Farinose flavonoids are associated with high freezing tolerance in fairy primrose(Primula malacoides)plants

Ryutaro Isshiki,Ivan Galisand Shigemi Tanakamaru

Institute of Plant Science and Resources,Okayama University,Kurashiki 710-0046,Japan.＊Correspondence:igalis@rib.okayama-u.ac.jp

INTRODUCTION

Unstable climate and extreme fluctuations in atmospheric temperatures due to global warming are likely to occur more frequently in the future,exposing plants to increasingly stressful and unpredictable growth conditions(Ahuja et al.2010;Albert et al.2013).Freezing damage in plants is one of the serious problems that regularly cause severe crop loss,for example,in fruit trees due to the irreversible damage of young buds and flowers after spring frosts(Rodrigo 2000).However,some plants were able to colonize low temperature niches,presumably due to evolution of efficient frost tolerance mechanisms(Shindo et al.2007).Occupying these niches and shifting phenology to the early spring allowed them to avoid competition of other non-adapted plants(Basler and Korner 2012).Indeed,freezing tolerance is one of the primary factors determining geographic distribution of land plants(Korn et al.2008).

The level of freezing tolerance in plants is highly correlated with the intensity of damage caused by the frosting event.In general,to minimize freezing damage,physiological changes such as accumulation of soluble materials,sugars,and amino acids,occur in plants to generate a negative osmotic potential and/or increases desaturation of cell membrane fatty acids(Walker et al.2010).For example,Klotke et al.(2004)reported that soluble sugar content was well correlated with the observed freezing tolerance in selected accessions of Arabidopsis thaliana plants.

Primula is one of the most widespread of all plant genera,possibly because of its successful adaptation to extreme environmental conditions(Richards 2003),and approximately 430 Primula species occur throughout the moister and cooler regions of the northern hemisphere.Primula malacoides Franchet,Bull(Primulaceae),known as fairy primrose,is a well-adapted plant species to higher altitudes;it grows as annual plant at altitudes approximately 2,000 m from inside Burma to Yunnan in China(Richards 2003).P.malacoides became a very popular garden plant with many horticultural varieties,some of which show high freezing tolerance in early springs.Interestingly,a number of Primula species are able to produce farinose flavonoids on their body surfaces,such as leaves or buds(Wollenweber 1984).Farinose flavonoids,with aglycones as major constituents,become visible to the naked eye as a white powder(farina)on the plant surface.From our empirical observations,farina on these plants occurs primarily during the winter and spring seasons,showing a potential but undocumented link between freezing tolerance and farinose flavonoid aglycones.

Flavonoids and anthocyanins are classified as plant secondary metabolites and are generally colored compounds(Buer et al.2010).They commonly occur in colored plant parts,such as flowers,where they serve as attractants to pollinators.In addition,flavonoids are involved in resistance of plants against various stresses.For example,flavonoids and other phenolics function as ultraviolet(UV)light screens in plants that are exposed to strong solar radiation(Harborne and Williams 2000;Buer et al.2010).Flavonoids are well known for their radical scavenging properties that can protect cells from unavoidable internal damage after deployment of defensive reactive oxygen species(ROS)during stress(Agati et al.2012).Recently,strong correlations between flavonoid content and freezing tolerance have been shown in selected Arabidopsis accessions(Korn et al.2008).Although less understood,flavonoids are also involved in regulation of plant development,for example,by regulating the transport of plant hormone auxin(Buer et al.2013)or seed germination(Jia et al.2012).

Because substantial amounts of flavone(2-phenyl-1,4-benzopyrone),one of the flavonoid aglycones,deposit on the leaves of P.malacoides during cold seasons,this plant provides a suitable model for investigating the function of farinose flavone in freezing tolerance.First,we asked if the amount of farinose flavone correlates with experimentally determined levels of freezing tolerance in P.malacoides,and if so,whether exogenous application of flavone could be used to protect other plants from freezing damage.We show that farinose flavone has a role,possibly an essential role,in the adaptation process of these plants to low temperatures,and application of flavone has a beneficial effect on isolated leaves of several fruit trees exposed to freezing temperatures.The mechanism by which flavone plays a role in freezing tolerance is discussed.

Figure 1.Farinose flavonoids on aerial parts of Primula malacoidesFlavonoids deposited on(A)calyx,flower stem,and(B)underside of leaf in flowering stage fairy primrose plant(P.malacoides cv.Uguisu)grown for 2 months at low non-freezing temperature.Flavone deposition appears as white mealy powder(farina).(C)Leaves of 4 month old P.malacoides cv.Lollipop plants cultivated continuously at 22°C and(D)after placing in 4°C for 4–5 weeks.(E,F)White crystals of secreted flavone developed around glandular trichomes on cold acclimated leaves.

RESULTS

Correlation between farinose flavone and freezing tolerance in P.malacoides cultivars

It is known that Primula species deposit various amounts of farinose flavonoids on their leaves.Such depositions are particularly visible during cold growing seasons;P.malacoides cv.Uigusu leaves and buds became heavily covered with farina during growth in temperature uncontrolled glasshouse(see Materials and Methods),even during mild non-freezing winter/spring seasons in Okayama Prefecture in Japan(Figure 1A,B).In the summer,flavonoid deposition could be induced by transferring plants from 22 to 4°C for approximately 1 month(Figure 1C,D;P.malacoides cv.Lollipop).Secreted flavonoids appeared as crystals of various sizes around short glandular trichomes on the leaves(Figure 1E,F).

To determine if the amount of farinose flavonoids is correlated with empirically observed varietal differences in freezing tolerance,we randomly selected several commercially available P.malacoides varieties and determined their leaf ice nucleation temperatures(INT).Simultaneously,surface components were collected by shortly dipping the leaves in acetone(30 s)and,after identification of flavone(2-phenyl-1,4-benzopyrone)as a major extract component by gas chromatography mass spectrometry(GC-MS)(Figure 2A),flavone content in the extracts was determined by standard GC coupled to a flame ionization detector(FID).

Leaf INTs were negatively correlated with the increasing flavone contents up to 10 mg/g fresh weight(FW)(Figure 2B;filled diamonds;linear regression r=0.617)but cultivars with extremely high flavone(＞10 mg/g FW;open diamonds)showed no further improvement of their INT values(Figure 2B;open diamonds).The maximal difference in INT between various P.malacoides cultivars was as high as 6.2°C,strongly suggesting a direct involvement of flavone in freezing tolerance.Next,we examined the importance of surface localization of flavone by experimentally removing and/or replacing the protective flavone layer on the leaves.

Flavone removal compromises freezing tolerance

Surface flavone was removed from the leaves of P.malacoides(cultivar Lollipop,highlighted by the arrow in Figure 2B)using 10%(v/v)ethanol solution-wetted tissue and leaves were immediately subjected to INT measurements.At the same time,EL measurements that reveal post-freezing damage were conducted with another independent set of samples as described in Materials and Methods.The leaves that had their surface flavone removed showed significantly higher INTs(early freezing)compared to control leaves with intact flavone(Figure 3A;ANOVA P＜0.05).Additionally,these leaves suffered more EL resulting from freezing damage compared to intact leaves(Figure 3B;ANOVA P＜0.01).However,in this experimental setup,it was possible that the leaves may have had suffered direct surface damage from the 10%(v/v)ethanol wipe that could cause changes unrelated to the role of flavone as freezing protectant.To exclude such a possibility,we used a commercially available grade of flavone(see Materials and Methods)and,after wiping leaves with 10%(v/v)ethanol as previously,sprayed the naturally sourced compound dissolved in ethanol again on the leaves at 4 mg/g FW concentration.After brief drying at room temperature,INT and EL measurements were conducted.While this flavone treatment restored the original high flavone status of the leaves,it included an ethanol wiping treatment.Corroborating the hypothesis of direct function of flavone as freezing protectant,reconstituted leaves had INTs and EL levels similar to control leaves without any treatments(Figure 3A,B;ANOVA P=0.70 and P=0.29,respectively).

Figure 2.Flavone identification and ice nucleation temperatures of leaves in selected Primula malacoides cultivars(A)Total ion chromatogram(m/z 50–250)and mass spectrum of flavone(2-phenyl-1,4-benzopyrone)found as major component in leaf surface extracts of P.malacoides exposed to cold treatment;the major molecular peak at m/z 222 was detected by mass spectrometry operating in electron impact mode after separation on capillary gas chromatography(GC)column.(B)Relationship between flavone content and ice nucleation temperature(INT)of the leaves in selected P.malacoides cultivars.INTs were determined for 10 individual leaves in each cultivar;flavone content was determined in pooled leaf extracts in three technical replicates by GC flame ionization detector.Linear regression was calculated for leaves containing less than 10 mg/g fresh weight(FW)flavone(black symbols).P malacoides cv.Lollipop used in the following experiments is highlighted by arrow.

Figure 3.Effect of flavone on ice nucleation temperatures and electrolyte leakage of Primula malacoides leaves(A)Effect of surface flavone on ice nucleation temperatures of the leaves(n≥6)using intact(control),flavone-deprived(removed),and reconstituted leaves with exogenously sprayed flavone(4 mg/g fresh weight(FW)in pure ethanol).(B)Effect of leaf surface flavone on reduced freezing damage measured as electrolyte leakage(n=8)after treatments described in(A).(C)Concentration-dependent relationship between leaf ice nucleation temperatures and exogenously applied flavone(n≥7).All leaves were deprived of natural flavone prior to spray by gently wiping the leaves with 10%(v/v)ethanol soaked tissue.Statistically significant differences in(A–C)were determined by ANOVA followed by Tukey’s honestly significant difference test(P＜0.05).

Typically,the frosting temperature(-8°C)-exposed flavone-deprived leaves treated with ethanol(mock)spray appeared macerated and watery after transfer to room temperature,in contrast to flavone(4 mg/g FW in ethanol)-treated leaves or leaves with natural farinose flavone,which appeared visually undamaged.Moreover,ethanol mocktreated leaves discharged a typical scent of green leaf volatiles,putatively released from the damaged cells.

Flavone concentration-dependent INT responses

In the previous experiment,a single dose of 4 mg/g FW of flavone was sufficient to restore the original INT values in flavone-deprived leaves.We then examined a dose dependency of flavone effect on INT using P.malacoides cultivar Lollipop.The ethanol-wiped leaves were sprayed with increasing concentrations of flavone dissolved in ethanol ranging 0–6 mg/g FW that corresponded to naturally observed flavone amounts on Primula leaves(Figure 2B).The INT values decreased with the increasing amounts of exogenous flavone(Figure 3C)and a statistically significant difference was found between 0 and 4 mg/g FW flavone concentrations applied on the leaves by Student’s t-test(P ＜ 0.05).Interestingly,higher amounts of flavone(6 mg/g FW)did not show any further improvement similar to naturally occurring flavone on the leaf surface(Figure 2B),suggesting the existence of relatively narrow optimal concentration of flavone that provides maximal freezing tolerance effect.

Mechanistic studies and application of exogenous flavone to other plants

The location of farinose flavone on the leaf surface does not support a role for the farina as a direct contributor to the osmotic potential in the leaves.A physical interaction of waterinsoluble(hydrophobic)flavone with the process of ice nucleation is therefore more likely to explain the effects of surface flavone on freezing behavior of the leaves.This assumption is supported by the rapid effects of physical removal and reconstitution of flavone shown in Figure 3.

To confirm physical(rather than physiological)effects of farinose flavones on the leaves,we measured INTs using vinyl chloride impregnated fiber cloth pieces soaked with water.Flavone was sprayed on one set of the sheets of approximate leaf size before INT measurements,while the control set remained untreated(Figure 4A).The difference of INTs between control and flavone-treated non-living material was-4.4°C with high statistically significant difference observed at P ＜ 0.001(Student’s t-test).

Next,we examined the potential effects of flavone application on other plant species.At first,flavone at empirically determined optimal concentration of 4 mg/g FW dissolved in ethanol was applied to leaves of selected fruit trees.INTs of flavone-treated leaves significantly decreased,providing freezing protection,in all fruit tree species(Figure 4B).In addition,after defrosting,less damage was observed,in regards to leaves turning brown,on flavonetreated apple leaves compared to non-treated controls(Figure 4B).A similar effect of flavone was observed using winter buds of peach tree for both INT and EL values(Figure 4C).These results suggested a practical potential of flavone application in frost damage protection of plants as discussed below.

DISCUSSION

We show that experimentally determined INT values of P.malacoides leaves for a range of cultivars are inversely correlated with the amounts of farinose flavone deposited on leaf surfaces.In addition,exogenously applied flavone to the leaves,which had the endogenous flavone artificially removed,ameliorated freezing tolerance of the leaves,pointing to external flavone as one of the primary factors in freezing tolerance.In addition,we found that exogenous flavone application improved freezing tolerance in several other plant species that normally do not secrete flavonoids,thus providing a new generally applicable plant-protection method against frost damage.

Most plants are able to increase their freezing tolerance after exposure to low but nonfreezing temperatures,engaging in an adaptive process known as cold acclimation(Xin and Browse 2000;Uemura et al.2003).This feature is particularly well developed in plants growing in cold climate or thriving during early spring,thus escaping from resource competition of non-adapted plants(Shindo et al.2007).The changes in freezing tolerance are generally associated with profound changes in gene expression to accommodate a substantial reprogramming of cell metabolism(Thomashow 1999;Hannah 2006)that leads to major physiological changes such as accumulations of osmolytes including soluble sugars,proline or betaine,and alterations in plasma membrane composition in plants(Guy 1990;Steponkus et al.1993).In addition to osmolytes,a possible role of flavonoids in freezing tolerance of plants has also been proposed(Hannah et al.2006;Korn et al.2008),but the exact mechanism remains unclear.

In general,plants phenolics that include a broad range of compounds such as various phenylpropanoids,flavonoids,and anthocyanins can protect plant cells against damage caused by ROS during,for example,drought or UV light irradiation stresses(Winkel-Shirley 2002;Buer et al.2010;Pollastri and Tattini 2011).Furthermore,flavonoids play important roles in deterring herbivorous insect and in antimicrobial resistance as phytoalexins(Harborne and Williams 2000;Matejic et al.2012).As introduced,the presence of various flavonoids was also closely correlated with levels of freezing tolerance in plants,pointing to yet another protective role of flavonoids in plants.Specifically,Vaclavik et al.(2013)reported that cold-tolerant accessions of A.thaliana possess high concentrations of kaempferol-3,7-O-dirhamnoside.A remarkably strong correlation between flavonol contents and freezing tolerance,in addition to sugars,was found in crosses between A.thaliana accessions of different freezing tolerance(Korn et al.2008).Apart from Arabidopsis,several Rhododendron cultivars showed higher total flavonoid contents,including flavonoid aglycones and glycosides,which was associated with significantly higher levels of freezing tolerance(Swiderski et al.2004).Kasuga et al.(2008)reported that flavonoids found in xylem parenchyma cells of the katsura tree(Cercidiphyllum japonicum)have strong anti-ice nucleation properties,causing survival at low temperatures.Although the exact mechanism for anti-ice nucleation effect of xylem parenchyma flavonoids has not been proposed,these findings suggest that various flavonoids may contribute to freezing tolerance in plants,although in contrast to P.malacoides,these were likely sequestered in vacuoles or cytosol,or localized in xylem parenchyma cells.

Figure 4.Application of exogenous flavone and protection mechanism(A)Effect of flavone application on small pieces of generic vinyl chloride coated fiber cloth(n=100)after the cloth was soaked with water and subjected to freezing.Ice nucleation temperatures were determined essentially the same way as for the living tissues(leaves).(B)(Upper panel)Effect of flavone application on fruit tree leaves(n≥8)with and without application of flavone at 4 mg/g fresh weight(FW)concentration.Flavone application significantly decreased the ice nucleation temperatures in all tree species.(Lower panel)Thawed apple leaves after freezing treatment(left,control leaf treated with pure ethanol;right,leaf treated with 4 mg/g FW flavone prior freezing).Control leaves showed extensive browning due to putative leakage and oxidation of phenolic substrates in the leaves.(C)(Upper panel)Effect of flavone application on ice nucleation temperatures of peach tree winter buds(n=10).Buds were sprayed with flavone at 4 mg/g FW concentration or mock-treated(control)before freezing test.(Lower panel)Effect of flavone application on electrolyte leakage caused by freezing damage of peach tree winter buds(n=8)treated as above.Statistically significant differences in(A–C)were determined by Student’s t-test(*P＜0.05,**P＜0.01,***P＜0.001).

After perception of initial stress stimulus,defense responses are often associated with changes in gene expression and proteosynthesis(Xu et al.2011;Koehler et al.2012;Yuan et al.2013).Therefore,gene expression patterns can provide useful hints to associate various metabolic pathways with defense against stress.For example,expression of several flavonoids synthesis-related genes is activated by oxidative stress after UV irradiation(Winkel-Shirley 2002).Similarly,among several Miscanthus ecotypes,the most frost-tolerant genotype showed highest elevation in phenylalanine ammonia-lyase(PAL)activity during cold acclimation(Domon et al.2013).Cold-hardened winter wheat cultivars also had elevated PAL activities and content of phenolic compounds(Olenichenko et al.2008).Moreover,it has been shown that ectopically enhancing the expression of flavonoid synthesis-related genes can lead to improved freezing tolerance in plants.Transgenic rape plants containing the Osmyb4 gene that controls flavonoid biosynthesis in rice(Oryza sativa)showed higher cold tolerance and flavonoid contents compared to wild-type plants(Gomaa et al.2012).In the view of yet unknown molecular mechanisms,flavonoids clearly work against freezing damage in plants.

Flavonoids are strong antioxidants and free radical scavengers in cells,especially active on plasma membrane lipids(Saija et al.1995;Martens and Mithofer 2005).Some of these endogenous effects can be attributed to particular structural features of flavonoids as discussed previously(Saija et al.1995;Hoekstra and Golovina 2002;Korn et al.2008).In our experiments,however,the flavone was secreted outside of P.malacoides cells and accumulated in large quantities as farina on the leaf surface after putative secretion by the specialized glandular trichomes(Sch?pker et al.1995),pointing to an alternative role of flavone from an intracellular antioxidant.

Our observations with non-living water-soaked materials sprayed with flavone(Figure 4A)suggest that direct physical,rather than plant physiological adaptations,are likely to be involved.Previously,flavonoid’s role as phytoalexins has been attributed to their hydrophobic properties(Martens and Mithofer 2005;Picman et al.1995).Therefore,protection effect in P.malacoides could be,as one explanation,related to putative hydrophobic interactions of flavone at the leaf surface.Tomato leaves coated with M96-018 hydrophobic kaolin particle film showed significantly lower ice nucleation temperatures compared to leaves treated with hydrophilic kaolin(Wisniewski et al.2002).In the same report,plants coated with M96-018 showed better survival rates compared to non-coated tomato after exposure to-3.0°C.

Another possible explanation could be that the flavone crystals on the surface act as ice nucleation centers,allowing moisture in the air to freeze and form a protective insulating barrier to cold.In support of this hypothesis,we observed that,when flavone was applied to the leaves in ethanol solution,it quickly dried as crystals on the surface of the leaves(data not shown).To this end,water sprays creating insulating ice layers are commonly used in horticulture to protect plants from freezing(Anconelli et al.2002).

Only a limited number of plant species are capable of flavonoid secretion,such as farinose ferns or trees like Betula nigra,Populus fremontii,P.maximowiczii,and Pityrogramma triangularis(Wollenweber 1984;Greenaway et al.1989;Valant-Vetschera and Brem 2006).Considering the useful properties of surface flavone(s),and a “magical” lesson learned from“fairy” primroses,we propose that exogenous application of flavone could be used as an efficient way to minimize freezing damage in crop plants.The flavone effect tested in several fruit trees was immediate,and in connection with increasingly reliable weather forecasts,flavone-based protection could be quickly deployed to prevent frost damage of the leaves or buds.However,several unresolved issues remain before practical introduction of the method.For example,identification of environment-friendly organic solvents and cost-effective production of flavone or its substitutes are required,and duration of flavone effect in a real environment should be examined.Our preliminary experiments showed that the exogenous application of other flavonoids,such as flavanon and chalcone also improved freezing tolerance,however,the effect was less pronounced compared to flavone(data not shown).Last but not the least,the molecular mechanism of flavone effect on ice nucleation should be resolved.

In the future,increased freezing damage in plants by frost events is predicted due to global warming,as sufficient protective cold acclimation could not be acquired by some plants(Albert et al.2013).It is therefore important to explore new alternative methods of plant protection such as the exogenous application of flavone described in this paper.

MATERIALS AND METHODS

Plants and materials

Commercially available gardening cultivars of Primula malacoides(family Primulaceae)were used in all experiments.The plants were grown in an air-ventilated,temperature uncontrolled glasshouse,and they were fertilized biweekly using recommended 1,000 fold dilution of Hyponex(Hyponex Japan)in water.Plants at flowering stage(＞12 weeks old)during winter/spring season(average air temperatures day,7–12°C;night-2 to 4°C)were used for experiments.Leaves 3–5 cm in length and similar age were selected.In addition,six fruit species(apple,Malus pumila “Tsugaru”;cherry,Prunus avium“Satonishiki”;grape,Vitis vinifera “Muscat”;orange,Citrus unshiu “Chusei”;peach,Amygdalus persica “Shimizu-hakuto”;and plum,Prunus mume “Oshuku”)were used in flavone sprays.Material from young trees was obtained from the nursery in Okayama Prefecture(Kurashiki,Japan)at the onset of winter bud formation.To determine the mechanistic action of surface flavone,small pieces of commercially available generic vinyl chloride coated fiber cloth,cut to leaf size,were used.

Application of exogenous flavone

Flavone(unsubstituted 2-phenyl-1,4-benzopyrone)that was determined to be the main component in the farinose layer on P.malacoides leaves by GC-MS operating in electron impact mode(Agilent 240 Ion Trap Mass Spectrometer system;Agilent)was used.In the reconstitution and leaf freezing protection experiments,commercial grade flavone(CAS:525-82-6;Tokyo Chemical Industry)was dissolved in pure ethanol and sprayed on the leaf surface at desired concentrations.The original farinose flavonoids were removed by gently wiping the leaves with 10%(v/v)ethanol solution in case of reconstitution.In every experiment,a fresh mass of the leaves was determined and the required amount of 1.8×10-3mol/L flavone solution dissolved in pure ethanol was calculated before spraying onto the leaf surface to achieve the required final concentration per g FW.Leaves were air dried at room temperature under a gentle airstream for approximately 5 min and used for measurements.Control leaves were sprayed with leaf mass-adjusted amounts of pure ethanol,dried,and used as mock treatments.

Estimation of freezing tolerance

Freezing tolerance was evaluated by measuring the INT and freezing-induced EL of the leaves.Ice nucleation was not seeded by the application of ice nucleators,in order to maintain natural conditions in the leaves.

Ice nucleation temperatures were measured by copperconstantan thermocouple thermometers attached to a data logger(Midi Logger GL820,Graphtec).Thermocouple thermometers were attached to the center of each leaf by sticky tape and hang-inserted into a Twinbird SC-DF25 Digital FPSC portable freezer(Shinyei).The cooling rate of the leaves was set to approximately-2.0°C/min and exotherms generated at freezing point of the leaves were detected as sharp increase of temperature on data plots.To investigate direct physical effect of flavone on freezing temperature,water-soaked vinyl chloride-coated fiber cloth pieces(400 mm2)were treated and measured identically to the leaves.

Freshly detached leaves were used to measure EL.After a brief rinsing with de-ionized water,leaves were then blot-dried on tissue paper and cut into 155 mm2pieces by a cork borer.The sections were placed in plastic Petri dishes,sealed,and inserted in a pre-cooled freezer.After the leaves had been maintained at-8°C for 60 min,dishes were removed from the freezer and left at 4°C overnight.To each tube,20 mL of deionized water was added and tubes were slowly agitated in the rotary shaker at 25°C for 4 h.The electrical conductivity(EC)of the solution was measured prior and after heating of the tubes to 100°C for 60 min.The freezing damage(%)was calculated as:(EC after freezing/EC after 100°C)×100.

Extraction of surface flavone and quantification by GC-FID

Freshly detached leaves(1 g)of P.malacoides were rinsed with acetone(20 mL)for 30 s,and flavone content in the extracts was determined by GC-FID using a GC-18A instrument(Shimadzu)equipped with a capillary column(30 m×0.25 mm i.d.,df=0.1 μm;DB-5MS,Agilent).Nitrogen was used as carrier gas with oven temperature programmed to isothermal run for 2.5 min at 200 °C,increased from 200 to 300 °C at a rate of 10 °C/min,and subsequently kept at 300°C for 2 min.The injector temperature was held at 200°C and FID detector temperature was 230°C.Flavone contents in the extracts were estimated from FID peak areas and the standard curve prepared from commercial grade of flavone(Tokyo Chemical Industry).

ACKNOWLEDGEMENTS

We acknowledge Drs.M.Mori and D.Yasutake(Kochi University),and Dr.B.Ezaki(Okayama University)for helpful discussions and comments.We thank Dr.J.T.Christeller for critically reading the manuscript and for his invaluable editorial help.

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