society of NSW Dietary Chemicals and Brain Function GRAHAM A. R. JOHNSTON Abstract Phytochemicals in our diet may play a vital role in maintaining the brain’s chemical balance by influencing the function of receptors for the major inhibitory neurotransmitter GABA. The flavonoids apigenin and epigallocatechin gallate, found in chamomile and green tea respectively, influence the way in which GABA receptors are modulated by drugs such as di- azepam. Resveratrol, a flavonoid-like polyphenol found in red wine, acts on a subtype of GABA receptors consistent with its action as a cognitive enhancer. Bilobalide from Ginkgo biloba, a herb used in cognitive therapy, also influences GABA receptors. a-Thujone, a terpenoid in the alcoholic beverage Absinthe, acts in a similar manner to bilobalide on GABA receptors. (+)-Borneol and other terpenoids from Valerian, a herb used to promote sleep, enhance the effects of GABA. The effects of these phytochemicals on GABA receptors are consistent with the overall actions of the beverages and herbal preparations that contain them, thus providing a rational basis for the use of these beverages and herbal preparations. These studies provide evidence that chemicals in our diet may influence brain function in a positive way. The chemical nature of these substances may lead to the development of new therapeutic agents for the treatment of anxiety, epilepsy, memory disorders and insomnia. Keywords: Brain function, chemicals, diet, balance, dosage THE BRAIN’S CHEMICAL BALANCE Two simple chemicals, glutamic acid and GABA (Figure 1), are responsible for most of the communication between nerve cells in the brain. Indeed, at a very simple level, brain func- tion may be thought of as a balance between excitation mediated by glutamic acid and inhi- bition mediated by GABA. All nerve cells in the brain have receptors for glutamic acid and GABA. Some 40% of nerve cells release glutamic acid as an excita- tory neurotransmitter, while a different 40% re- lease GABA as an inhibitory neurotransmitter. The balance between these two chemical trans- mitters is vital to normal brain function. An excess of excitation over inhibition results in an EMTHSONTA JUN 1 7 2005 CIBKANiL- over excited brain (as in Figure 1) that can be manifested as anxiety, agitation, exhilaration, convulsions and death. On the other hand, an excess of inhibition over excitation can be man- ifested by depression, anaesthesia, coma and death. ‘he particular manifestations of such imbalances in the brain depend on what neu- ronal circuitry is involved. Ethanol is an example of a chemical that acts on both sides of the brain’s chemical bal- ance. The CNS depression that results from in- gestion of ethanol is due to a reduction in excita- tion mediated by glutamic acid acting on a sub- type of glutamate receptors known as NMDA receptors and to an enhancement. of inhibition mediated by GABA acting on GABAaj recep- tors. 58 JOHNSTON HoN COOH COOH Glutamic acid Exitation GABA Inhibition 1) Figure 1: The brain’s chemical balance between excitation mediated by the major excitatory neurotransmitter, glutamic acid, and inhibition mediated by the major inhibitory neurotransmitter, GABA. GABA RECEPTORS GABA (whose name is derived from the old chemical name, y-aminobutyric acid) acts on three main types of receptor to influence brain function. GABA,g and GABAc receptors are fast acting receptors that belong to the group of receptors called ligand-gated ion channels (LGICs) (Chebib and Johnston, 2000). GABA acts as the ligand gating these receptors to open channels specific for chloride ions, allowing these ions to flow rapidly into nerve cells mak- ing them more negative and thus harder to ex- cite. GABAg receptors act more slowly, induc- ing metabolic changes in nerve cells and belong to the group of receptors called G-protein cou- pled receptors (GPCRs) (Bowery et al. 1997). The study of GABA receptors has been rev- olutionised by the introduction of recombinant receptor technology whereby receptors cloned from human brain are expressed in cells that do not normally express such receptors (Barnard et al. 1998). The recombinant receptors so formed may be studied in relative isolation using stan- dard electrophysiological methodology. Since all GABA receptors are made up of protein sub- units, recombinant receptors of known subunit composition may be studied. The most common way to study recombi- nant GABA receptors is to express them in oocytes from the South African frog, Xenopus laevis following injection of either DNA or RNA cloned from human brain and coding for par- ticular GABA receptor protein subunits. These oocytes have the necessary cellular machinery to make the human proteins and assemble them on the surface of the oocytes as functional GABA receptors. ‘The oocytes are approximately one millimetre in diameter and readily penetrated by glass microelectrodes. Using 2-electrode voltage clamp methodology, the effects of chem- icals on the function of these GABA receptors may be assessed in a convenient quantitative manner. For example, using recombinant recep- tor technology, the effects of anti-anxiety agents such as diazepam (Valium) on GABA recep- tors can be easily shown to be restricted to a specific sub-group of GABAg receptors. The technology is not restricted to the study of pure chemicals — relatively crude mixtures of chem- icals can be studied, for example to follow the purification of chemicals acting on GABA re- ceptors from extracts of herbal products. FLAVONOIDS AND TERPENOIDS Flavonoids are polyphenolic chemicals widely distributed in the plant kingdom particularly in flowering plants. Flavonoids are responsi- ble for many of the brilliant colours of fruits DIETARY CHEMICALS AND BRAIN FUNCTION 59 and vegetables and are important constituents of red wine, green tea and many herbal prepa- rations (Aherne and O’Brien, 2002). Chemi- cally, flavonoids are C15 compounds based on the chromane ring structure. Flavonoids have been studied extensively as anti-oxidants and oestrogens (Collins-Burow et al. 2000). Many of them show anti-cancer and anti-viral proper- ties (Le Marchand, 2002). There is an extensive literature on the effects of flavonoids on GABA receptors (for a recent review see Marder and Paladini (2002), dating from the discovery of some plant derived isofla- vans in bovine urine that inhibited the bind- ing of diazepam to brain membranes (Luk et al. 1983). In the present context of actions on GABA receptors, the following flavonoids are of interest: the flavone apigenin; the isoflavone genistein; the flavanone naringenin; and the fla- OH OH O Apigenin, a flavone found in chamomile tea and related beverages. OH O Naringenin, a flavone found in grapefruit. Figure 2: Some representative flavonoids van, epigallocatechin gallate (Figure 2). Terpenoids are also widespread in plants, es- pecially in what are known as essential oils that can be extracted from plants and have a wide range of uses from perfume constituents to paint thinners. Terpenoids are oxygenated products formally derived from C5 isoprene units and are classified by the number of C5 units in their structure. ‘Thus monoterpenoids have 2xC5 units, sesquiterpenoids 3xC5 units, diterpenoids 4xC5 units and triterpenoids 6xC5 units. In the present context of actions on GABA recep- tors, the following terpenoids are of interest: a-thujone, (+)-borneol, bilobalide and picrotox- inin (Figure 3). Picrotoxinin is widely used ex- perimentally as a non-competitive antagonist of GABAjs and GABAcg receptors, however, its convulsant action restricts its therapeutic use (Chebib and Johnston, 2000). OH Genistein, an isoflavone found in soy products, including tofu. OH OH OH (-)-Epigallocatechin Gallate, a flavan found in green tea. 60 Hz. Es _CHs a-Thujone, a monoterpene from Artemisia absinthium JOHNSTON H3C CH CH3 OH (+)-Borneol, a monoterpene from Valerian officinalis Bilobalide, a sesquiterpenoid from Ginkgo biloba Picrotoxinin, a sesquiterpenoid from Anamirta cocculus Figure 3: Some terpenoids that influence GABA receptors DIETARY CHEMICALS AND BRAIN FUNCTION 61 APIGENIN FROM CHAMOMILE TEA The lead compound for our investigations was apigenin (Figure 2), a flavonoid with a known anti-anxiety action found in chamomile tea. Chamomile tea is used widely to treat anxi- ety and insomnia. Current therapeutic drugs used for the treatment of anxiety and insom- nia such as the benzodiazepines Valium and Serepax act at GABAg receptors in the brain, increasing chloride flow into neurones, result- ing in decreased neural activity. There were divergent reports of the effects of apigenin on GABAag receptors. Viola et al. (1995) con- cluded that apigenin is a benzodiazepine ago- nist, like diazepam. However, Avallone et al. (2000) concluded that apigenin was a benzodi- azepine inverse agonist (the exact opposite of diazepam). Viola et al. (1995) based their conclusion that apigenin is a benzodiazepine agonist on the ability of apigenin to displace the bind- ing of radiolabelled benzodiazepines from rat brain membranes, coupled with benzodiazepine- like effects in a rodent model of anxiety. How- ever, binding studies do not reliably distinguish between agonists, antagonists and inverse ag- onists. Indeed, on the basis of similar bind- ing studies, Dekermendjian et al. (1999) con- cluded that apigenin was a benzodiazepine an- tagonist (that is, it binds to the benzodiazepine site, blocking the binding of benzodiazepine ag- onists and inverse agonists, without having any effect on GABA responses itself). Avallone et al. (2000) used electrophysiological recordings from rat neurones. This allowed a more di- rect investigation of the activity of apigenin and showed that apigenin inhibited currents due to GABA, an effect which was blocked by the ben- zodiazepine antagonist, flumazenil. This fits the profile of a benzodiazepine inverse agonist. However, Avallone et al. (2000) did find some behavioural effects of apigenin which could be consistent with an action as a benzodiazepine agonist. As part of her PhD studies, Erica Camp- bell in our research group investigated the ac- tion of apigenin on recombinant GABA recep- tors. She used electrophysiological recordings from Xenopus laevis oocytes injected with re- combinant human RNA for the most common subtype of GABAag receptor (a@)(272,) in the brain. The actions of GABA at these receptors are enhanced by a variety of modulators includ- ing barbiturates, benzodiazepines, ethanol, and neuroactive steroids. She showed the action of apigenin on the GABAag receptor is more complex than sug- gested by earlier studies. The effects of api- genin were biphasic dependent on the dose used. Moderate doses of apigenin inhibited the ac- tions of GABA, diazepam and the steroid al- lopregnanalone, whereas low apigenin concen- trations enhanced the effects of diazepam only (Figure 4). These effects are unlikely to be due to a simple action at the benzodiazepine site, suggesting a new site on the GABAag receptor. While the inhibitory actions of apigenin at moderate doses were consistent with it acting as a benzodiazepine inverse agonist, the abil- ity of apigenin to enhance the enhancing action of diazepam was novel. At low doses, apigenin had no direct effect on the action of GABA on these recombinant receptors. The presence of diazepam was necessary in order to observe the enhancing effects of apigenin. Thus apigenin appeared to be modulating the action of a mod- ulator, an action not previously described in the pharmacological literature. Apigenin might be described as a second order modulator that influences the modulatory action of diazepam as a first order modulator on the activation of GABAag receptors. The second order modulation of GABA, receptors by apigenin requires the presence of GABA and a first order modulator acting at a benzodiazepine site. The sedative and anx- iolytic actions of apigenin observed in rodents (Avallone et al. 2000, Viola et al. 1995) can be interpreted on the basis of apigenin potentiating the action of endogenous benzodiazepine-like agents in the brain. Evidence for the physiolog- ically relevant presence of such agents, termed 62 JOHNSTON endozepines, has come from the discovery of a mutant GABAas receptor in childhood absence epilepsy and febrile seizures that has diminished sensitivity to benzodiazepines with little other alteration in GABAg receptor function (Wal- lace et al. 2001). Genistein (Figure 2), an isoflavone found in soy products, did not show the biphasic ef- fects of apigenin. Genistein, a structural isomer 2 ess 5s 15 BOF ag aS (eD) eg I S ga ro 4 383 Sa cure Las 7 e335 Sp cod 0 ~0.5 0.01 0.1 of apigenin, showed only the GABA4g antago- nist action of apigenin. In addition, a dihydro derivative of apigenin, naringenin (Figure 2), a flavanone found in grapefruit juice and other cit- rus products, also showed only the GABA, an- tagonist action of apigenin. Thus, the second order modulatory action of apigenin is struc- turally specific. Enhancement Inhibition 10 100 Apigen Concentration (uM) Figure 4: Effects of apigenin on the enhancement by diazepam of the action of GABA on recom- binant GABAg receptors (Campbell et al. 1999). EPIGALLOCATECHIN GALLATE FROM GREEN TEA Epigallocatechin gallate (Figure 2, EGCG) is the most abundant flavan in green tea (Camel- lia sinensis). It is found in all teas made from C’. sinensis but not in many other food products (Arts et al. 2000b). Green tea is known to have many beneficial effects, including prevention of cancer, lowering of blood pressure and lipids, and acting as an antioxidant. EGCG has been shown to contribute to these effects and, in ad- dition, has been shown to have neuroprotective properties. Erica Campbell investigated the actions of EGCG on recombinant GABA receptors (Campbell et al. 1999). She found that it shared the same biphasic action of apigenin, enhancing the action of diazepam at low concentrations and inhibiting at higher concentrations. In both the enhancement and inhibition phases, EGCG was at least 10 times more potent than apigenin. DIETARY CHEMICALS AND BRAIN FUNCTION 63 (+)-Catechin and (-)-epicatechin, the most abundant flavans in nature, being found in many foods (Arts et al. 2000a, Arts et al. 2000b), did not influence recombinant GABA receptors, showing that the basic flavan ring structure is not sufficient for either of the actions of EGCG observed on recombinant GABAag receptors. The biphasic actions of apigenin and EGCG emphasise the importance of dose in drug ac- tion. Our experiments show that low doses of apigenin and EGCG can enhance the activation of GABA receptors under the right conditions and thus could produce sedation and relief of anxiety. On the other hand, higher doses have the opposite effect and thus are likely to pro- duce stimulation. The second order modulatory action of api- genin and EGCG might have therapeutic possi- bilities. Low doses of these substances could reduce the therapeutic dose of diazepam and related benzodiazepines, while higher doses might reduce the effectiveness of such benzo- diazepines. The possibilities of interactions be- tween benzodiazepine medication and the con- sumption of chamomile and green tea need to be considered, particularly as chamomile tea may be used as a home remedy for those conditions for which benzodiazepines are frequently pre- scribed. RESVERATROL FROM RED WINE The relatively low incidence of coronary heart diseases in France, despite intake of a high- fat diet, — the “French Paradox” -— has been attributed to the consumption of red wine containing high levels of polyphenolic com- pounds (Mojzisova and Kuchta 2001, Sun et al. 2002). Resveratrol (3,4’,5-trihydroxystilbene, Figure 5) is one of the most interesting polyphe- nolic compounds found in red wine. It has been shown to have estrogenic (Turner et al. 1999) and neuroprotective effects (Bastianetto et al. 2000). OH Resveratrol, a stilbene found in red wine from Vitis vinifera OH Figure 5: Structure of Resveratrol In view of the structural similarities be- tween resveratrol and apigenin, we investigated its effects on recombinant GABA receptors ex- pressed in oocytes. To our surprise, resvera- trol showed little action on GABAag receptors but was a GABAc receptor antagonist (Camp- bell and Johnston, 2003). Resveratrol non- competitively inhibited the effects of GABA (1uM) at GABAc receptors with an [C50 of 72 uM, while having no significant effect at doses up to 100uM on the effects of GABA (40 4M) at GABAg receptors. This is the first report of a non-competitive antagonist show- ing some selectivity for GABAc over GABAa 64 JOHNSTON receptors, the widely used non-competitive an- tagonist picrotoxinin being some 30 times more potent at GABA,j than at GABAc receptors (Chebib and Johnston, 2000). We have a patent on the use of GABAg re- ceptor antagonists to enhance cognitive activ- ity and stimulate memory capacity (Johnston et al. 1998). Thus it was interesting to note that resveratrol has also been patented for the treat- ment of mild cognitive impairment (Wurtman and Lee, 2002) based on its ability to increase the expression of soluble amyloid precursor pro- tein. Using resveratrol as a lead compound, we are examining structural analogues to see if we can develop more potent and selective com- pounds acting on GABAc receptors. Recently we discovered a range of very promising activ- ities, including the ability to enhance GABAc receptor activity, in a series of compounds syn- thesized in the 1970s by David Collins and col- leagues in Veterinary Physiology at ‘The Univer- sity of Sydney as antiestrogenic and antifertility agents (Collins et al. 1971). There is great interest in drugs to treat memory impairment in disorders such as Alzheimer’s disease and schizophrenia. The use of such “Smart Drugs” in healthy people to in- crease their cognitive ability raises a variety of ethical, legal and social issues (Rose 2002). Resveratrol and related stilbenoids are found in a variety of plants and herbs. Major di- etary sources include grapes, wine, peanuts and soy (Burns et al. 2002). These compounds are also found in Itadori tea which has long been used in Japan and China as a traditional rem- edy for heart disease and stroke. For people who do not wish to consume alcohol, Itadori tea may be a substituent for red wine as a di- etary source of resveratrol (Burns et al. 2002). For those who prefer white wine to red, French winemakers have created a Chardonnay, called Paradoxe Blanc, that is enriched in polyphenols and has been shown to be effective in reducing oxidative stress in diabetic rats (Landrault et al. 2003). As noted above, ethanol itself enhances the effectiveness of GABA acting on GABAg recep- tors and there is evidence that moderate con- sumption of alcoholic beverages is beneficial to health. However, other substances in these bev- erages, such as resveratrol, are likely to con- tribute to the overall beneficial effects. Re- cently it has been reported by Aoshima and col- leagues (Hossain et al. 2002) that the fragrance of whiskey is able to enhance the effectiveness of GABA acting on GABAag receptors. Several components of the fragrance showed this prop- erty, the most potent being ethyl phenylpropi- onate, which was shown to have an anticonvul- sant action in mice on inhalation. Enhancement of GABA action was also found in extracts of other alcoholic drinks such as wine, sake, brandy and sochu. Hossain et al. (2002) noted “Although these fragrant components are present in alcoholic drinks at low concentrations (extremely small quantities compared with ethanol), they may also modu- late the mood or consciousness of the human through the potentiation of the GABAag recep- tor response”. Aoshima and colleagues have shown that various perfume constituents and aromatherapy agents potentiate GABAag recep- tors (Aoshima and Hamamoto 1999, Aoshima et al. 2001). Clearly there are many interesting and inno- vative ways to explore the possibilities of influ- encing cognitive function. BILOBALIDE FROM GINKGO BILOBA Extracts of Ginkgo biloba leaves are widely em- ployed as herbal medicines to treat symptoms associated with mild-to-moderate dementia, im- pairment of other cognitive functions associated with ageing and senility and related neurosen- sory problems (Diamond et al. 2000). A study has indicated that the cognition-enhancing ef- fects of the Ginkgo leaf extracts may be partly mediated by bilobalide via GABA receptors (Sasaki et al. 1999b). Enhanced hippocampal pyramidal neuronal excitability has been shown to correlate with learning and memory (Power DIETARY CHEMICALS AND BRAIN FUNCTION 65 et al. 1997). Bilobalide (Figure 3), a sesquiter- penoid isolated from Ginkgo biloba leaves, has been shown to enhance this excitability in rat hippocampal slices, an action proposed to in- volve blockade of GABAergic neurotransmis- sion (Sasaki et al. 1999b). In collaboration with Sasaki and colleagues, Shelly Huang and Rujee Duke in our research group have shown that bilobalide is a potent an- tagonist of the action of GABA on recombinant GABA,g and GABAc receptors (Huang et al. 2003). Bilobalide was only marginally less po- tent than picrotoxinin in these actions but there were subtle differences between the actions of bilobalide and picrotoxinin. These findings strongly support the proposal by Sasaki and col- leagues (Sasaki et al. 1999b) that the observed enhanced neuronal excitability in hippocampal slices was due to its blockade of GABAergic neu- rotransmission. Bilobalide and picrotoxinin share common structural features, including a hydrophilic cage and lipophilic side chain. However, bilobalide and picrotoxinin have opposite actions upon systemic administration to animals. Bilobalide is an anticonvulsant (Sasaki et al. 1999a,b) whereas picrotoxinin is a potent convulsant (Jarboe et al. 1968). There are, however, only minor differences in their activities at recom- binant GABAag and GABAc receptors. Bilob- alide has been shown to increase GABA lev- els in the hippocampus and cerebral cortex of mice (Sasaki et al. 1999a). This increase may override the GABA, antagonist action of bilob- alide that would be expected to produce convul- sions and result in the overall anticonvulsant action. Nonetheless, the induction of epilep- tic seizures by Ginkgo extracts has been noted in rare cases (Granger, 2001). The anticonvul- sant/convulsant actions of bilobalide need fur- ther investigation and may provide vital clues as to the safe use of Ginkgo extracts in the treat- ment of mild cognitive deficits. Ginkgo leaves were used traditionally in Japan to protect books against harmful worms and insects before the introduction of modern insecticides. Like picrotoxinin, bilobalide is a potent insecticide (Ahn et al. 1997), an action likely to be due to blockade of insect GABA re- ceptors. THUJONE FROM ABSINTHE Absinthe was the favoured drink of artists and writers in Paris at the end of the 19th cen- tury. ‘The emerald green liqueur made famous by Van Gogh, Toulouse-Lautrec and their col- leagues was banned in France and most other countries by 1915 due to its ability to cause convulsions, hallucinations and psychotic dis- turbances. The toxic component of absinthe has been identified as the monoterpenoid a-thujone (Fig- ure 3). It is also the active ingredient of worm- wood oil and some other herbal medicines and is reported to have antinociceptive, insecticidal, and anthelmintic activity. Hold et al. (2000) showed that a-thujone acted like picrotoxinin as a GABAag receptor non-competitive antago- nist. ‘They showed that a-thujone was rapidly metabolised to less active metabolites and con- cluded that “a-thujone in absinthe and herbal medicines is a rapid-acting and readily detox- ified modulator of the GABA-gated chloride channel”. Matthew Roper in our research group has shown that a-thujone is a non-competitive in- hibitor of the action of GABA on recombinant a1 Goyer GABAag and p; GABAc receptors ex- pressed in oocytes. Like picrotoxinin, a-thujone was about 30 times more potent at GABA, than at GABAc receptors. Furthermore, site- directed mutagenesis studies showed that muta- tions in the second membrane-spanning region of the wildtype GABAc receptors influenced the potency of a-thujone and picrotoxinin in a simi- lar manner indicating that both convulsants in- teract with the same amino acid residues on the GABAcg receptor. Many plant-derived essential oils, such as wormwood, have been known for over a cen- tury to have convulsant properties. Burkhard et al. (1999) reported on case studies of plant- related toxic seizures related to use of these oils 66 JOHNSTON for therapeutic purposes. ‘They noted that “the literature shows essential oils of 11 plants to be powerful convulsants (eucalyptus, fennel, hys- sop, pennyroyal, rosemary, sage, savin, tansy, thuja, turpentine, and wormwood) due to their content of highly reactive monoterpene ketones, such as camphor, pinocamphone, thujone, cine- ole, pulegone, sabinyl acetate, and fenchone.” They went on to state “Nowadays the wide use of these compounds in certain unconventional medicines makes this severe complication again possible” . Absinthe is now available in a less potent form that contains less than 10 parts per mil- lion of a-thujone, whereas traditional absinthe contained more than 250 parts per million. BORNEOL FROM VALERIAN Valerian (Valeriana officinalis) is a medicinal plant used widely throughout the world. Ex- tracts of the dried underground parts of the plant are used to relieve anxiety, restlessness and nervous sleep disorders. There is evidence of its use by the ancient Greeks, Romans and Chinese for healing purposes. Early herbalists and physicians such as Hippocrates, Galen and Culpeper noted the sedative and digestive prop- erties of valerian, advocating its use as a mus- cle relaxant, diuretic, expectorant and wound healer (Plushner, 2000). Today there are over 400 commercially available products containing valerian in Germany, more than 80 in the United Kingdom and more than 30 available in Aus- tralia (Houghton, 1999; Shohet et al. 2001). Valerian extracts may be considered to be a “herbal Valium”, given that they have a benzodiazepine-like action reducing the latency of sleep onset and increasing the depth of sleep and the perception of well-being. These extracts contain a large number of constituents, many of which are thought to be active at GABA re- ceptors. Compounds that have been identified include acids (valerenic acid and isovalerenic acid), ketones (valeranone), alcohols (valerenol, maaliol), aldehydes (valerenal) and valepotri- ates (valtrate, isovaltrate). Valerian extracts also contain various flavonoids, alkaloids, tan- nins and amino acids. Renee Granger in our research group ob- tained 2kg of the dried underground parts of Valeriana officinalis from Newton’s Herbal Pharmacy in Sydney. She extracted this with hexane, ethyl acetate, methanol and methanol:water (1:5), and fractionated the ex- tracts using silica gel chromatography. ‘This procedure produced more than 450 fractions, which were assessed using thin layer chromatog- raphy and functional studies on recombinant re- ceptors many fractions influenced GABA action on GABAag and GABAg receptors. Dried valerian root normally contains 0.3- 0.8% volatile oil, including borneol, valerenic acid, valeranone and kessy] glycol. These essen- tial oil fractions proved very difficult to purify, sO pure compounds were purchased and tested on recombinant receptors. This produced a very surprising result. (+)-Borneol, the natural enantiomer found in Valerian, produced a 12 fold enhancement of the action of 104M GABA on recombinant GABAag receptors under conditions whereby di- azepam would give at best a 2 fold enhancement (Figure 6; Granger et al. 2002). While relatively high concentrations of (+)-borneol were needed (EC50 400 uM), this degree of enhancement is unprecedented. Under these conditions, (-)-borneol pro- duced a 4 fold enhancement (EC50 450 uM), isoborneol a 7 fold enhancement (EC50 680uM), while the structurally related monoterpenes camphor, bornyl acetate and p- cymene each produced an approximately 3x potentiation (with EC50’s around 300 uM). While many general anaesthetics, barbitu- rates and benzodiazepine are known to pro- duce up to 2 fold enhancement of the re- sponse of GABAag receptors to GABA (Belelli et al. 1999b), the general anaesthetic etomidate is known to cause much larger enhancements, particularly at mutated GABA receptors, e.g. etomidate produces a 10 fold enhancement of GABA responses at GABAc receptors where the wild type isoleucine residue at position 307 DIETARY CHEMICALS AND BRAIN FUNCTION 67 had been mutated to a serine (Belelli et al. 1999a). (+)-Borneol represents an intriguing struc- tural lead for the development of a new class of therapeutic agents acting on GABA receptors. Purified (+)-borneol has been used for medicinal purposes in China and Japan (Hattori, 2000). I) oO (+)-Borneol A (-)-Borneol 2 5 10 a ox eee faa) oO < 5 0 2s 5 2 Bes tH A, 2 I see 3 ee DO oe Se ee eee la a Ele tS = 0 Borneol is a common constituent of the essential oil component of many plants and thus a com- ponent of many herbal preparations. On the basis of our studies, (+)-borneol would be ex- pected to have antianxiety, anticonvulsant and sedative properties. " na a Log Dose (uM) Figure 6: Dose-response curve for the potentiation of the response to 10 uM GABA by (+)- and (-)-borneol at recombinant GABAg receptors (Granger et al. 2002). NATURAL VERSUS SYNTHETIC Natural products derived from plants provided the first medicines. These were complex mix- tures of chemicals. ‘The active principles in these mixtures were isolated and developed as single chemical entities to use as drugs and from which to develop new therapeutic agents. The development of aspirin from the salicylates found in Willow bark is a classic example of this. Natural products continue to be an im- portant source of new drugs. There are sophisti- cated laboratories in many countries, including Australia, using high throughput technology to screen extracts of natural products for desired biological activities. Herbal preparations are by their nature mix- tures of chemicals. It is a basic tenet of herbal medicines that the whole is more than the sum of the parts. The various chemicals in herbal preparations are considered to act together in a synergistic way to effect treatment of partic- ular disorders. This is in direct contrast to the “magic bullet” approach of single chemical en- tity drugs designed to hit a particular target in a highly selective manner. Both approaches have 68 JOHNSTON their role in promoting health and well-being. There is a widespread belief on the part of the general public that natural substances are inherently superior to synthetic substances with regard to efficacy and safety in matters related to human health. This question has been ad- dressed recently by a task force of the Inter- national Union of Pure and Applied Chemistry (Topliss et al. 2002). A comparison of the char- acteristics of natural and synthetic substances used in a variety of therapeutic drugs, herbal medicines, vitamins and nutrients shows a sim- ilar range of favorable and unfavorable effects. It was apparent that molecular structure and dose determine the effect of chemicals on hu- man health, not whether they are of natural or synthetic origin. Natural chemicals such as many flavonoids have been consumed in our diet for countless generations. This suggests that they would be unlikely to have serious adverse effects se- vere enough to prevent their use as therapeutic agents. However, it is likely that the overall bal- ance of flavonoids and related natural chemicals in our diet is of vital importance, given the ex- amples in this review of such chemicals having opposing actions on GABA receptors. Recom- binant receptor technology offers the means to assess the overall effects of complex mixtures of chemicals on the functioning of key receptors. Such technology is expected to play an increas- ingly important role in the quality control of herbal preparations and “functional foods”. Herbal preparations can have significant in- teractions with therapeutic drugs, for example by altering the metabolism of these drugs and thus influencing their potency and duration of action (Izzo and Ernst 2001). It is important that health care professionals ask their patients about their use of herbal products and consider the possibility of herb-drug interactions. Food- drug interactions are also important (Sorensen 2002) as many naturally occurring substances influence the cytochrome p450 enzymes that play such an important role in drug metabolism. Grapefruit juice is a classic example. It con- tains substances, including the flavonoid narin- genin (Figure 2), that inhibit the p450 enzyme CYP3A4 resulting in higher bioavailability of drugs with a high firstpass metabolism (Fuhr 1998). While there may be a place for grape- fruit juice as a drug-sparing agent, more re- search is needed and drugs possibly influenced by the consumption of grapefruit juice should be appropriately labelled. CONCLUSIONS GABA receptors in our brain are susceptible to a wide variety of chemicals consumed in the diet. Our GABA receptors exist in a milieu of substances that influences their function, of- ten in opposing ways. ‘The balance between the effects of these substances will determine at any particular point in time how the recep- tors respond to GABA, their natural neuro- transmitter. This review has summarised the effects of some substances found in four bever- ages (chamomile and green tea, red wine, ab- sinthe) and two herbal preparations (Gingko and Valerian) that have significant actions on recombinant GABA receptors consistent with the overall actions of the beverages and herbal preparations. The chemical nature of these sub- stances may lead to the development of new therapeutic agents for the treatment of anxi- ety, epilepsy, memory disorders, and insomnia. Does the concept of dietary substances influenc- ing brain function indicate that we have entered an era of neuraceuticals? These studies provide a chemical basis for some of the effects that these beverages and herbal preparations have on brain function, and may lead to rational improvements in their qual- ity control and use, especially in combination with other agents known to influence GABA re- ceptors, such as alcohol, anaesthetics and ben- zodiazepines. 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PCT International. Application, 2002, WO 02/28141 A2. Professor Graham Johnston AM The Adrien Albert Laboratory of Medicinal Chemistry, Department of Pharmacology The University of Sydney DO6, NSW 2006 Australia Phone: 612 9351 6117, Fax: 612 9351 2891 e-mail: grahamj@Qmail.usyd.edu.au This paper is based on the 33°? Liversidge Lecture presented before the Royal Society of New South Wales at the Frencham School, Mit- tagong, 22nd November 2002. (Manuscript received 29.1.2003) Journal & Proceedings of the Royal Society of New South Wales, Vol. 135, p. 73-83, 2003 ISSN 0035-9173/03/020073-11 $4.00/1 lonic Combating Mechanisms and their Comparative Effects on Seed Hardening under Simulated Supra-Optimal Environmental Conditions M. A. KADER Abstract Heat extremes and limited moisture are two of the most dominant environmental factors impacting stand establishment of rainfed sorghum (Sorghum bicolor L. Moench). Hardening is the process of exposing plants to gradual levels of stress and acclimation to foster better re- sponse to post-treatment stress factors. Three experiments were carried out under phytotron and germination cabinet conditions to test the effects of osmotic soaking of sorghum seeds with sodium chloride (NaCl) on germination and growth under simulated heat stress. The hypothesis was that the NaCl treatment forms an acclimation to stress by inducing hormonal responses to ionic toxicity caused by salt. This acclimation would lead to a lowered degree of response when the seed is exposed to future stress; namely, heat and/or drought. Independent variables included NaCl concentration, treatment duration, storage and genotype, whereas ger- mination and growth were dependent variables. Further experiments tested various methods of achieving the initial acclimation “signaling” whilst reducing ionic toxicity through combat- ing ions. Longer soaking treatment durations (2-3 days) and higher NaCl concentrations (16 g NaCl 1~*) were detrimental to germination in comparison to lower concentrations (8 g NaCl 1~') and shorter durations (1 day). An interaction between concentration and duration of treatment existed where high concentrations performed better at lower treatment durations and vice versa. Combining 10 g NaCl 17! with 5g calcium sulphate 1~’ to combat ionic toxicity produced a greater advancement of germination than NaCl alone. Drying duration of seeds did not affect subsequent germination nor did storage for 10 days to 1 month. The effects of osmotic conditioning are discussed and could have potential for improving sorghum success rates in harsh arid environments. Keywords: ions, acclimation, stress, sorghum, germination INTRODUCTION The fate of sorghum (Sorghum bicolor L. Moench) seeds sown into dry or gradually dry- ing seedbeds that hamper emergence is not well known. Even though some seeds may germi- nate and give rise to seedlings, the majority will fail to emerge (Al-Mudaris and Jutzi 1998). This clearly sets back stand establishment and subsequent yield due to thermo and other forms of dormancy (Silvertown 1999). Treating seeds with osmotic solutions before sowing, also termed “osmoconditioning”, has been shown to improve germination and seedling emergence in a range of species (Heydecker and Gibbins 1978, Brocklehurst and Dearman 1984). The use of sodium chloride (NaCl) as the osmotic agent in such treatments has also been investigated and shown to yield enhanced germination pat- terns in sorghum (Al-Mudaris 1998). Both the concentration of NaCl and treatment duration seem to play major roles in the response exhib- ited by seeds. However, little is known of the role of both factors or of the effect of various salt mixtures on germination and early seedling growth of treated seeds. This is especially the case when ionic toxicity is taken into account as this is the single most important factor af- fecting NaCl usage as a priming agent. Ionic combating by way of adding extra elements to the NaCl solution may aid in reducing the neg- 74 ative effects of Nat and Cl~ ions (Al-Mudaris and Jutzi 1999). Conditioning whole plants to stress by grad- ual exposure to limited and increasing levels of the particular stress factor has been found to alleviate part of the stress at later stages due to an early peaking of abscisic acid (ABA) produc- tion and a better hormonal balance in favour of Kinetin (Al-Mudaris 1998a). The concept of conditioning seeds to heat stress by expos- ing them to pre-germination salinity stress may aid in “hardening” seeds and improving subse- quent germination and seedling growth under heat stress. This paper investigates the effects of NaCl concentration, treatment duration, salt mix- tures and storage on germination and growth parameters in four sorghum genotypes. It also evaluates the effect of combining NaCl with other salts in advancing the “hardening” ef- fect to improve germinative performance under drought and/or heat stress by way of ionic com- bating. MATERIALS AND METHODS Effect of Salt Concentration, Osmoconditioning Treatment Duration and Drying Seed lots of the sorghum variety IRAT 204 were used in this set of experiments. Seed tests revealed a 1000 seed weight of 34g, a mois- Time (hh.mm) Temperature (°C) 24.00 — 05.00 15 06.00 — 08.00 Le 09.00 — 12:00 29 13.00 — 19.00 43 20.00 — 21.00 20 22.00 — 23.00 23 Relative Humidity (%) 65 62 50 38 D0 58 KADER ture content of 15%, viability of 100% and ger- minability of 98.0% following International Seed Testing Association (ISTA) rules (ISTA 1993). Previous work (Al-Mudaris and Jutzi 1998 and 1998a) showed the upper range of posi- tive responses to NaCl to lie between 10 and 20g NaCl 1~!. Therefore, two concentrations around this range were used. These were 8 and 16g NaCl 1~!. A water-soaked (distilled water) wet control was also evaluated. Sodium chloride solutions were prepared at the concentrations mentioned, and seeds soaked in them at 10°C in the dark inside glass beakers for one of three durations; namely 1, 2 or 3 days (d). The wet control was also soaked in water for the same periods of time. The 10°C incubation temper- ature proved low enough to prevent premature germination (visible signs of radicle emergence after imbibition) during soaking in water. After treatment, seeds were either sown fresh without drying or surface dried by expos- ing to an airflow of 25°C for 3 hours (h) inside an incubator and sown dry. Seeds were sown in batches of 100 in 1000mL polystyrene trays filled with equal volumes of sieved sand (weight basis) and irrigated to weight with 200mL of water, initially, and as they lost one third of their weight thereafter. Trays were weighed daily at 7:00am. Experiments were conducted in a 18m? walk-in phytotron (Heraeus-Voetsch, Germany) set at the environmental conditions shown in Table 1. ‘These conditions were an at- tempt to simulate likely heat stress during the course of a day (Al-Mudaris 1998a). Light (33 klux) Absent Activated Activated Activated Activated Absent Table 1. The course of temperature, relative humidity and light activation in the phytotron during a 24 hour cycle. IONIC COMBATING MECHANISMS IN SEED 79 Treatment combinations were replicated four times each at 100 seeds per replicate (3 treatments x 3 durations x 2 drying treatments x 4 replicates = 72 experimental units). Trays were arranged in a Randomized Complete Block Design (RCBD). Emergence counts were taken daily for 12d and from them the final germina- tion percentage (FGP), mean germination time (MGT) and germination index (GI) calculated. MGT and GI were calculated following Orchard (1977) and Benech Arnold et al. (1991), respec- tively. The MGT is a measure of the mean time taken for a seed lot to germinate, while the GI assigns maximum weight to a higher number of seeds germinating earlier (Al-Mudaris 1998). Data were arcsin transformed (Yang et al. 1999 and Houle et al. 2001) and analysed in ANOVA using the General Linear Model (GLM PROC) of the SAS® statistical package for Windows®. At 12 days of age seedlings were collectively harvested from each tray, separated into shoots and roots, and, after washing roots under run- ning tap water, dried at 80°C for 4d in a reverse cycle oven (Conviron Industries, Canada). Av- erage dry weights of shoots (DWS) and roots (DWR) and the shoot: root ratio (SRR) were obtained and averaged. Also, after harvest, the contents of each tray were emptied into a sieve with 2mm openings, and germinated but unemerged seeds retrieved and counted (here- after termed GUE). Those that had neither ger- minated nor emerged were classified as non- germinated seeds and represented the difference between emerged and germinated unemerged seeds (Munir et al. 2001). These were sub- jected to a tetrazolium test of viability following ISTA (ISTA 1993) to verify their viability sta- tus, but data was not analysed. Individual seeds were also studied under a dissecting microscope for further viability and anatomy notes follow- ing Hidayati et al. (2001). Germination and growth data were exposed to one-way and two- way analysis of variance (ANOVA) procedures for single factors and interactive factors of seed treatment x duration (Weber and D’Antonio 1999). Effect of Salt Concentration, Osmoconditioning Treatment Duration and Storage The same NaCl treatments mentioned above (0, 8 and 16g NaCl 1~') were used in combi- nation with the same treatment durations (1, 2 or 3d). After treatment, IRAT 204 seeds were dried back at 25°C for 24h and stored at 22°C and 50 to 52% relative humidity for 30d in the dark. Thereafter, seeds were retrieved from storage and sown in batches of 100 in 1000 mL polystyrene trays lined with creased fil- ter paper. Each tray was moistened with 40 mL of a polyethylene glycol solution (PEG) (Fluka Chemie, Germany) and covered with a lid to minimise evaporation. The solution had an os- motic potential (w,) of -10 bar (circa -1.0 MPa) simulating drought (Marschner 1995, Dodd and Donovan 1999). The PEG molecular weight (m.w.) was 10,000. Trays were placed in an in- cubator set at 42/25°C (day/night, 12h/12h). Germination counts were taken daily for 12d and from them the FGP, MGT and GI calcu- lated. Statistical arrangements were similar to the first experiment with experimental factors being treatment and soaking duration applied in RCBD (Dodd and Donovan, 1999). Effect of Salt-Based Mixtures and Drying Duration Three sorghum varieties were tested in this trial. ‘These were IGSV 112, ICSV 7¢45-and Me5=-1. Seed lot tests revealed germinability, moisture content and viability levels comparable to those of IRAT 204 sorghum used in the previous two experiments. The hypothesis to be tested here was that mixing NaCl with other minerals might pro- vide both the stress acclimation (hardening) ef- fect and a mineral uptake effect, thus improving seed performance under stress. The “softening” effect of other elements in the mixture might, thus, lead to a balance and a combating of Na* and Cl~ ions. A basic 10g NaCl 17! (wv, of -7.7 bar) (Knauer Osmometer, Germany) treat- 76 KADER ment was mixed with calcium sulphate or one of three fertilizers as follows: T,: Dry Control (dry, untreated seeds) Do 10s NaGitie: 4 og CaSO,4.2H2O ie (calcium sulphate) 10g NaCl 1~! + 7.5¢ Urea fertilizer 17! (water-soluble nitrogen-source fertilizer) 10g NaCl 1—! + 10g NPK fertilizer 1~* (slow release compound fertilizer with 6% N, 12% P2Os and 18% K20O) 10g NaCl 1~! + 15g DAP fertilizer 1~! (di-ammonium phosphate, soluble phosphorous source) 10g NaCl 17? Ts: dare Te: Ee: Seeds were soaked in the above mentioned solutions for $d’ at 18°C: in ‘thedark. “The concentrations were based on earlier work with sorghum (Al-Mudaris 1998). After treatment, seeds were dried back at 25°C for 7, 14 or 21h and stored for 1 month at 24°C in the dark. Subsequently, seeds were sown in batches of 100 in polystyrene trays at 40/20°C (day/night temperatures, 12h/12h) under a PEG-induced (m.w. 10,000) drought of -3.3bar (0.3 MPa). The change in temperature down to 40/20°C was based on observation of partial fungal infec- tion at the 42/25°C level in the previous trial. Again, the FGP and MGT were calculated in addition to the coefficient of velocity of germi- nation (CVG) (Jones and Sanders 1987). The CVG increases when the number of germinated Seed FGP MGT GI Treatment (%) (day) Wet Control 78.8 a BS a 278.6 a 8g NaCl 17! Vora 3 OM 262.2 a 16g NaCl 17! 62.1 a 335. b 228.0 b seed increases and the time required for ger- mination decreases. Theoretically, the highest CVG possible is 100 and would occur if all seeds germinated on the first day (Jones and Sanders 1987). Statistical procedures were the same as those for the second experiment. RESULTS AND DISCUSSION Effect of Salt Concentration, Osmoconditioning Treatment Duration and Drying The 16g NaCl 17! treatment reduced the FGP and GI in comparison to the wet control and the 8g NaCl1l7! treatment. Both salt soaks reduced the time needed to germinate as seen from lower MGT values (Table 2). Neither growth param- eters (DWS, DWR, and SRR) nor the number of GUE seeds differed between treatments. The longer the duration of soaking in NaCl solutions, the lower the final germination percentage and germination speed, and the higher the number of GUE seeds (Table 3). Drying seeds, on the other hand, did not affect germination charac- teristics (FGP, MGT and GI) but reduced the DWR and, thus, increased the SRR (Table 4). The interactive effects of NaCl concentra- tion, soaking duration and drying on germina- tion and growth characteristics of IRAT 204 re- vealed no significant interactions between treat- ment combinations regarding the parameters studied (data not shown) DWS DWR SRR GUE (mg) (mg) (seeds) 6.6 a 94a 0. 75sa; A a 6.6 a Gilea 0.75 a A Dua, 6.3 a 8.6 a 0.84 a 6.0 a Table 2. Effect of NaCl treatments on germination and growth characteristics of sorghum IRAT 204 under phytotron conditions. Means in columns followed by similar letters are not significantly different (a < 0.05). FGP: Final Germination Percentage, MGT: Mean Germination Time, GI: Germination Index, DWS: Dry Weight of Shoot, DWR: Dry Weight of Root, SRR: Shoot : Root Ratio and GUE: Germinated Unemerged Seeds. IONIC COMBATING MECHANISMS IN SEED 77 Treatment FGP(%) MGT GI DWS DWR SRR GUE Duration (day) (mg) (mg) : (seeds) (days) i 79.4 a S020 296.8 a 6.7 a 8.6 a 0.84 a 2010 2 70.0 b 4.0a 242.5-b 6.3 a 98a 0.69 a 5.0: a 3 64.7 b 3.7 ab 229.5 b 6.5 a 8.7 a 0.80 a 5.9 a Table 3. Effect of duration of soaking in NaCl solutions on germination and growth characteristics of sorghum IRAT 204 under phytotron conditions. Means in columns followed by similar letters are not significantly different (a < 0.05). FGP: Final Germination Percentage, MGT: Mean Germination Time, GI: Germination Index, DWS: Dry Weight of Shoot, DWR: Dry Weight of Root, SRR: Shoot : Root Ratio and GUE: Germinated Unemerged Seeds. Drying Treatment FGP MGT GI DWS DWR SRR GUE (%) (day) (mg) (mg) (seeds) No Drying (Fresh Sown) 72.7 a 3.6 a 263.0a 6.54 10.0 a 0.69 b 46a Drying (25°C, 3h) 70.1 a 3.9 a 249.5a 65a 8.1 b 0.86 a Orca) Table 4. Effect of drying after seed treatment on germination and growth characteristics of sorghum IRAT 204 under phytotron conditions. Means in columns followed by similar letters are not significantly different (a < 0.05). FGP: Final Germination Percentage, MGT: Mean Germination Time, GI: Germination Index, DWS: Dry Weight of Shoot, DWR: Dry Weight of Root, SRR: Shoot : Root Ratio and GUE: Germinated Unemerged Seeds. Effect of Salt Concentration, Osmoconditioning Treatment Duration and Storage The germination pattern of treated seeds after storage revealed that in both the Wet Control and 8g NaCl 1~! treatment, a soaking dura- tion of 2 days yielded better FGP values than 3 or 1 day soaking treatments, respectively. At the apparently high 16 g NaCl 17! level, soaking seeds for 1 day only was superior to soaking for 2 or 3 days (Table 5). Neither the MGT nor the GI were clearly affected by treatment and duration interactions. However, the highest GI value (best germination percentage and germi- nation speed relationship) was observed in seeds treated with 8g NaCl 17! for 2 days. Effect of Salt-Based Mixtures and Drying Duration The interactive analysis of seed treatment, genotype and drying duration did not reveal significantly different germination percentages. From Table 6 it can be seen that the low- est FGP was observed in the NaCl + DAP fertilizer treatment. All seed osmocondition- ing treatments reduced the MGT (seeds ger- minated faster) and increased the CVG (bet- ter germination percentage and rate) over un- treated seeds. This means that seed osmocon- ditioning increased overall germination speed. Genotypes differed in their response to osmo- conditioning with M35-1 performing in an in- 78 KADER ferior manner in comparison to ICSV 112 and the germination parameters studied (data not ICSV 745 as far as the FGP, MGT and CVG _ shown) pointing to the possibility of drying were concerned (Table 7). The duration of dry- seeds after treatment in order to improve stor- ing, on the other hand, did not affect any of age life. Seed Treatment Duration (days) FGP (%) MGT (day) GI Wet Control 1 78.6 be 4.2 ab 255.0 b-d ya 86.6 ab O.2' a Z210rored 3 t2.6.C 4.7 ab 223.6 cd 8g NaCl 17} 1 bard 34 b 201.0 cd 94.6 a 3.6 b 348.0 a 3 86.6 ab 4.0 ab 298.3 ab 16g NaCl 17! 1 84.0 be 4.4 ab 275.3 be 58.0 d 4.1 ab 200.0 cd 3 54.0 d 4.1 ab 185:3 a Table 5. Interactive effects of NaCl concentration and soaking duration on the germination of sorghum IRAT 204 seeds after storage for 10d under ambient conditions. Means in columns followed by similar letters are not significantly different (a < 0.05). FGP: Final Germination Percentage, MGT: Mean Germination Time and GI: Germination Index. Seed Treatment FGP (%) MGT (day) CVG Dry Control (4.2) 3.7 a PASTE NaCl (10g/1) + Calcium Sulphate (5g/l) 81.2 a 29K 36.1 ab NaCl (10g/1) + Urea Fertilizer (7.5 g/1) 66.4 c 2,9) be 33.9 ab NaCl (10 g/l) + NPK Fertilizer (10 g/1) 7120) o¢ Dae 36.8 a NaCl (10g/1) + DAP Fertilizer (15 g/1) 56.8 d Sw 6) 32) (ib NaCl (10 g/1) 68.7% be Zales 36.4 ab Table 6. Effect of NaCl and NaCl-based seed osmoconditioning treatments on germination char- acteristics of sorghum varieties ICSV 112, ICSV 745 and M35-1 after storage for 1 month at 30°C. Means in columns followed by similar letters are not significantly different (a < 0.05). FGP: Final Germination Percentage, MGT: Mean Germination Time and CVG: Coefficient of Velocity of Germination. Genotype FGP (%) MGT (day) CVG 1eSV 112) 2b 2.9 b Son hoe ICSV 745 84.4a 2.9 b 34.9 a M35-1 53.6 ¢ 3.2 a BIE) Table 7. Effect of genotype on germination characteristics of sorghum after storage for 1 month at 5 O- Means in columns followed by similar letters are not significantly different (a < 0.05). FGP: Final Germination Percentage, MGT: Mean Germination Time and CVG: Coefficient of Velocity of Germination. IONIC COMBATING MECHANISMS IN SEED 79 From the results of the first experiment, which was carried out under simulated condi- tions in the phytotron, it is clear that NaCl concentration is decisive in the post-treatment germinative response seeds exhibit. While 8g NaCl 1~! improved germination, 16g NaCl 17! retarded it in terms of FGP and GI. Both con- centrations of salt, however, increased germina- tion speed over water-soaked controls, but nei- ther affected seedling growth or the fate of seeds which did not germinate. The longer the dura- tion of soaking, the less positive effects were ob- served. Additionally, longer soaking durations (2 or 3 d in comparison to 1 d) almost doubled the number of germinated but unemerged seeds (Table 3). This response observed in fresh sown seeds was also observed in the second experi- ment where seeds were dried back and stored. The 8g NaCl 17! and water-soaking treatments were better than 16g NaCl 17! except that a 2-day treatment duration was observed to be better than 1d for water and 8g treatments, whereas | d was more suited for 16 g treatments. Again, 8g NaCl 1~! combined with a 2d treat- ment duration advanced germination as seen from higher GI values. This shows that stor- age of treated seeds at ambient temperatures for a duration of 30d does not negatively affect performance of pre-storage-hardened seed. The fact that longer treatment durations in- creased the number of germinated unemerged seeds (first experiment) and reduced overall ger- mination (16g NaCl for 2 or 3d in the second experiment) may be a direct effect of NaCl tox- icity to seeds. That seeds germinate but do not emerge reflects a reduced vigor and/or abnor- mality (microscopic evidence, data not shown) since all seeds were planted at the same depth of 2cm. Moisture supply was also regulated by weight and so the effect of external factors seems unlikely. A replication of the same treatments was conducted in a separate experiment and seeds/seedlings retrieved at 1, 3, 5 and 7 days after sowing. These were analysed for Nat and Cl~ content and showed levels up to five times as high as non-treated seeds/seedlings. The re- sults of this experiment will be reported in a subsequent paper, but would indicate that ionic toxicity occurred. An analysis of hormonal lev- els would point to a sharp increase in ABA lev- els during soaking treatments and a mild in- crease during and after heat shock (Kader, un- published data). This is where the hardening effect is likely to have taken effect, but at ex- cessive NaC] levels a germination block would have occurred due to the shortage in Kinetin and GA3 as a direct result of this physiologi- cal shock (Al-Mudaris 1998, Kader and Jutzi 2002). This points to the possibility that hard- ening can only provide alleviation from stress at moderate stress levels and becomes a stress it- self at higher degrees of heat, drought or salinity (Noe and Zedler 2000). The observation that 1d was better for 16g NaCl 1~! treatments tends to confirm the toxi- city hypothesis, since 1d would not be enough to inflict such stress due to the uptake of water by the seed at high rates during the first day of treatment (Al-Mudaris and Jutzi 1998ab). Bussell and Gray (1976), in their work on os- moconditioning tomato seeds at -5 to -15 bars, observed that the length of soaking period had little effect at low osmotic potentials but not at high potentials. The 8 and 16g NaCl 1~! treat- ments measured -6.02 and -11.1 bar on the os- mometer, respectively. This means that longer durations would expose the seeds to more os- motic stress. Soaking sorghum in water alone, on the other hand, has been reported to increase germi- nation speed as soaking period increased (Har- ris 1996). This has also been reported for pep- per seed treated with the non toxic, inert man- itol (Georghiou et al. 1987). The problem, then, lies within the nature of the osmoticum used. Seeds coming into contact with NaCl solutions have been found to have higher Cl7 concentrations in their different components, the accumulation of which, in the embryo (Es- echie 1995) and endosperm (Al-Mudaris, 1998), was associated with germination failure. Ful- bright (1988) observed reduced germination in Indiangrass (Sorghastrum nutans cv. Cheyenne, Lometa) at 0.12mol NaCl 1~! (6.9g NaCl 17?), 80 KADER whereas Shanmugasundaram and Kannaiyan (1989) found 1.0% NaCl (10g NaCl 1~*) to give the highest germination percentage in pear! mil- let (Pennisetum glaucum L. R. Br.) in compar- ison to 2.5% (25g NaCl 1~*). Increasing the salt concentration of a so- lution in contact with sorghum seeds reduces a-amylase and protease activity, reducing and non-reducing sugar contents and the rate of re- serve protein mobilization (Khan et al. 1989). Additionally, if a seed takes up solutes or mobi- lizes its reserves in an osmotically active form, its own water potential will be reduced although physiological processes may be inhibited both by the low water potential and the toxicity of ions (Bannister 1978). Ells (1963) reported that the priming effect of seed treatment with nutri- ent solutions (including 2% NaCl) is not due to the salts, nor to the amount of water retained by the seed from the treatment, but rather to certain enzymatic activities which take place within the seed while it is being held in a moist condition. Reduced emergence, after seed germination (germination in the seedbed, but lack of emer- gence above the soil surface) was observed in the phytotron trial. If the radicle emerges from the testa and the soil water content is reduced below the initial soil water content due to dry- ing, emergence is reduced (Helms et al. 1996). This could have been the case where trays were re-irrigated by weight only when they lost one third of their initial moisture content. This would have been more deleterious to seeds os- moconditioned for longer periods due to their higher initial moisture contents and would have led to a loss of the capacity to emerge (Peske 1983). Combining NaCl with calcium sulphate im- proved seed response as seen from the third ex- periment, whereas other mixtures did not raise the final germination percentage but improved germination speed over controls. This may be due to the fact that the fertilizers used were, in themselves, salts and as such contributed to the ionic stress discussed above. A notable point is that of solubility. Calcium sulphate was not totally soluble at the rates used, whereas sodium chloride was. The NPK fertilizer was also only partly soluble, whereas urea and DAP were more soluble. Mixing sodium chloride with calcium prob- ably increased Cat? content of seeds (Bharati and Vaidehi 1989, Al-Mudaris 1998, Kader, un- published data). Calcium has been found to play a major role in tolerance to NaCl where tol- erant genotypes of vegetables contained higher Cat? reserves in their seeds (Guerrier 1983). It has also been noted as modifying seed re- sponse to heat shock in maize (Gong et al. 1997) due, probably, to increasing membrane stabil- ity (Marschner 1995). Amazallag et al. (1997) also reported that leaf malformations in plants exposed to high NaCl concentrations were pre- vented by the addition of Cat? to the nutrient solution. Drying duration did not affect seed response to osmoconditioning. ‘This agrees with the re- sults of Emmerich and Hardegree (1996) who found that the germination of four warm-season grasses was not affected by length of the de- hydration period. It is also in line with the work of Brocklehurst and Dearman (1984) who found that drying vegetable seeds after treat- ment did not interact with either the priming chemical used or the species tested and those of Al-Mudaris and Jutzi (1998ab) and Al-Mudaris (1998a). Drying the seed slowly by control- ling humidity may impact germination rates achieved through conditioning (Mueller 1996). The loss of cell membrane integrity during dry- ing is repaired when seeds are allowed to imbibe water, albeit after a certain period (Knypl and Khan 1981). It follows that the rate of dry- ing (Dell Aquila and Trito 1990) and its timing after initial imbibition (Kutschera 1995) play the major roles (Al-Mudaris and Jutzi 1999 and 1999a). Both the degree of drying and its timing used here seem to fall within the range which does not alter osmoconditioning effects. ‘This ranged between 14 and 19.5% moisture content following treatment and after drying prior to storage. IONIC COMBATING MECHANISMS IN SEED 81 CONCLUSIONS The three experiments were carried out under varying temperature and drought stress situ- ations and after storage at ambient tempera- tures. The response of sorghum seeds to os- moconditioning in all three cases was gener- ally more affected by the priming agent itself, its concentration and the duration of treatment than by drying or storage. ‘This drying and stor- age is of great practical significance in the field, for if hardening is to be practiced, convenience in handling seeds must be fostered. This is dif- ficult to achieve in moist batches of seed, which would be prey to fungal infection and render sowing a difficult task. In conclusion, it would appear that seed hardening of sorghum via an NaCl-based soak has some potential to improve performance un- der post-treatment supra-optimal environmen- tal conditions like drought or heat stress. The decisive factor in this is the thin line between this hardening actually inducing germination and it being an additional plant stress factor itself. REFERENCES Al-Mudaris, M. 1998. Notes on various param- eters recording the speed of seed germina- tion. Journal of Agriculture in the Tropics and Subtropics, 98, 147-154. Al-Mudaris, M. 1998a. Studies on osmotic con- ditioning seed treatments to enhance germi- nation and early seedling growth of sorghum and pearl millet under drought, heat and combined stress conditions, Tectum Verlag Publishers, Marburg, Germany, 225p. Al-Mudaris, M. and Jutzi, S. 1998. Emergence and growth characteristics of sorghum and pearl millet intercropped with French beans after PEG-based seed priming under green- house and phytotron conditions. Journal of Agriculture in the Tropics and Subtropics, 98, 31-42. Al-Mudaris, M. and Jutzi, S. 1998a. The influ- ence of genotype, priming material, tempera- ture and osmotic potential of priming solution on imbibition and subsequent germination of sorghum and pear] millet seeds during and af- ter treatment. Journal of Agriculture in the Tropics and Subtropics, 98, 133-145. Al-Mudaris, M. and Jutzi, S. 1998b. The effect of incubation temperature during presowing seed treatments on the subsequent germina- tion behavior in Sorghum bicolor and Pen- nisetum glaucum. German Journal of Agron- omy, 2, 131-134 Al-Mudaris, M. and Jutzi, S. 1999. The in- fluence of fertilizer-based seed priming treat- ments on emergence and seedling growth of Sorghum bicolor and Pennisetum glaucum in pot trials under greenhouse conditions. Jour- nal of Agronomy and Crop Science 182, 135- 142 Al-Mudaris, M. and Jutzi, S. 1999a. Osmot- ically primed seed and seedling reactions to variations in day/night temperature. Jour- nal of Agronomy and Crop Science, 182, 217- 22\e Amzallag, G., Seligmann, H. and Lerner, H. 1997. Leaf malformation during early devel- opment in Sorghum. Evidence for an em- bryonic developmental window. Physiologia Plantarum, 99, 470-476. Bannister, P. 1978. INTRODUCTION TO PHYS- IOLOGICAL PLANT ECOLOGY, Second Print- ing, Oxford, Blackwell Scientific, UK. Benech Arnold, R., Fenner, M. and Edwards, P. 1991. Changes in geminability, ABA con- tent and ABA embryonic sensitivity in devel- oping seeds of Sorghum bicolor (L.) Monech induced by water stress during grain filling. New Phytologist, 118, 339-347. Bharati, P. and Vaidehi, M. 1989. ‘Treatment of sorghum grains with calcium hydroxide for calcium enrichment. Food and Nutrition Bul- letin, 11, 53-56. Brocklehurst, P. and Dearman, J. 1984. A comparison of different chemicals for osmotic treatment of vegetable seed. Annals of Ap- plied Biology, 105, 391-398. Bussell, W. and Gray, D. 1976. Effects of pre- sowing seed treatments and temperatures on 82 KADER tomato seed germination and seedling emer- gence. Scientia Horticulturae, 5, 101-109. Dell Aquila, A. and Trito, V., 1990. Ageing and osmotic priming in wheat seeds: effects upon certain components of seed quality. Annals of Botany, 65, 21-26. Dodd, G. and Donovan, L. 1999. Water po- tential and ionic effects on germination and seedling growth of two cold desert shrubs. American Journal of Botany, 86, 1146-1153. Ells, J. 1963. The influence of treating tomato seed with nutrient solutions on emergence rate and seedling growth. Proceedings of the American Society for Horticultural Science, 83, 684-687. Emmerich, W. and Hardegree, S. 1996. Partial and full dehydration impact on germination of 4 warm-season grasses. Journal of Range Management, 49, 355-360. Esechie, H. 1995. Partitioning of chloride ion in the germinating seed of two forage legumes under varied salinity and tempera- ture regimes. Communications in Soil Sci- ence and Plant Analysis, 26, 19-20. Fulbright, T. 1988. Effects of temperature, wa- ter potential, and sodium chloride on Indian- grass germination. Journal of Range Man- agement, 41, 207-210. Georghiou, K., Thanos, C. and Passam;’ H. 1987. Osmoconditioning as a means of coun- teracting the ageing of pepper seeds during high-temperature storage. Annals of Botany, 60, 279-285. Gong,”'M., ‘Yong=Jun, L., ‘Dai,’ XxX.) Tians iM. and Li, Z. 1997. Involvement of calcium and calmodulin in the acquisition of heat-shock induced thermotolerance in maize seedlings. Journal of Plant Physiology, 150, 615-621. Guerrier, G. 1983. Germination in vegetables and oil plants in the presence of NaCl. Seed Science and Technology, 11, 281-292. Harris, D. 1996. The effects of manure, geno- type, seed priming, depth and date of sow- ing on the emergence and early growth of Sorghum bicolor (L.) Moench in semi-arid Botswana. Soil and Tillage Research, 40, 73-88. Helms, T., Deckard, E., Goos, R. and Enz, J. 1996. Soil moisture, temperature, and drying influence on soybean emergence. Agronomy Journal, 88, 662-667. Heydecker, W. and Gibbins, B. 1978. The “Priming” of seeds. Acta Horticulturae, 83, 213-215. Hidayati, S., Baskin, J. and Baskin, C. 2001. Dormancy-breaking and germination require- ments for seeds of Symphoricarpos orbicula- tus (Caprifoliaceae). American Journal of Botany, 88, 1441-1451. Houle, G., Morel, L., Reynolds, C. and Siegel, J. 2001. The effect of salinity on different developmental stages of an endemic annual plant, Aster laurentianus (Asteracea). Amer- ican Journal of Botany, 88, 62-67. ISTA, 1993. International Rules for Seed Test- ing. Seed Science and Technology, 21. | Jones, K. and Sanders, D. 1987. The influence of soaking pepper seed in water or potassium salt solutions on germination at three tem- peratures. Journal of Seed Technology, 11, O7- 102. Kader, M. and Jutzi, S. 2002. Temperature, os- motic pressure and seed treatments influence imbibition rates in sorghum seeds. Journal of Agronomy and Crop Science, 188, 286-290. Khan, A., Azmi, A. and Ashraf. M. 1989. In- fluence of NaCl on some biochemical aspects of two sorghum varieties. Pakistan Journal of Botany, 21, 74-80. Knypl, J. and Khan, A. 1981. Osmocondi- tioning of soybean seeds to improve perfor- mance at suboptimal temperatures. Agron- omy Journal, 73, 112-116. Kutschera, U. 1995. KURZES LEHRBUCH DER PFLANZENPHYSIOLOGIE. Wiesbaden, Quelle und Meyer Verlag. Marschner, H. 1995. MINERAL NUTRITION OF HIGHER PLANTS (Second Edition), London, Academic Press. Mueller, D. 1996. Germination and root growth of 4 osmoconditioned cool-season grasses. Journal of Range Management, 49, 117-120. Munir, J., Dorn, L., Donohue, K. and Schmitt, J. 2001. The effect of maternal photoperiod IONIC COMBATING MECHANISMS IN SEED 83 on seasonal dormancy in Arabidopsis thaliana (Brassicaceae). American Journal of Botany, 88, 1240-1249. Noe, G. and Zedler, J. 2000. Differential ef- fects of four abiotic factors on the germina- tion of salt marsh annuals. American Journal of Botany, 87, 1679-1692. Orchard, T. 1977. Estimating the parameters of plant seedling emergence. Seed Science and Technology, 5, 61-69. Peske, S. 1983. Germination and emergence of soybean seeds as related to moisture stress. Dissertation Abstracts International, 44, 668 B. Shanmugasundaram, V. and Kannaiyan, M. 1989. Effect of concentration of seed hard- ening chemicals on physiological characters of pearl millet (Pennisetum typhoides Stapf and Hubb). Journal of Agronomy and Crop Sci- ence, 163, 174-176. Silvertown, J. 1999. Seed ecology, dormancy and germination: A modern synthesis from Baskin and Baskin. American Journal of Botany, 86, 903-905. Weber, E. and D’Antonio, C. 1999. Germi- nation and growth responses of hybridising Carpoprotus species (Azzoaceae) from coastal California to soil salinity. American Journal of Botany, 86, 1257-1263. Yang, J., Lovett-Doust, J. and Lovett-Doust, L. 1999. Seed germination patterns in green dragon (Arisaema dracontium, Araceae). American Journal of Botany, 86, 1160-1167. M. A. Kader Director, Consultica Worldwide, PO Box 3089 Tamarama NSW 2026 Australia m.kader@mbox.com.au (Manuscript received 5.11.2002) (Manuscript received in final form 04.02.2003) he fH ag: el: Ai iD Journal & Proceedings of the Royal Society of New South Wales, Vol. 135, p. 85-86, 2003 ISSN 0035-9173/03/020085-2 $4.00/1 Edgeworth David Medal 2002 PROFESSOR MARCELA BILEK Professor Marcela Bilek was appointed Pro- fessor of Applied Physics at Sydney University in 2000. She graduated from Sydney University with the University Medal in Physics in 1991 and has since worked in a number laboratories on projects that include atomic scale computer simulation, plasma processing, thin film mate- rials and surface modification. In 1993 she was awarded the Minerals, Metals and Materials So- ciety Reduction Technology Prize for her suc- cessful computer simulation of bubble stirring effects in aluminium cells, an achievement of considerable economic importance to the alu- minium smelting industry. A Peterhouse College and Cambridge Com- monwealth Trust scholarship enabled her to obtain a PhD from Cambridge University in plasma technology for the fabrication of thin solid films. She continued similar work at Cam- bridge under an Emmanuel College Research Fellowship. Specialised materials, increasingly needed in microelectronics, biomaterials and op- tics, are enhanced by these methods. Much of this work has been done in collaboration with research groups in Australia. She was the first to accurately model the transport of cathodic arc plasmas through mag- netic filters, enabling the removal of microparti- cles from beams and produce beams with homo- geneous cross sections for uniformly processing large wafers. Her models for the structure of both hydrogenated amorphous carbon and hy- drogenated silicon carbide, based on quantum mechanical treatment of the bonding electrons, has been confirmed by numerous experimental observations. One socially significant example of her current research is the surface coating of materials to be implanted in the human body as prosthetic devices. Biocompatibility, adhesion and corrosion resistance is always a problem in the body which sees such devices as foreign and attempts to remove them. Her plasma implan- tation methods promise to significantly extend the useful life of such devices. She has raised more than a million dollars for research since her Sydney appointment, con- vened a conference on biological effects of mi- crowave radiation, delivered the 14th Pollock Lecture, been awarded a Young Tall Poppy Award 2001 by the Australian Institute of Po- litical Science and the 2002 Malcolm McIntosh Prize for Physical Scientist of the Year. JCK 86 CLARKE MEDAL The Clarke Medal for 2002 PROFESSOR ROBERT HILL Professor Robert Hill is a Senior Research Fellow in the School of Earth and Environmen- tal Sciences at the University of Adelaide and is Head of Science at the South Australian Mu- seum. He is a graduate of the University of Adelaide. He completed his Ph.D. on Tertiary plant macrofossils in 1981, and his D.Sc. on the interaction between climate change and the evolution of the living Australian vegetation in 1997. In 1979 he accepted a position as Tutor in Botany at James Cook University, and in 1980 was offered a lecturing position in the Depart- ment of Botany at the University of Tasmania where he remained until 1999, being promoted to Professor in 1993. He was Head of the School of Plant Science for 6 years prior to his depar- ture, and was awarded Professor Emeritus sta- tus by the University of Tasmania Council in 2000. In 1999 he returned to the University of Adelaide to take up his current position. Professor Hill has had a lifetime interest in the evolution of the vegetation in Australia and Antarctica. He has published more than 125 refereed journal papers, 35 book chapters, sev- eral symposium papers and has edited or co- edited four books, including The History of the Australian Vegetation (Cambridge University Press), Ecology of the Southern Conifers (Mel- bourne University Press), The Ecology and Bio- geography of Nothofagus Forests (Yale Univer- sity Press), and Vegetation of Tasmania (Aus- tralian Biological Resources Study). Professor Hill is President and a Fellow of the Australian Institute of Biology and a Fel- low of the Linnean Society of London. His cur- rent research interest is the adaptation of the Australian vegetation to increasing aridity dur- ing the last 30 million years. He is developing a research program on the impact of fire on the Australian vegetation during this time period. He is best known for his research on the fossil history of the southern beech, Nothofagus, and the southern conifers. His work on the fossil his- tory of Nothofagus has been critical in refining our understanding of its evolution and has led to a major revision of our understanding of the biogeography of this critical southern genus. JCK Journal €& Proceedings of the Royal Society of New South Wales, Vol. 135, p. 87-88, 2003 ISSN 0035-9173/03/020087-2 $4.00/1 Biographical Memoir Te hi SIR ARTHUR RODEN CUTLER, V.C., AK, GMEG., K.C.V.0., C.B.E., K: St.Ji, B.Ec (Syd.), L1.D (Hon.) (Syd.}, D.Sc. (Hon.) (NSW & Newcastle) 1916-2002 Arthur Roden Cutler, known as Sir Roden, was born on 24 May, 1916, at Manly, son of Arthur William Cutler and Ruby Daphne (née Pope) of Bathurst. The Cutler family arrived in New South Wales as free settlers in 1833. The Roden fam- ily, of which his mother was a direct descendent, arrived even earlier (1827) with the army. Roden Cutler was educated at Sydney Boys’ High School and Sydney University, graduat- ing B.Ec in 1935. At the University he excelled in sports, gaining Blues from both Sydney and the Australian Universities in swimming, and reprinted with permission of the Australian War Memorial participating in water polo and shooting. As a teenager he made an heroic surf rescue, risking his life against a large shark. He joined the Public Trust Office of NSW on graduating, taking leave in 1940 to join the A.LF. (2/5 Field Regiment). Roden Cutler’s war service was relatively short, but spectacu- lar, and became legendary. In Syria on 19 June, 1941 he showed “exceptional courage” in driving the enemy back and establishing outposts which were important factors in capturing Merdijay- oun. Three weeks later (6 July), at Damour, he went forward against heavy machine-gun fire, was severely wounded, and subsequently had his leg amputated. He was awarded the Victoria Cross on 28 November, 1941. Between 1942-43 he was a member of the Commonwealth Aliens Classification and Advi- sory Committee, Assistant Deputy Director of 88 BIOGRAPHICAL MEMOIR the Security Service of NSW (1943), and from 1943 to 1946 Assistant Commissioner of the Repatriation Department. He was also NSW State Secretary for the R.S.L, 1942-43. In 1946 he married Helen Gray Annetta Morris (d. 1990) and a new career path opened with his appointment as High Commissioner to New Zealand (to 1952), to Ceylon (now Sri Lanka), and the equivalent position (Australian Minister) to Egypt between 1955-56. Then fol- lowed two years in Canberra where he was Chief of Protocol in the Department of External Af- fairs, Secretary-General of a S.E.A.T.O. Meet- ing, and ACT President of the RSL. In 1959 he moved overseas again as High Commissioner to Pakistan, with a brief inter- lude in the Somali Republic, and then to New York, where he was Australian Consul-General from 1961-65, and a delegate to the United Na- tions. His final overseas posting was as Ambas- sador to the Netherlands, 1965-66. His association with this Society began in 1966, when he was appointed Governor of New South Wales, a post he held until 1981, the longest-serving Governor of the State. During this time he was also Administrator of Australia (acting Governor-General) on six occasions. He accepted the Council’s invitation to joint Pa- tronage (with the Governor-General), and was a strong supporter (with his wife) of the Soci- ety, during a period of turmoil, with the move from Science House and a considerable drain of the Society’s finances. At the Centenary Dinner in 1966 Sir Ro- den stressed the importance of scientific work in modern times, and the contribution made by Australian scientists. Sir Roden’s speech at the 1974 Dinner, held at the Sydney Opera House, was memorable, marked as it was by some humorous compar- isons with former Governors who had been asso- ciated with the Society (and its ancestors), but with discussion of more serious Society matters. He said “The Royal Society of New South Wales may not be as widely known as it deserves, nor may its functions be fully understood”. While there was encouragement that membership had increased in the previous year, “the real value is in the learned qualifications of your member- ship, not in the total number”, and the medals were held in high regard. “The Society’s task is to bring a balance into people’s assessment of the advantages and lim- itations of scientific progress. You need to en- courage research and investigation, and occa- sionally express a word of warning”. He com- mended a “most valuable function” of the So- ciety, the “keeping of a library,” which he saw in its final open arrangement when opening the the Science Centre in 1977. Sir Roden displayed in his speeches a good knowledge of the workings of the Society, de- spite the many other calls on his time, and his widespread interests. He died on 21 February, 2002 after a short illness, and is survived by his second wife, Lady Jane C. Cutler and four sons from his first marriage. The Society was repre- sented at the State Funeral. DFB Journal € Proceedings of the Royal Society of New South Wales, Vol. 135, p. 89-90, 2003 ISSN 0035-9173/03/020089-2 $4.00/1 Index to Volume 135 A Aeronautica Antipodean. D.A. CRADDOCK, Presidential Address 2002 1 Aerial and Below Ground Biomass Production of Acacia as Influenced by Organic Waste Substrates During Nursery-Stage Seedling Growth. KADER, H.A., OMARI, M.A. and HATTAR, B.I. 1 Awards, Citations for 2001 37-43 Awards, Citations for 2002 85-86 Annual Report of Council for the year ended 31 March 2002 49 B BILEK, Professor Marcela, Edgeworth David Medal for 2002 85 Biomass Production Dy, Biographical Memoirs Samuel Warren CAREY AO 45 Andrew John CORBYN 47 Sir Roden CUTLER 87 C CRADDOCK, David A. Aeronautica Antipodean. Presidential Address 2002 1 Clarke Medal 2001 43 Clarke Medal 2002 86 CAREY AO, Samuel Warren, Obit 45 D Deposition of Trace Elements from the Atmosphere in the Sydney Region. D.J. SWAINE 20 E Edgeworth David Medal for 2001 39 Edgeworth David Medal for 2002 85 H HATTAR, B.I., KADER, M.A. and OMARI, M.A. Aerial and Below Ground Biomass Production of Acacia as Influenced by Organic Waste Substrates During Nursery-Stage Seedling Growth. 17 HILL, Professor Robert, Clarke Medal for 2002 86 K KADER, M.A., OMARI, M.A. and HATTAR, B.I. Aerial and Below Ground Biomass Production of Acacia as Influenced by Organic Waste Substrates During Nursery-Stage Seedling Growth. ii continued on next page 90 INDEX O OMARI, M.A., HATTAR, B.I. and KADER, M.A., Aerial and Below Ground Biomass Production of Acacia as Influenced by Organic Waste Substrates During Nursery-Stage Seedling Growth. Obituary: Andrew John CORBYN Obituary: Sir Roden CUTLER P Presidential Address 2002 PARKER, Michael William, Walter Burfitt Prize for 2001 PACKHAM, Gordon Howard, Clarke Medal for 2001 R RICHARDSON, Samantha Jane, Edgeworth David Medal for 2001 S SWAINE, D.J. Deposition of Trace Elements from the Atmosphere in the Sydney Region. Society’s Medal 2001 T Trace Elements in Sydney Region W Walter Burfitt Prize for 2001 WILLIAMS, P.A., Society’s Medal for 2001 Al 37 JOURNAL AND PROCEEDINGS OF THE ROYAL SOCIETY O F NEW SOUTH WALES Volume 135 Parts 1 to 4 (Nos 403 — 406) 2003 ISSN 0035-9173 PUBLISHED BY THE SOCIETY PO BOX 1525, MACQUARIE CENTRE, NSW 2113 Issued June 2002 (Parts 1 and 2), April 2003 (Parts 3 and 4) THE ROYAL SOCIETY OF NEW SOUTH WALES OFFICE BEARERS FOR 2002-2003 Patrons His Excellency the Right Reverend Dr Peter Hollingworth AC, OBE, Governor General of the Commonwealth of Australia. Her Excellency Professor Marie Bashir, AC, Governor of New South Wales. President Mr D.A. Craddock, BSc(Eng) NSW, Grad.Cert. Management UWS. Vice Presidents Prof. P.A. Williams, BA (Hons), PhD Macq. Dr W.E. Smith, MSc Syd, MSc Oxon, PhD NSW, MInstP, MAIP. Mr C.F. Wilmot Hon. Secretary (Gen.) vacant (acting Hon. Sec. Prof. P.A. Williams) Hon. Secretary (Ed.) Mrs M. Krysko von Tryst, BSc, Grad.Dip.Min.Tech NSW, MAusIMM. Hon. Treasurer Prof R.A. Creelman, BA, MSc, PhD Hon. Librarian Dr E.V. Lassak, MSc, PhD NSW, ASTC; FRACI Councillors Mr J.R. Hardie, BSc Syd, FGS, MACE. Prof. J. Kelly, BS¢ Syd, PhD Reading, DSc NSW Ms K. F. Kelly, BSc(Hons) Mr M.F. Wilmot, BSc Prof M.A. Wilson, PhD, DSc. Auck, FRACI, C.Chem. Southern Highlands Rep. Mr C.M. Wilmot The Society originated in the year 1821 as the Philosophical Society of Australasia. Its main function is the promotion of Science by: publishing results of scientific investigations in its Journal and Proceedings; conducting monthly meetings; organising summer science schools for senior secondary school students; awarding prizes and medals; and by liason with other scientific societies. Special meetings are held for: the Pollock Memorial Lecture in Physics and Mathematics, the Liversidge Research Lecture in Chemistry, the Clarke Memorial Lecture in Geology, Zoology and Botany, and the Poggendorf Lecture in Agricultural Science. Membership, as an Ordinary, Associate or Absentee Member, is open to any person whose application is acceptable to the Society. An application must be supported by two members of the Society. Subscriptions for the Journal only are accepted. The Society welcomes, from members and non-members, manuscripts of research and review articles in all branches of science, art, literature and philosophy for publication in the Journal and Proceedings. Manuscripts from non-members must be communicated through a member. ISSN 0035-9173 (© 2003 Royal Society of NSW. The appearance of the code at the top of the first page of an article in this journal indicates the copyright owner’s consent that copies of the article may be made for personal or internal use, or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per-copy fee through the Copyright Clearance Centre Inc., 222 Rosewood Drive, Danvers, Massachusetts, 01923, USA (CCC Online http://www.copyright.com) for copy- ing beyond that permitted by sections 107 and 108 of the US Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Responsibility for interpretations, opinions, reproductions and data published on behalf of authors rests with the relevant authors, not with the Royal Society of New South Wales. CONTENTS Vol. 135 Parts 1 and 2 CRADDOCK, DAVID, A. Antipodean Aeronautica, Presidential Address 2002 KADER, M.A., OMARI, M.A. & HATTAR, B.I. Aerial and Below Ground Biomass Production of Acacia as Influenced by Organic Waste Substrates During Nursery-Stage Seedling Growth SWAINE Deposition of Trace Elements from the Atmosphere in the Sydney Region AWARDS The Society’s Medal 2001 Edgeworth David Medal 2001 The Walter Burfitt Prize for 2001 The Clarke Medal for 2001 BIOGRAPHICAL MEMOIRS Samuel Warren Carey AO Andrew John Corbyn ANNUAL REPORT OF COUNCIL FOR THE YEAR ENDED 31%* MARCH 2002 Vol. 135 Parts 3 and 4 JOHNSTON, GRAHAM A. R. Dietary Chemicals and Brain Function KADER, M. A. Jonic Combating Mechanisms and their Comparative Effects on Seed Hardening under Simulated Supra-Optimal Environmental Conditions AWARDS Edgeworth David Medal 2002 The Clarke Medal for 2002 BIOGRAPHICAL MEMOIR Sir Arthur Roden Cutler, V.C., AK, K.C.M.G., K.C.V.O., C.B.E., K. St.J., INDEX TO VOLUME 135 ye 20 ov 73 85 86 S7 89 NOTES NOTES 32 os NOTICE TO AUTHORS Manuscripts should be addressed to The Hon- orary Secretary, Royal Society of New South Wales, PO Box 1525, Macquarie Centre, NSW 2113. Manuscripts submitted by a non-member (through a member) will be reviewed by the Hon. Editor, in consultation with the Editorial Board, to decide whether the paper will be further considered for publication in the Journal. Manuscripts are subjected to peer review by an in- dependant referee. In the event of initial rejection, manuscripts may be sent to two other referees. Papers, other than those specially invited by the Editorial Board on behalf of Council, will only be considered if the content is substantially new ma- terial which has not been published previously, has not been submitted concurrently elsewhere nor is likely to be published substantially in the same form elsewhere. Well-known work and experimental pro- cedure should be referred to only briefly, and exten- sive reviews and historical surveys should, as a rule, be avoided. Letters to the Editor and short notes may also be submitted for publication. Three, single sided, typed copies of the manuscript (double spacing) should be submitted on A4 paper. Spelling should conform with “The Concise Oxford Dictionary” or “The Macquarie Dictionary”. The Systéme International d’Unites (SI) is to be used, with the abbreviations and symbols set out in Aus- tralian Standard AS1000. All stratigaphic names must conform with the In- ternational Stratigraphic Guide and new names must first be cleared with the Central Register of Australian Stratigraphic Names, Australian Geo- logical Survey Organisation, Canberra, ACT 2601, Australia. The codes of Botanical and Zoological Nomenclature must also be adhered to as necces- sary. The Abstract should be brief and informative. Tables and Illustrations should be in the form and size intended for insertion in the master manuscript - 150 mm x 200 mm. If this is not readily pos- sible then an indication of the required reduction (such as ‘reduce to 1/2 size’) must be clearly stated. Half-tone illustrations (photographs) should be in- cluded only when essential and should be presented on glossy paper. Maps, diagrams and graphs should generally not be larger than a single page. However, larger figures may be split and printed across two opposite pages. The scale of maps or diagrams must be given in bar form. Half-tone ilustrations should be included only when essential and should be presented on glossy paper. All tables and illustrations should be numbered con- secutively with Arabic numerals in a single sequence and each must have a caption. References are to be cited in the text by giving the author’s name and year of publication. References in the Reference List should be listed alphabetically by author and then chronologically by date. Titles of journals should be cited in full — not abbreviated. MASTER MANUSCRIPT FOR PRINTING The journal is printed from master pages prepared by the ATRX typesetting program. When a paper has been accepted for publication, the author(s) will be supplied with a guide to acceptable electronic format for the submission of the revised manuscript. Galley proofs will be provided to authors for final checking prior to publication. REPRINTS An author who is a member of the Society will re- ceive a number of reprints of their paper free. Au- thors who are not a members of the Society may purchase reprints. iN CONTENTS 3 9088 01308 49 Vol. 135 Parts 3 and 4 JOHNSTON, GRAHAM A. R. Dietary Chemicals and Brain Function KADER, M. A. Ionic Combating Mechanisms and their Comparative Effects on Seed Hardening under Simulated Supra-Optimal Environmental Conditions AWARDS Edgeworth David Medal 2002 The Clarke Medal for 2002 BIOGRAPHICAL MEMOIR Sir Arthur Roden Cutler, V.C., AK, K.C.M.G., K.C.V.O., C.B.E., K. St.J., INDEX TO VOLUME 135 ADDRESS Royal Society of New South Wales, PO Box 1525, Macquarie Centre, NSW 2113, Australia http://nsw.royalsoc.org.au DATE OF PUBLICATION April 2003 | SONIAN INSTITUTION LIBRARIES i ov 13 85 86 87 89