Annals of Botany 66, 655-663, 1990 655

The Effect of Pollination and Ethylene on the ColourChange of the Banner Spot of Lupinus albifrons

(Bentham) Flowers

A. D. STEAD* and M. S. REIDDepartment of Environmental Horticulture, University of California, Davis, California 95616, USA

Accepted: 6 June 1990

A B S T R A C T

In Lupinus albifrons flowers the banner spot of the standard is initially coloured white or pale yellow. Twoto three days after reaching the stage of full flower opening, this banner spot develops a pinkish blush andis deep magenta after a further 24 h. The development of this pigmentation is accelerated by exposure toethylene in a concentration- and time-dependent manner. Flowers with a pinkish banner spot produced thegreatest amounts of ethylene and production was much lower in flowers which had either completed thecolour change or in which the banner spot colour remained unchanged. Treatments such as stigma removalor pollination increased the rate of ethylene production. Dissection of the flowers showed that while thebanner spot is changing colour there is no change in the rate of production of ethylene from the standard,i.e. from the banner spot or surrounding tissue. The major sites of production at this time are the keel andpistil.

Isolated flowers withered within 2 d of removal from the plant and therefore did not show any changein the colour of the banner spot unless exposed to ethylene. The increase in banner spot pigment was aboutfourfold when isolated flowers were exposed to ethylene (0-24 /i\ I"1): however, the increase was less thantwofold when isolated standards were exposed to ethylene (0-27 /i\ I"1). Application of silver thiosulphate(STS) to intact isolated flowers, as a 1 h pulse prior to ethylene exposure, partially prevented the pigmentaccumulation, whilst a continuous supply of STS reduced the ethylene-induced colour change by approx.50%. Low concentrations of cycloheximide (CHI) (0-01 mg ml"1) reduced the accumulation of pigment inthe banner spot of ethylene-treated flowers, and higher concentrations (1-0 mg ml"1) completely preventedthe ethylene-induced colour change.

Key words: Ethylene, flower senescence, Lupinus albifrons, pollination.

INTRODUCTION (Whitehead, Halevy and Reid, 1984) or byaccelerating abscission of the corolla in Digitalis

The senescence of many flowers has been shown to (Stead and Moore, 1983).be influenced by pollination (Halevy, 1986). Whilst species exhibiting either corolla wiltingUsually, pollination accelerates the pattern of or abscission have received considerable attention,senescence observed in unpollinated flowers. For probably because of the horticultural implicationsexample, pollination causes the wilting of car- (Woltering and van Doom, 1988), those whichnation (Nichols, 1977) or Petunia flowers (Gilissen, show changes in the colour of all or part of the1977) or the abscission of the corolla in Digitalis flower have been somewhat neglected. The oc-(Stead and Moore, 1979). In each of these species, currence of such colour changes does, however,ethylene production increases following appear to be widespread with examples from overpollination. Furthermore, exposure to exogenous 50 Angiosperm families (M. Weiss, pers. comm.).ethylene mimics the effect of pollination by causing Subtle pigmentation changes after pollination inwilting of Vanda orchids (Burg and Dijkman, parts of the inflorescence of, for example,1967), carnations (Nichols, 1968) or Petunia Phalaenopsis, after pollination were reported by

Curtis (1943) and in Lantana camara by Mathur* Present address and correspondence: Department a n d Mohan Ram (1978) and Mohan Ram and

of Biology, Royal Holloway and Bedford New College, Mathur (1984). In some Cymbidium blossoms,Egham Hill, Egham, Surrey TW20 OEX, UK. anthocyanins accumulate in the labellum after

0305-7364/90/120655 + 09 $03.00/0 © 1990 Annals of Botany Company

656 Stead and Reid—Ethylene and Pollination in Lupins

pollination or treatment with ethylene (Arditti,Hogan and Chadwick, 1973). However, in speciesother than orchids, the role of ethylene inpollination-induced colour change has not beenstudied.

In this study we have examined the accumulationof anthocyanins by flowers of Lupinus albifronsfollowing pollination or treatment with ethylene,and also the production of ethylene, both by intactflowers and isolated parts of the flower, in order todetermine the role of ethylene in the pollination-induced response seen in many species of Lupinus(Wainwright, 1978; Schaal and Leverich, 1980).

MATERIALS AND METHODS

Individual flowers were removed from theflowering spikes of Lupinus albifrons (Bentham)that were growing in the arboretum at theUniversity of California, Davis, CA. Flowers wereremoved at specific developmental stages (Fig. 1),the youngest flowers had the keel and standardtightly folded with no colour development (I);older flowers were taken just as the standardcommenced opening (II). The youngest (i.e. closestto the top of the spike) flower with a fully reflexedstandard was taken as the third stage (III). Thefourth stage was similar but taken from lowerdown the spike and was therefore older (IV).Occasionally there were younger flowers higher upthe spike in which the banner spot had alreadystarted to change colour. Older flowers wereremoved when the banner spot was turning pink(V), and finally when the spot had undergone acomplete change to magenta (VI). Older flowerswere selected on the basis of the visual appearanceof the standard, either partially or fully wilted(VII) or completely withered (VIII).

At the completion of each experiment, thebanner spot was excised from the standard andplaced in a minimal volume of methanol containing1 % (v/v) concentrated HO. Storage, if necessary,was at 4 °C. A minimum of eight samples (eachwith either four or five banner spots) were takenfor each treatment. Extraction of the material wasalso in acidic methanol and, after recording thevolume of each extract, the absorbance was readon a Cary 210 spectrophotometer at the wavelengthof maximum absorbance (535 nm). The majorpigment content of the banner spot was identifiedby one- and two-way paper chromatography ascyanadin and contents calculated using the molarextinction coefficient for cyanadin (2-95 xlO4;Harborne, 1967).

For the measurement of ethylene production,individual flowers were placed in small glass vialswith the cut end of the pedicel in water, left for

1-2 h for any wound-induced ethylene productionto diminish and then enclosed with rubber 'Suba-seals' and left at 20 °C under low light. Aftersufficient time for a detectable amount of ethyleneto accumulate in each tube (usually 2 h) a 1 ml gassample was removed and analyzed with a Variangas chromatograph using an alumina column at70 °C and a sensitivity to >001/ill"1 ethylene.Ethylene production was calculated by referenceto the peak height of known calibration standards.For the measurement of ethylene production fromisolated flower parts, a similar procedure was usedafter first allowing the production of wound-induced ethylene to subside.

For measurement of the effect of exogenousethylene, flowers were removed when the standardwas fully unfolded but before there was any colourchange of the banner spot. Flowers were rested onsmall vials with the pedicel immersed in water andplaced inside tanks with a flow (approx. 27 1 h"1)of either ethylene-free air or appropriateconcentrations of ethylene. Initial experimentsassessed the effect of exposure for 10 h to variousconcentrations of ethylene (0-1-2 fi\ I"1); otherexperiments assessed the effect of varying thelength of exposure (0-24 h) to ethylene (0-27 fil I"1).In experiments using isolated standards, thesewere placed on damp filter paper and then placedinside the appropriate ethylene-containingenvironment.

Silver thiosulphate (STS, 4 HIM), an inhibitor ofethylene action (Veen, 1979), was applied either asa pulse (1 h) or continuously. Cycloheximide(CHI), an inhibitor of protein synthesis, wasapplied continuously at concentrations between001 and 1 mg ml"1. The effect of these inhibitorson the colour change of the banner spot wasstudied either in the presence or absence of ethylene(0-24 fiM'1).

RESULTS

During the normal development and senescence offlowers of L. albifrons the banner spot undergoes adramatic colour change, from an initially white orpale yellow colour to a deep magenta (Fig. 1). Thepigment content of the banner spot of youngflowers, before any visible colour change, was1370 ±541 ngmg"'(n= 10). For flowers in whichthe spot had changed colour, the concentration ofcyanadin was 565-3 ± 82-3 ng mg"1 (n = 10). Themagnitude of the response varied little betweenflowers of the same plant but more widely betweenplants, probably because the initial colour andextent of the banner spot varied considerablybetween plants. Tagging of individual flowers wasused to determine the length of time between the

Stead and Reid—Elhylene and Pollination in Lupins 657

FIG. 1. A flowering spike of L. albifrons, with the various flowering stages indicated.

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658 Stead and Reid—Ethylene and Pollination in Lupins

300

•= 200

30 60

Time (mins)

9 0 120

FIG. 2. Ethylene production from intact, isolated flowersof L. albifrons at various times after removal from theflowering spike. Ethylene production was measured after90 min enclosure and the data plotted as the mean (n =22) hourly rate of production (±s.e.) against the time of

commencing enclosure.

identified floral stages. Thus, the standard startedto unfold approximately 3 d after the first stageand this was complete within 1 d. The banner spotcommenced its colour change about 2 d later andthe colour change was complete within 1 d. Theflowers persisted for a further 2 d with little sign ofsenescence. However wilting commenced at aboutthis time, some 8 d after the first floral stage, andthe flowers withered within 2 d.

Measurement of floral ethylene productionimmediately after removal of individual flowersfrom the plant showed that the initial burst ofethylene production due to wounding had subsidedwithin 2 h (Fig. 2). After allowing wound-inducedethylene production to dissipate, the production ofethylene by the lupin flowers was relatively low forall stages except those in which the banner spotwas either beginning to change, or had recentlychanged, colour (Fig. 3). Dissection of flowerswith fully opened standards, but with varyingdegrees of banner spot pigment development,indicated that prior to any colour change theproduction of ethylene was similar from the

750

500

250

10

Fio. 3. Ethylene production by intact, isolated flowers ofL. albifrons at various stages of floral development;relative position of data points adjusted to account forthe differences in time between each floral stage. Theflowers were removed from open pollinated plants.

Vertical bars represent standard errors.

standard and the remainder of the flower. How-ever, when anthocyanin was accumulating, eth-ylene production by the standard was unchangedwhilst that of keel plus ovary, and the ovary alone,increased ten-fold. Therefore, the major sites ofethylene production at this stage did not includethe standard (Table 1). Further dissection of thekeel, ovary and style showed that the productionfrom these tissues was approximately equal at thetime of the banner spot colour change, eventhough in the younger flowers the ovary producedless than the keel (Table 1). Treatments such aspollination, stigma removal or stigma squeezing,all resulted in increased production of ethylene bythe flowers (Table 2).

In response to exogenous ethylene, the bannerspot of isolated flowers changed colour to thatusually associated with the natural development/senescence of the intact flower. The response wasconcentration dependent, with a significant ac-cumulation of pigment occurring in response tothe lowest concentration of ethylene (0-05 ft,] I"1)(Fig. 4). When exposed to 0-24 ft\ I'1 ethylene therewas no detectable colour change after 6 h but after

TABLE 1. The production of ethylene (pi flower'1 h~l) by intact flowers and dissected parts of flowers ofL. albifrons at various stages of colour development of the banner spot. Values are the means of 20 replicate

flowers ±s.e.

Banner spot colour

YellowPinkRed

Intact flower

331-1 ±62-41383-2 ±385-8288-6 ±35-2

Standard

173-5 ±26-2139-5 ±22-938-0 ±4-3

Keel + pistil

167-6±2SH)1109-8 ±359-2428O± 161-5

Pistil

47-2 ±6-5450-8 ±143-0

85-6 ±37-9

Stead and Reid—Ethylene and Pollination in Lupins 659

T A B L E 2. The effect of pollination, stigma damage or style removal on the production of ethylene (piflower'1 h~l)from isolated flowers ofL. albifrons at differing times after treatment. All flowers were fullyopen but taken prior to any visible change in the banner spot colour. Values are the means of 16 replicate

flowers ±s.e.

Treatment

Time after treatment

2 h 20 h 44h

ControlPollinatedStigma squeezedStyle removed

40-6 ±5-8459-2 ±85-9445-9 ±54-8838-9 ±119-9

20-9 ±3-6I23-9± 13 1337-9 ±83-5245-7 ±48-0

203-8 ±21-5191-4±26-l581-8 ±82-0252-2 ± 5 0 4

800

0-4 0-8

Ethylene concentration {p\ r 1 )

I 2

FIG. 4. The effect of a 10 h exposure to varyingconcentrations of exogenous ethylene on the accumu-lation of pigment in the banner spot of L. albifronsflowers removed from the plant soon after full floweropening and therefore prior to pollination. Values are

the mean of 11 flowers ±s.e.

10 h, the pigment concentration was more thandouble that of the untreated controls (Fig. 5). Thebanner spot of isolated standards responds simi-larly to the intact isolated flower, although themagnitude of the response to exogenous ethylenewas greatly reduced (Table 3).

The use of STS, an inhibitor of ethylene action,revealed that a short pulse (1 h) was sufficient toreduce the colour development that was alwaysseen in water-treated controls upon exposure toethylene (Table 4). Continuous STS treatment,throughout the exposure to ethylene was moreeffective than the pulse treatment at inhibiting thedevelopment of the pigmentation (Table 4).Experiments in which the effect of STS wasinvestigated in the absence of applied ethylenewere inconclusive because flowers held in waterrarely showed any colour change; the flowerswithered within 2 d of removal from the plant with

24

T i m e ( h )

FIG. 5. The effect of various lengths of exposure to0.27 /t\ I"1 ethylene on the accumulation of pigment inthe banner spot of flowers similar to those used in Fig. 4.The concentration of pigment in flowers treated withethylene-free air for 24 h is shown by the hatched bar.

Values are the mean of 11 flowers ±s.e.

T A B L E 3. The effect of exposure (10 h) to ethylene(0-27 nl ml'1) on the colour development of thebanner spot in isolated, but otherwise intact flowers,compared to detached standards. All flowers werefully open but taken prior to any visible change inthe banner spot colour. Values are the means of sixreplicate groups each of four banner spots ±s.e.

Pigment concentration (ng mg~')

In air In ethylene

Isolated standardsIntact flowers

139-8 ±39-0129-4 ±29-5

207-2 ±48-5420-7 ±46-8

660 Stead and Reid—Ethylene and Pollination in Lupins

T A B L E 4. The effect of STS or cycloheximide onethylene-dependanl accumulation of pigment in thebanner spot of isolated flowers. Ethylene exposurewas for 16 h at 0-24 nl ml'1. Values are the means

of six groups each using five banner spots ±s.e.

Treatment Pigment concentration (ng mg ')

WaterSTS (1-h pulse)STS (continuous)CHI (0-01 mgmT1)CHI (0-1 mg ml-1)CHI (1 mgml"1)

816-0 ±67-2620-9 ±86-0454-9 ±58-9385-3 ±38-5239-9±41-513fX)±21-5

T A B L E 5. The effect of STS or cycloheximide onethylene production from intact, isolated flowers ofL. albifrons. All measurements taken 12 h aftertreatment and, with the exception of those treatedwith an STS pulse, held in the treatment solutionsthroughout. Ethylene production measured as piflower'1 h~l with a minimum of ten replicates per

treatment

TreatmentEthylene production

(pi flower"1 h"1)

WaterSTS (1-h pulse)STS (continuous)CHI (0-01 mgml"1)CHI (01 mgml-1)CHI (1 mg ml"1)

83-0 ±6-3117-6± 18-9465-2 ± 113-4155-4±21-0161-7±26-310O-8±9-5

the banner spot remaining pale yellow. When leftunpollinated on the plant however, several dayselapsed between the flower opening fully andanthocyanin accumulation commencing.

Cycloheximide was more effective than STS atpreventing the ethylene-induced colour change ofthe banner spots; concentrations as low as001 mg ml"1 reduced the accumulation of pigment,whilst higher concentrations ( l m g m r 1 )completely prevented the ethylene-induced de-velopment of banner spot colouration (Table 4).Both STS and CHI treatment caused an increasein the rate of ethylene production, relative towater-treated flowers (Table 5).

DISCUSSION

The colour change of the banner spot of the flowerof L. albifrons is the result of a three- to fivefoldincrease in anthocyanins. The extent of the increasedepends on the individual plant, since the initial

size and colour of the banner spots variesconsiderably from plant to plant. This situationalso occurs in Digitalis, where the pigment contentof the entire corolla is very variable (Stead andMoore, 1977). This colour change appears to beaccelerated by pollination and probably detersfurther insect visits, therefore avoiding unnecessarywastage of pollen. Although the banner spotcolour changes, individual flowers do not wilt orabscind for a further 2 d and the persistence ofthese flowers maximizes the overall attractivenessof the inflorescence to pollination vectors, a speciesof Bombus (Wainwright, 1978; Schaal andLeverich, 1980). Close observations of theseflowers show that bees do not visit flowers oncethe banner spot has changed colour (data notshown). In other species, the response to pol-lination is very different. In Digitalis, the corollasabscind within 24 h of successful pollination (Steadand Moore, 1979), whilst in Petunia the corollaloses turgjdity and wilts (Gilissen, 1977). Similarly,the characteristic petal in-rolling seen in senescentcarnations is also accelerated by pollination(Nichols, 1977). Furthermore, in all of thesespecies, the senescence of the flower is associatedwith increased ethylene production which occurssooner in pollinated flowers. In each of thesespecies the application of ethylene also causespremature flower senescence (Nichols, 1968; Steadand Moore, 1983; Whitehead et al., 1984). Thepresent study suggests that the colour change seenin L. albifrons may also depend on ethylene,although in a manner that differs from otherspecies.

The endogenous rate of ethylene production iscomparatively low from floral buds or fully openflowers with pale banner spots, being less than100 pi flower"1 b~ l, or considerably less than1 nl g"1 f. wt. h"1. However, in those flowers inwhich the banner spot can be seen to be changing,the rate is nearly tenfold higher. In Digitalis, at thetime of corolla abscission, the increase in ethyleneproduction is also about tenfold, although the rateof production prior to pollination is considerablygreater than that recorded in L. albifrons (Steadand Moore, 1983). In those flowers in which thecolour change is complete, the rate is reduced toabout 150 pi flower"1 h"1. In contrast to Petunia{Whitehead et al., 1984) and Dianthus (Nichols,1968), there is no evidence of a peak of ethyleneproduction associated with petal wilting, eventhough the petals of L. albifrons do wilt some 2 or3 d later, after the banner spot has changed colour.

Banner spot colour change can be induced byapplication of ethylene; exposure to 0-05 /tl I"1

almost doubled pigment concentration, relative toflowers held in air, after only 10 h. Exposure to

Stead and Reid—Ethylene and Pollination in Lupins 661

higher concentrations caused a greater response,while 1-2/ill"1 for 10 h was sufficient to effect acomplete colour change, i.e. a fourfold increase inpigment content. By choosing an intermediateconcentration of 0-27 fi\ I"1 ethylene, the colourchange was followed over a 24-h period. There wasno change in the pigment content of the bannerspot of flowers held in ethylene-free air for 24 hwhile those in ethylene also showed no increasewhen held in ethylene for 6 h or less. However,after 10 h, the pigment content was significantlyincreased and colour development was completeafter about 18 h exposure. At this time, theconcentration of cyanadin was equal to that offlowers allowed to age naturally, either pollinatedor unpollinated, on the plant.

The increase in ethylene production was derivedalmost entirely from the keel and ovary, as the rateof ethylene production from the standards declinedslightly when the banner spot started to changecolour. Furthermore, once the colour change wascomplete, the production of ethylene by thestandard decreased significantly. Measurements ofethylene production from either the keel plus pistilor the pistil alone, showed that there was a tenfoldincrease in production from these tissues. Theslight increase in the total ethylene produced fromthe dissected flower parts, relative to the intactflowers, is probably due to the persistence ofwound-induced ethylene production. From thesedata it is clear that the increase in ethyleneproduction, seen during the banner spot colourchange, is entirely due to the increases seen fromthe keel and pistil and not from the standard.Moreover, whilst the pistil contributed nearly halfof the ethylene produced by the keel and pistiltogether, the fresh weight of the pistil wasapproximately only one-tenth that of the keel: therate of production from the pistil was thereforeapproximately 100 nl g f. wt"1 h"1. This situationis identical to other species studied in which thepistil, and in particular the style, has the highestrate of ethylene production per unit fresh weight ofall the floral tissues (Hall and Forsyth, 1967;Nichols, 1977; Suttle and Kende, 1978). Pol-lination of isolated flowers resulted in a tenfoldincrease in the amount of ethylene produced 2-4 hfollowing pollination: this rate of ethylene pro-duction however, is still only half that recordedfrom flowers at the time the banner spotcommenced changing colour (Fig. 3). Similarincreases occur when the stigma is squeezed but alarger increase is detected after style removal.Although the stylar tissues undoubtedly possess ahigh capacity for ethylene production per unitweight, the tissue is very small when compared tothe much larger ovary, and thus the removal of

34

this tissue has little effect on the ethylene pro-duction from the remainder of the flower. In fact,the higher rate of ethylene production recordedfollowing style removal may be the result ofincreased tissue damage as removal of the style inthese flowers is particularly difficult. In Petuniatoo, stigma squeezing and style removal bothhasten corolla wilting and cause increased rates ofethylene production, with style removal againgiving the greatest response (Lovell, Lovell andNichols, 1987).

Whilst exogenous ethylene induces a colourchange of the banner spot in intact flowers, albeitisolated from the plant, the response of isolatedstandards was considerably less marked. Therewas only a 50% increase in the banner spotpigment content of isolated standards, relative toair-treated controls, but more than a threefoldincrease in the pigment content of the same area oftissue when the isolated flowers remained intact. Itwould therefore seem that either the keel and/orpistil are a source of pigment precursor, or someother factor which is involved in the sensitivity ofthe banner spot to applied ethylene. Halevy,Whitehead and Kofranek (1984) reported that inCyclamen, unpollinated flowers produce very littleethylene and show corolla discolouration andwilting. The senescence of such flowers is notaccelerated by exogenous ethylene. The corollas ofpollinated Cyclamen flowers do, however, abscindand this process is accompanied by a large increasein ethylene production. Furthermore, abscissioncan be hastened if the pollinated flowers areexposed to ethylene, thus pollination in some wayrenders these flowers sensitive to ethylene. InPetunia, the response of the corolla to appliedethylene is also dependent on a 'sensitivity' factor.Exposure to ethylene soon after pollination is lesseffective at accelerating senescence than appli-cation a few hours later and yet, in terms of theethylene production the flowers are identical(Whitehead and Halevy, 1989). The identity of thissensitivity factor has yet to be proven althoughdata of Whitehead and Halevy (1989) suggest thatit may be a fatty acid. If a similar situation shouldexist in L. albifrons then the removal of thestandard from the remainder of the flower couldremove the source of such a sensitivity factortherefore rendering the banner spot less sensitiveto applied ethylene.

In many situations a change of colour has beenascribed to changes in vacuolar pH since antho-cyanin pigments are well known to be pH-sensitive.In Lathyrus hirsutus, the change in petal colour isaccompanied by a rise in the pH of the expressedsap (Pecket, 1966). The cause of this pH changemay be the breakdown of the tonoplast and the

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662 Stead and Reid—Ethylene and Pollination in Lupins

mixing of the vacuolar and cytoplasmiccompartments as has been suggested in Ipomoeaand Tradescantia (Matile and Winkenbach, 1971;Suttle and Kende, 1978). There is, however, noevidence of the loss of membrane integrity inLupinus as the flowers persist, without wilting, fortwo to three days after the colour change iscomplete. This, together with the observation of alag in the accumulation of pigment in response toexogenous ethylene and the inhibition bycycloheximide, suggests that the pigment issynthesised de novo during the banner spot colourchange.

In common with other studies (Yang and Riov,1982) the effect of CHI was not due to a decreasein endogenous ethylene production, in fact the rateof production increased as the result of treatmentwith CHI (Table 5). As expected STS, a potentinhibitor of ethylene action (Veen, 1979), partiallyreversed the effect of exogenous ethylene, revealingthat STS was probably acting at the cellular levelto antagonise ethylene action. In many othersystems where STS has been shown to overcomethe effect of exogenous ethylene (Nichols andKofranek, 1982; Hoyer, 1986; Joyce, Reid andEvans, 1990) it has also been found to reversesimilar physiological changes associated withincreased ethylene production, thus lending sup-port to the view that the endogenous ethylene wasa causal agent. Although we were unable to testSTS on excised lupin flowers in the absence ofapplied ethylene because their life was too short,its activity in the presence of applied ethylene addscredence to the view that natural ethylene promotesthe colour change of the lupin banner spot.Furthermore, no attempt was made to quantifythe amount of silver taken up. In Hibiscus(Woodson, Hanchey and Chisholm, 1985), STSwas only effective at preventing senescence ifapplied to immature petals and it may be that thetranspiration by the mature flower, and thereforethe uptake of solution, was such that insufficientsilver was present at the site of ethylene action tototally prevent the effect of applied ethylene.

Pollination of isolated flowers also failed tocause banner spot colour change, possibly becausethe flowers wilted too soon. It was noticeablehowever, that the rate of ethylene production fromthese flowers never attained the level of productionassociated with flowers removed from the plant atthe stage when the banner spot colour waschanging, suggesting that the isolated flowers wereeither unable to maintain a comparable rate ofethylene production (to that achieved while re-maining attached to the plant), or that pollinationof isolated flowers was ineffectual. Certainly pollen

germination, and pollen tube growth, occurred butthe rate was not compared to that achieved in situ.

The present study indicates that, in commonwith other species, pollination can hasten theageing process of L. albifrons flowers and bringabout the characteristic colour change in thebanner spot. This change appears to be broughtabout by ethylene as the banner spot pigmentaccumulation is accelerated by ethylene andincreased ethylene production occurs concomi-tantly with colour development. The site ofsynthesis of the increased ethylene production ishowever, not the banner spot nor the immediatelyadjacent tissues of the standard, but rather the keeland ovary. It therefore seems probable that thelocalised increase in ethylene production in thesetissues induces the formation of an, as yet,unidentified pigment precursor which is thentranslocated to the banner spot where it contributesto the observed colour development.

ACKNOWLEDGEMENTS

This work was supported by the award to ADS ofa Post-harvest Physiology Fellowship from theUniversity of California, Davis, and a travel awardfrom the Wain Foundation (AFRC).

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