Plant Physiol. (1981) 67, 1245-12490032-0889/81/67/ 1245/05/$00.50/0

Flowering in Xanthium strumariumINITIATION AND DEVELOPMENT OF FEMALE INFLORESCENCE AND SEX EXPRESSION

Received for publication May 28, 1980 and in revised form November 26, 1980

MAGGY LEONARD, JEAN-MARIE KINET, MONIQUE BODSON, ANDREE HAVELANGE, ANNIE JACQMARD, ANDGEORGES BERNIERLaboratoire de Physiologie Vegetale and Centre de Physiologie Vegetale Appliquee (I. R. S. I. A.), Departmentde Botanique, Universite de Lie'ge, Sart Tilman, B4000 Liege, Belgium

ABSTRACT

Vegetative plants of Xanthium strumarium L. grown in long days wereinduced to flower by exposure to one or several 16-hour dark periods. Thedistribution of male and female inflorescences on the flowering shoot wasdescribed, and a scoring system was designed to assess the development ofthe female inflorescences. The time of movement of the floral stimulus outof the induced leaf and the timing of action of high temperature wereshown to be similar for both the apical male and lateral female infloresc-ences.

Strong photoperiodic induction of the plants favored female sex expres-sion, while maleness was enhanced by exogenous gibberellic acid. Theproblem of the control of sex expression in Xanthium is discussed inrelation to the distribution pattern of male and female inflorescences onthe flowering shoot and to the state of the meristem at the time of thearrival of the floral stimulus.

Flowering in the Chicago strain of Xanthium strumarium L., anabsolute short-day plant, has been extensively investigated (16,18). However, most studies were devoted to the initiation anddevelopment of the terminal staminate inflorescence, and little isknown about the formation of the lateral pistillate inflorescences.Their gross morphological and anatomical development has beendescribed in a few studies (4, 8, 9, 12), and fragmentary physio-logical aspects have been considered in some other reports (12, 13,19, 20).The available information on factors that affect sex expression

in cocklebur is also very scarce. Effects of photoperiod (12) andnutrition (13) have been described, and it was reported thatexogenous GA3 decreased the male to female ratio in a day-neutral strain (21).The aim of the present work was to start a comparative physi-

ological study of the initiation and development of the male andof the female inflorescences in the Chicago strain of cocklebur.Since GA is known generally to increase the tendency to malenesswhen applied to monoecious and dioecious plants (5), we alsoinvestigated the effect of GA3 on sex expression in Xanthium.As a basis for these studies and to facilitate the measurement of

the flowering response at the sites where pistillate inflorescencesare produced, we elaborated a floral-state scale similar to thatdesigned by Salisbury (15) for the staminate inflorescence.

MATERIALS AND METHODS

Growth Conditions. Seeds of X strumarium L. were isolatedfrom mature burs and allowed to germinate at 35 C in a moist

mixture of sand and leaf mold (1:3). After 3 weeks, the seedlingswere transplanted singly into 8-cm pots, and after 10 weeks, theplants were transplanted into 12-cm pots. They were grown at aconstant temperature of 20 C under overhead light from whitefluorescent lamps (ACEC LF-40 w/2 4,300 K) at an irradiance ofabout 19 w m- . Plants were supplied weekly with NPK fertilizer.The plants were kept vegetative by growing them in 20-h days.

When 7 weeks old, they were induced to flower by 1-, 8-, orcontinuous 16-h long nights. For the 32 h immediately precedingthe start of an inductive treatment, the plants were subjected tocontinuous light. At the time of transfer to continuous light, theywere defoliated except for the leaves shorter than 1 cm and thehalf-expanded one. All buds in axils below this leafwere removed,while buds in axils of the half-expanded and higher leaves wereleft in place. Following the inductive treatment, the plants werereturned to 20-h days until examination. Ten plants were used foreach experimental treatment, and each experiment was repeatedat least once.

Application of GA3. GA3 was applied in aqueous 0.3 mmsolution by spraying the whole plants to run off with a handatomizer. In preliminary experiments, this concentration wasfound to give the optimal effect on sex expression, and thisapplication method was found to be superior to spraying only thesole, half-expanded leaf or treatment of the roots by watering orof the shoot apex by application with a small cotton plug. Controlplants were sprayed with distilled H20. Tween 20 (polyoxyethyl-ene-sorbitan monolaurate; 0.1%) was present in all solutions assurfactant.

Recording Methods. The main axis and side axes of the plantwere considered. The side axes were numbered acropetally fromthe induced leaf (Fig. 1). On the basis of the sex of the terminalinflorescence of the side axes, the whole flowering shoot can bedivided into three zones (Fig. 1): a basal zone A, in which allterminal inflorescences are male; an intermediate zone B, in whichall terminal inflorescences are female; and an apical zone C, inwhich all terminal inflorescences are male.

In studies of initiation and development of the female infloresc-ence, the terminal bud of the side axes 8 and 9, which generallyproduced a pistillate inflorescence, were dissected 40 days afterthe start of the inductive treatment, and the floral stage wasclassified according to the scale described in Figure 2 and TableI. For comparison, the terminal staminate bud of the main axiswas also dissected at the same time, and the floral stage wasclassified according to Salisbury (15).

In experiments on sex expression, the production of male andfemale inflorescences by each plant was recorded 2 months afterthe start of the inductive treatment. The terminal bud of the mainaxis, as well as the terminal and axillary buds of side axes, wasexamined. Side axes 2 and 3 were not taken into account becausetheir development was generally reduced due to removal of their

1245

Plant Physiol. Vol. 67, 1981

ZoneC_

ZoneB

ZoneA

11

9

7

5

3

12

10

8

6

4

STAGE 0 STAGE 1

STAGE 4

STAGE 6

Inducedleaf

STAGE 2

fb

STAGE 3

STAGE 5

STAGE 7

0.0 0.5 1.0 mmI l

FIG. l. Distribution of staminate and pistillate inflorescences on aplant induced by a single long night. Observations were restricted to theterminal bud of the main axis and the terminal and axillary buds of sideaxes. 3, Staminate inflorescence; 9, female inflorescence; 0, undifferen-tiated meristem; 1 to 12, side axes numbered acropetally from the induced'leaf. Explanations of zones A, B, and C are given in the text.

subtending leaf just before induction (see "Growth Conditions").A primordium was classified as male (d) when it had reachedstage 3 on Salisbury's scale and female (9) when it had reachedstage 1 on the scale in Figure 2. To avoid confusion between stageO of the female scale and stages 0, 1, and 2 of the male scale, allprimordia at these stages were classified as undifferentiated (asfar as sex expression is concerned). As opposed to these meristems,all male and female primordia of higher stages were referred to asdifferentiated inflorescences.

RESULTS

Initiation and Development of the Female InflorescenceScale for the Female Inflorescence. A scoring system was de-

signed to assess the development of the pistillate inflorescence. Toestablish this scale of flowering, we followed the development ofthe terminal bud of side axes 8 and 9. The stages of developmentof the pistillate inflorescence of Xanthium are described in TableI and illustrated in Figure 2. The floral stage was a linear functionof time after induction; it was also positively correlated with thedegree of induction (3).

Translocation ofthe Floral Stimulus. The timing of export of thestimulus for female flowering from the induced leaf was done bycutting the base of petioles at successive intervals following com-pletion of a single 16-h inductive night. After 8 h, sufficientstimulus had left the induced leaf to permit a flowering responsein the terminal buds of side axes 8 and 9 (Fig. 3). The floral stagesreached by plants defoliated after 72 h were the same as those ofplants with the induced leaf left in place.

Effect of High Temperature. At various times before, during, orafter a single long night, plants were subjected to high temperature

FIG. 2. States ofdevelopment ofthe pistillate inflorescence of Xanthium(see Table I).

(35 C) for 8 h. This did not damage the plants but stronglyinhibited induction of both male and female inflorescences (Fig.4). The initiation of the apical staminate inflorescence was com-pletely inhibited by high temperature treatment during the secondhalf of the long night and nearly so by treatment during the firsthalf. At other times, the inhibitory effect of high temperature wasfar less marked. The initiation of pistillate inflorescences wasinhibited in the same way as, and even more strongly than, thatof the apical male inflorescence. The inhibition was completewhen the high temperature was given at any time during the longnight.Sex ExpressionEffect of the Number of Photoinductive Cycles. Increasing the

number of inductive long nights from 1 to 8 reduced the totalnumber of meristems per plant and the proportion of undifferen-tiated meristems but markedly enhanced the proportion of femaleinflorescences (Table II). Sex expression in plants kept underinductive conditions until the end of the experiment (40 inductivecycles) did not differ from that in plants subjected to eightinductive cycles, probably because after eight cycles all floralmeristems were determined male or female.

Effect of GA3. Different batches of plants were sprayed with 0.3mM GA3 once at different times before or after a single inductivenight.The total number of meristems per plant was enhanced by the

GA3 treatment (Table III). This increase was associated withadditional nodes on the main axis (Fig. 5) and also on the sideaxes as shown below. The number of nodes on the main stem wasincreased when GA3 was applied before the long night or withinthe first 2 days following it (Fig. 5) whereas the total number ofmeristems was affected by GA treatment given until 8 days afterthe end of the long night (Table III). This different time depend-ence indicates that the production of additional nodes by theterminal meristem of the main axis ceases before that by the

1246 LEONARD ET AL.

1- 2

I

FLOWERING AND SEX EXPRESSION IN XANTHIUM

Table I. Criteriafor Floral Stages of Pistillate Buds

Meristem MeristemStage Criteria Width Height

mm

0 Vegetative; meristem small and 0.18 ± 0.02a 0.11 ± 0.02flat

I First visible bract primordia at the 0.26 + 0.02 0.18 ± 0.03base of a big, round infloresc-ence primordium; wider thantall

2 First small spine primordia; inflo- 0.32 ± 0.03 0.21 ± 0.02rescence primordium flattenedat the apex

3 Inflorescence primordium with an 0.45 ± 0.03 0.25 ± 0.04apical depression, completelycovered by spine primordia

4 Development of two beaks 0.50 ± 0.07 0.38 ± 0.05through rapid growth of the re-ceptacle margins

S Elongation and bending of the two 0.69 ± 0.04 0.64 ± 0.06beaks

6 Top of the inflorescence primor- 0.97 ± 0.12 1.0 ± 0.16dium pubescent; inflorescenceprimordium as tall as wide

7 Elongation of the spines; infloresc- 1.02 ± 0.10 1.37 ± 0.19ence primordium taller thanwide

a Means with 95% confidence limits.

8-

7.

5-

4

to

L2

1-

Terminal Oeinf loreacenceof main axis:

---o---

---

_ x

0 4 8 16 24 36 48 72

Time of defoliation(hours from end of the long night)

FIG. 3. Translocation of the floral stimulus from the induced leaf asdemonstrated by cutting off this leaf at various times after the end of theinductive long night. ---, Stages reached by the nondefoliated controlplants.

oa 4-

a

u3-

2-

1-

0-

1247

---O---

-24 -16 -8 0 +8 +16 +24

Time of high temperature interruption'(hours from end of the long night)

+32

FIG. 4. Flowering response to high temperature interruptions appliedfor 8-h intervals at various times before, during, or after a 16-h inductivenight. Plotted points are the midpoints of the exposure. ---, Stagesreached by the nontreated control plants.

Table II. Effect of the Number of Inductive Cycles on Number ofMeristems per Plant and Sex Expression

Number Total Num- Undifferen- Differentiated

of Induc- ber of Men- tiated Meri- Inflorescences

tive Cycles stems per stemstiveCycles Plant s

1 36.8 13.9 35.3 64.78 29.2 0.3 15.5 84.5

40 24.3 0 15.6 84.8

terminal meristems of the side axes.

GA3 also enhanced the proportion of male inflorescences perplant. This effect was apparent after applications from 4 daysbefore to 8 days after the inductive dark period, but it was notapparent after later applications (Table III). In response to thehormonal treatment, several plants developed terminal staminateinflorescence on all side axes. The earlier the treatment, the greaterthe proportion of such plants (Fig. 6).Concomitant with the enhanced maleness, plants treated with

GA3 2 to 4 days before or 2 to 6 days after the long night producedan increased proportion of undifferentiated meristems, indicatingthat the enhanced maleness may be attributed, at least partly, toless effective induction.A careful analysis of the effect of the timing of the GA3

treatment on the number of side axes, separately in the three zones

of the flowering shoot as defined in Figure 1, indicated that theeffect of GA3 on sex expression involved both the development ofmale inflorescences instead of female ones and the developmentof supernumerary male inflorescences.A number of morphological modifications of the terminal inflo-

rescence of side axes in zone B also frequently occurred in GA3-treated plants, the most common being the formation of bisexual

Plant Physiol. Vol. 67, 1981

. . . . . . . .

LEONARD ET AL.

Table III. Effect of the Timing of GA3 Treatment on the Number ofMeristems and Sex Expression in Plants Exposed to a Single 16-h Long

Night

Total Undif- DifferentiatedNumber Inflorescences

feren -

Time of GA3 Treatment of Men- tiatedstems Mer-per MemsPlant stems

days % %Untreated plants 41.6 17.8 39.8 60.2

Before end of long night4 72.6 34.1 77.7 22.32 69.1 34.0 69.1 30.91 61.7 10.5 63.9 36.1

After end of long night0 60.8 17.4 65.1 34.92 66.7 33.9 66.7 33.34 71.4 38.0 66.6 33.46 56.0 29.5 62.3 37.78 45.8 10.3 44.5 55.515 42.5 9.4 40.8 59.2

14-0

v 130.0

E2 12

* GA3

0\

----0-----Owe

Control

I II I

-4 -2 0 +2 +4 +6 +8 +15

Time of GA3 treatment(days from end of the long night)

FIG. 5. Effect of the time of a 0.3 mm GA3 treatment before or after asingle 16-h long night on the total number of nodes on the main axis.Control plants were treated with H20 at the end of the long night.

inflorescences (i.e. a fertile female inflorescence with isolated maleflorets between the spines), sterile female receptacles withoutbeaks and ovules, or male inflorescences in the middle of a sterilefemale receptacle with some additional isolated male florets be-tween the spines.

DISCUSSION

Both the floral stage system of Salisbury (15) for the apical budand the scale for female inflorescences that we have establishedare so designed that the stages fall on straight lines when plottedagainst time after induction or degree of induction. These stagesare a measure of the rate of early inflorescence development. Astage system for the pistillate inflorescence qualitatively similar toours, though less elaborate, was already used by Caff (1).The translocation pattern of the floral stimulus for the male

apical meristem is consistent with previous results (7, 17). For theterminal female inflorescence of side axes 8 and 9, the transloca-tion of the floral stimulus out of the induced leaf starts at the sametime as it does for the male inflorescence, indicating that an

1001

80-

E 60-0to

c 40-0

0O20

oj

0

GA3

0

* a0O

0Control \

O * *Ia

-4 -2 6 +2 +4 +4 +4 +

Time of GA3 treatment(days from end of the long night)

-1t15

FIG. 6. Effect of the time of a 0.3 mM GA3 treatment before or after asingle 16-h long night on the percentage of plants having terminal maleinflorescences on all side axes. Control plants were treated with H20 atthe end of the long night.

identical stimulus-or an identical last-moving component-isinvolved in the initiation of both kinds of inflorescences.

Application of high temperature at different times before, dur-ing, or after the inductive dark period does not allow us todiscriminate between the processes involved in the initiation ofthe terminal male inflorescence and those involved in that offemale inflorescences. The time course of the action of heat onmale flowering is consistent with previous observations in Xan-thium (10, 17), except that in the present experiment there is astrong inhibitory action of the high temperature treatment givenduring the first half of the inductive long night.

Exogenous GA3 affects sex expression in Xanthium by enhanc-ing the tendency to maleness, as in a number of other monoeciousand dioecious species. This result differs from that of Von Witsch(21) who reported that femaleness was promoted by GA3 appli-cation in a day-neutral line of Xanthium. The reasons for thisdiscrepancy are unknown. A promotive effect of GA3 on thedevelopment of the male inflorescence in Xanthium was previouslyreported (2, 6).The question arises is to how sex expression is controlled in

Xanthium. Based on our observations that conditions that promoteflowering in this species also increase femaleness (Table II), onepossibility is that a balance between two different substances atthe level of each individual meristem is the critical factor. Onesubstance whose production increases with the degree ofinduction(i.e. the floral stimulus or one of its components) favors femaleness;the other substance, which might be a gibberellin, enhances male-ness. The most important problem encountered with this conceptis that it cannot be used to interpret the pattern of distribution ofmale and female inflorescences, as shown in Figure 1. It does notexplain, e.g. why the terminal meristem of the main axis alwaysdevelops into a male inflorescence, although this meristem isexpected to receive the most floral stimulus. It is established thatthe dominant apical bud drains most assimilates moving in theplant (1 1) and that the floral stimulus moves with the assimilates(22).

Another possibility is that the critical factor in sex determination

1248 Plant Physiol. Vol. 67, 1981

FLOWERING AND SEX EX]

is the state of the meristem at the time of the arrival of the floralstimulus. Parker and Borthwick (14) and Lance (9) reported that,in cocklebur, female inflorescences arise only from meristemswhich have not previously produced leaves, whereas male inflo-rescences arise from meristems which have already initiated atleast one leaf primordium. Current observations of GA3-treatedplants reveal, however, that female inflorescences may arise frommeristems which have already produced foliaceous appendages(Fig. 7). Whether these appendages are true leaves or not is

FIG. 7. Flowering response of X. stnumarium induced by a single 16-hlong night and treated eight times with GA^ (0.3 mm). GA3 applicationswere made by days 0, 2, 5, 9, 12, 16, 19, and 23 after the end of the longnight. The arrow shows a terminal pistillate inflorescence on a side axiswith several foliaceous appendages. Note the elongate internodes and thelanceolate leaves induced by GA3.

PRESSION IN XANTHIUM 1249

unknown. Thus, the question is still unresolved as to how sexexpression is controlled in Xanthium, and further work is neededto clarify this problem.

Acknowledgments-The authors wish to thank Dr. J. A. D. Zeevaart, MSU-DOE,East Lansing, MI, for supplying burs of Xanthium strumarium and reading themanuscript. They appreciate the able technical assistanc6 of Mr. G. Sylvestre. One ofus (M. L.) is grateful to the Institut pour l'Encouragement de la Recherche Scienti-fique dans l'Industrie et l'Agriculture (I.R.S.I.A.), Belgium, for the award of aresearch fellowship. Financial support from the Belgian government, through theprogram of "Action de Recherche Concertee," is gratefully acknowledged.

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5. FRANKEL R, E GALUN 1977 Pollination Mechanisms, Reproduction and PlantBreeding. Springer-Verlag, Berlin

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Plant Physiol. Vol. 67, 1981