quantitative assay of photoperiodic floral inhibition and stimulation

Quantitative Assay of Photoperiodic Floral Inhibition andStimulation in Biloxi Soybean' 2

Murray W. Coulter3 and Karl C. HamnerSpace Science Center, Institute of Geophysics and Planetary Physics,

and Department of Botany and Plant BiochemistryUniversity of California, Los Angeles 24, California

Evidence has accumulated to suggest the involve-ment of an endogenous rhythm in the photoperiodicflowering response of long and short day plants. Re-cent investigations with Biloxi soybean have shownthat light breaks applied during the 64-hour experi-mental dark period of a 72-hour, or tridiurnal, cycleare stimulatory, inhibitory or innocuous dependingupon their time of occurrence during the cycle (2).High intensity light applied in the range between thetwenty-second to thirty-fifth hour and the forty-seventh to fifty-sixth hour in a tridiurnal cycle wasshown to be stimulatory to flowering, whereas lightapplied in areas surrounding the 16-, 40- and 64-hourpoints was inhibitory. It was concluded, therefore.that the 8-hour light period which initiated each 72-hour cycle established an endogenous rhythm whichcompleted an oscillation each 24 hours and which hadalternate 12-hour phases of sensitivity to light. Lightis stimulatory if applied during the first 12 hours, orphotophil phase, of the oscillation, but is inhibitoryif it occurs during the last 12 hours, or photophobephase. Even a very brief exposure to light duringcertain of the photophobe phases may completelyinhibit flowering, and light during the photophilphases may stimulate flowering to such an extentthat, in some cases, nearly every bud on the plantresponds. In the following experiments it was pre-sumed that floral repression due to light applied inphotophobe phases might be counterbalanced by ap-plication of additional light during photophil phases.In other words, by balancing floral inhibition andstimulation, it might be possible to obtain a more ac-curate quantitative measure of the inhibitory orstimulatory effects of light applied during the dif-ferent phases. The degree of floral repression dlueto an inhibitory light treatment, therefore, would hereflected by the differential between the floral re-sponse of a balanced treatment and that for thestimulatory treatment alone. Furthermore, various

Received February 17, 1965.2 This work was supported in part by National Sci-

ence Foundation Grant G-23893 and also by subsidy fromthe UCLA Space Science Center under the NationalAeronautics and Space Administration sustaining GrantNsG 237-62.

3 Present address: Department of Biology, TexasTechnological College, Lubbock, Texas.

873

levels of response can be obtained by controlling theamount of floral stimulation. The usefulness of thistype of analytical approach, however, is highly de-pendent upon the occurrence of a functional relation-ship between the stimulatory treatment and the re-sulting number of flowers.

Materials and Methods

Biloxi soybean (Glycine max L. Merr.) seed, ob-tained from Dr. H. A. Borthwick, were planted in asupplemented soil mixture and grown in the green-house under long day conditions for about 6 weeksprior to photoperiodic treatments. The basic experi-mental treatment consisted of a 72-hour, or tridi-urnal, cycle in which an 8-hour photoperiod was fol-lowed by a 64-hour experimental dark period. Mo-difications of this treatment were made by the ap-plication of light interruptions given either separatelyor in combinations at various times during the longexperimental period. Modified treatments were com-pared with the basic tridiurnal control as well as 24-hour and 48-hour cycle controls in which the 8 hourphotoperiod was followed by uninterrupted darknessin each respective cycle. Unless otherwise indicated,each cycle of a specified treatment was repeated 7times in direct succession. All light treatments weregiven with high intensity (1500-2000 ft-c) light ob-tained from cool-white fluorescent tubes at approx-imately 280 and intervening dark periods were main-tained at about 22°. Following the specified ex-perimental treatments, plants were returned to thelong day conditions in the greenhouse until time fordissection. Ten plants were used for each treatmentalnd the floral responses were scored as the numberof nodes with flowering buds per 10 plants. Theequipment used and the details of our methods forgrowing and treating plants have been described else-where (2).

Relationship Between Numinber of Inductive 72-Houtr Cycles anid Floral Response in Biloxi Soybean.The following experiment was designed to determineif the floral response to tridiurnal and to modifiedtridiurnal cycles was proportional to the number ofinductive cycles given.

Experiment I. Plants were moved from the longday conditions of the greenhouse to the experimentaltreatment area and separated into lots labeled A, B

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PL4.ANT I]N'llYS1010(;i '

and C, respectively. A diag-raniatic exaiiiple of thetreatments given in each cycle of lot A, B alnd C isshown in figure 1. The treatmiienits for lot A con-sisted of various ntumbers of cycles, each cycle con-sisting of the basic treatmiienit wN,ith 04 hours of con-tinluous darkness following the 8-lhouir photolperiod.One group of 10 plalnts was remiioved after each 72-hour cycle until 10 treatmenits receiving from 1 to10 cycles had beeni made. After each cy-cle, the comn-pleted treatments were returned to the long day con-ditions of the greenhouse where they remained unitiltime for dissection approximately 6 weeks later. Adirect functional relationshil) with a regression coef-ficient of 17 exists between the floral responise andlthe number of tridiurinal cycles giveni from 5 to 8(curve A, fig 2). The direct relationship for plantsreceiving more than 8 cycles of treatmiient is lost.presumably (ltue to factors associated with the ex-

tremiiely long dark period. Few lplants will with-stanl(l the rigor of more thlnl 1() tri(liuirnial cycles ofthis type.

The remaining pots labeled B and( C were used todetermine if the direct relationslhip between the treat-ment and the floral response would be mainitaieledwhen the tridiurnal cycle was modified. Pots in lotB were separated into 8 different treatmenit groupsand given a varying number of cycles in which a 2-hour light break at the 16-hour point was followed by2 separate 4-hour high intensity light breaks applie(lat the 24-and 48-hour points in each cycle. Thistreatment constitutes a severe modification of thebasic tridiurnal cycle, but the purpose here was todetermline if a direct relationship between number ofcycles of treatment anid response was maintained.Pl'ants of lot B receivinig frolmi 1 to 8 cycles of this

CYC LE T ME IN HOURS

0 8 16 24 32 40 48 56 64 72

APh7OOpiod [xP-ri-ntol Do,k Period

-m-

2 hr ..,h b, tory P- t, b.t4on

4 hr SJpple.ent., y

-a

c

ONE CYC LE OF IREATMENI

FIG. 1. Diagram of tridiurnal treatment cycles usedin experiment I. Different numbers of cycles of A, B,and C were given to determine if there were corre-sponding functional relationships.

140

120

z

3

0

0

z

0

* PIt ,.Ybt S dt the 16 24 & AS hr pts

* Irt., b.tlI, the 24 & 40 hrI p ts

2 3 4 5 6 7 8 9 10C Y C L E S

FIG. 2. Floral responise curves to differenit nuimibersof tridiurnal treatment cycles. The niature of the treat-ment cycles for A, B, and C are designatedl in figure 1.The vertical lines through each point represenlt thestandard errors.

ino(lified tridiurnlal treatmsent were ruin simultanieouslywith the conitrols in lot A with the samiie conditionsof temperature and light intensity. Although thefloral responses indicated a higher degree of vari-ability, curve 13 of figure 2 demonstrates that thereis a linear relationship from 4 to 8 cycles which isnot siginificantly different from the basic tridiurnalcontrol plotted as curve A.The plants in lot C were given highly stimulatory

treatments in order to see if the direct relationshipbetween flowering and the inductive treatmenit wouldbe maintainied for higher levels of flowering. The 2treatmenlts were given 3 and 7 72-hour cycles respec-tivelv, in which 4 hour light perturbations were initi-ated at the 24- and 48-hour points of each cycle. Asillustrated by curve C in figure 2, the relationship be-tween the response and the nulmiber of treatmentcycles parallels the other curves even at high levelsof flowering. As might be expected. however, thevariability is greater at higher levels of flowering,and it would be difficult to distinguish small (if-ferences for responses in this range.

The results of experiment I indicate that thereis a (direct relationship) betweeni inductive treatmlenitsgiven in 72-hour or tridiurnal cycles and( the numberof flowering noldes that result. 'I'his relationishi) ismaintaine(l with miiodified tridiurinal treatmeints foraverage floral responses fallilng in a ranige of fromii8 to 103 flowering nodes per 10 plants. TI'he regres-sioIn coefficient for flowering in this range resultingfrom the number of cycles of inductive treatmieiit isapproximately 17 and is linear between 5 and 8 cyclesof treatment when the standard 8-hour photoperiodof each cycle is followed by 64 hours of continuousdarkness. It is concluded that the floral responise ofBiloxi soybean to variotis tridiurnal treatments couldserve as a quantitative index for measuriing the

I87/4

* 72 hr C.. ro.I (8Oh, oh t aplr,.d 64 hrs da.,

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('Otl,Tl-TR AND) 11AAMNAROO\ NTITAI ASSAX' OF FLORALIN, lIBTION

degree of floral inductioni and should reveal any

stimulatory or inhibitory effects of modified treat-ments given (luring that cycle.

Inihibitory Inifliuenlcc of Light Give,l at the 16-Houtr Poinit in ai 72-Hour) C cle. The following ex-

periment was designed to study the inhibitory in-fluence of light given at the 16-hour point in a tri-diurnial cycle. This was done by giving variouscombinatioins of 2- and(l 4-hour light breaks at the 24-hour points following eitlher 30-minute or 2-hourperturbations at the 16-hour poinlt.

Experiniienit II. Pots were move(d from the long(lay conditions of the greenhouse and split into 3 lotsof ;5 which were labeled A, B and C respectively.Using 5 pots per treatmenit, each lot was furtherlabeled for treatim,ents from zero to 10. Lot Aserved as treatmlenit controls with no inhibitory liglhtgiveen at the 16-hour point. Lots B and C were

,given identical treatmielnts for eachl group except that30 miniutes and( 2 hours of light, respectively, were

given at the 16-hour point. A treatment diagramsumnmariziing all treatirents given is presented infigure 3. Individlual comparisons within the experi-ment, however, should be mzade separately due tothe complexity of the experiment. The inhibitoryeffect of light at the 16-hour poiInt may be seen bvcomparing simlilar treatnment groups from each lotseparately. Light at the 16-hour point cancels floralinduction even when 2 hours of supplementary lightis applied at the 24- or 48-hour points, as shown bycomparing each lot for treatmlenits 3 alnd . Fourhouirs of supplenientary light giveni at the 48-hourpoint will partially overcome the inhibitory effectsof the 16-hour point interruption, but 4 hours of lightgiven at the 24-hour point is not sufficieint.

B, comnparilng treatmenlts 4. 6 and 10 of each lot.it is clear that the stimulation due to supplenientarylight at the 24- and 48-hour points is not completelv(utle to the (luration or total ami0ounlt of light, butrather the (lurationl and the timne at which the light isgiven.. In treatment 6. for example. two 2-hour lightbreaks at the 24- and 48-hour points give mlore ef-fective stimuitilatioll to flowerinig thani a single 4-hourlight treatmlenit iniitiate(d at the 24-hour point. Thistreatmenit, however, is less effective thlan a iingl(de 4-

lhouir light apl)licatioln iliitiate(l at the 48-hour point.A comiiparisonl of treatmlenits 0 ainid () of each lot

itl w\hich (loubl)le sul)l)lemlelntary light breks are -isveli.agtin (lemonstrates that 4-houtr supplemientary liglhttreatmiielnts are imiore effective than 2-hour treatmiienitsboth in stimiiulatinig flowering and( overcominig the ill-lil)itorv effects of a light break given at the 16-hourpoint. A similar coi11parison miiay le ma(le by relat-ing treatments /7 and 8 of each lot to treatmelnts 6 or

9. Treatment groups 7 an(d 8, however, show thatlonger durationis of light are mlore effective in floralstimulationi xxhein given at the 48-hotur point thanlgiven at the 24-hour point, even though the cycle isbroken at both 24- and 48-hour points 1y light treat-

menits. The same is noted 1y a comnparison of treat-ments 4 and 10, and 3 and 5.

Figure 3 demonstrates that light initiated at the16-hotir point is inhibitory in every case andthat a 2-hour light interruptioni initiatedl at thispoint is more inhibitory than 30 minutes. l)ue to theconsistency in the amlouint of floral repression ob-tained due to either a 30-miniute or a 2-hour lightinterruptioni at the 16-hour point, regression analysisfor selectedl treatmenits of each lot was madle in whichthe floral response was compared to the total amiioulntof supplementary light given. As as the case inexperiment I where the floral response was comparedto the number of treatmiienit cycles, the regression lines

for floral responses falling between approximately 8and 110 flowers were parallel for all comparabletreatments, thus indicating a direct funlctional rela-tionship. Using only those treatmenits in which thenumber of flowers falls within the linear response

range. a comparison of the actual amlounlt of floralrepression dlue to an inhibitory treatnment at the 16-hour poiInt vas made. In table I, treatimieints of lotA and B are compared, and the floral reductioni dueto a 30-minute light interruption for each treatmentis designated. The mean value given below the table

Tr-atm*ntt40.

A-

A- 2A -3A-4A-5

A-6

A-7A-8A -9

A -10

B-

rn-2

B-3r-4B-S

B-6r-7e-8e-9e-10

c-I

C-2C-3

c-4c-5C-6

C-7

C-8c-9

c-10

A -21- 30B-61-67

an-91-97

B- 101 -107c -62-68c-92 -98

P r og o-m of Tr*-t m*ntsF lo_-or

Cycle T.m- Hours -

0 8 16 24 32 40 48 56 64 72

63

39

~~~~~56f_ _ 111

44

84-~ 1)0

102

12769

0

0

0

-__ 250

~~~~~~4_ 230

_64

0

0

0

0

4~~~~~~~~~~~~~~~~~~~0

o_ ~~~~~~~~~~~~~~~~4_ _20

0 -660-4_ _ o0 77

0

0-71

One CYCl f 'T -et-et

FI(;. 3. Treatment diagrablm of cycles usedl in experi-ment II. Treatmenlts 1 to 10 of lot A, B, and C are

identical except for the application of light interruptionsat the 1 6-hour poilnt. The ad(litionial treatmiients repre-sented at the bottom of ths page are cycle controls inwhiclh different numiibers of cycles of selecte(d treatmenitswYere given.

875

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Table I. -Reduction of FIowiers duie to a 30-Minitte Liyiht Intcrruftion Givten at the 16-Hour Poinit

Hrs of supplementaryTreatmenit light given at

no. 24-hr pt 48-hr pt

68479

4()24

22444

Floral response*No light at 16-hr pt 30-min light at 16-hr pt

Lot A Lot B

86102111110127

411253064

Avg floral decrease = 80.0 -+ 5.3

* Treatments of lot A are identical to those of lot B except that a 30-minute light break was applied at the 16-hourpoint of the cycle for lot B treatments.

Table II. Reduction of Flowers I)ue to a 2-Hour Iutterrutptioui Given at the 16-floutr Point

Hrs of supplementaryTreatmenit light given at

no. 24-hr pt 48-hr pt

68479

4024

22444

FloralNo light at 16-hr pt

Lot A

84102111110127

resl)onse*2 hr light at 16-hr pt

Lot C

1~~~~

246

34

Avg floral decrease =

* Treatments of lot C are identical to those of lot A except that a 2-hr light break was applied at the 16-hrpoint of the cycle for lot C treatmenits.

Table III. Flor(al Inihibitioni antd Stim-ulation I),c to Hiigh Initensity Liglht Breaks in a 72-Hour Cycle

Duration of light break

30 mmi2 hr

X2 hr2 hr4 hr2 hr4 hr

X 2 lhr* 4 hr

Time of initiation in cycle

16-hr point16-hr point36-hr point24-hr point24-hr point48-hr point48-hr point56-hr point56-hr point

Avg floral deflection

-80.0- 97.4-52.1-2-20.4+ 32.2- 34.1+ 64.7d- 21.4+ 40.0

* Values derived from experiment III. See figure 3.

inidicates that 30 minutes of light giveni at the 16-hour point will reduce the floral response by 80 -+- 5.3flowers. Table II gives a similar coml)arison fortreatments of lot A and C which indicates a mean

(lifferenitial of 97.4 1.4 floweris (Itle to a 2-hourlight break at the 16-hour point. In comparing thefloral reduction caused by 30 minu.ttes vs. 2 hours oflight at this point, a calculated value of t in(licatesthat the differenice between the 2 imeanis is signiificantat the 2 % level.

In floral stimulation, 4 hours of light giveneither at the 24- or 48-hour point is more effectivethan 2 hours, regardless of whether inhibitory lightbreaks are given at the 16-hour point or not (fig 3).It is furthermore apparent that supplementary light

initiated at the 48-hour poinlt is more effective forfloral stimuitilationi than the samiie amount of lightinitiated at the 24-hour point. (Quantitative analysesof the degree of floral stimiiulation in this experimentcause(1 by appllications of light at the 24- and 48-hourpoints have beenimade separately by the samiie methoddemonistrated in tables f and( 1T and( are summililarizedin table ITI.

Treatmelnt groups in lot I) were giveni differentnumbers of cycles for various treatmlents of lot A.B and C. as specified at the bottom of figure 3. Thelast digit of each treatment number indicates the num-

ber of cycles of the designated treatment which were

given. In treatments A-21-30, therefore, 1 to 10cycles of treatment A-2 were applied and constitute

Differenice

8091868063

Difference

8310010710493

97 -+- 1.4

5.31.46.34.75.64.55.03.24.1

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(OlULTEIR AND 11A NAERQUANTITT;IV ASS;AY 01F FLORAL INmII IrTION7

10 separate treatmlents. The individual responses ofeach treatment villiinot be given here, but regressionanalysis has been applied for every appropriate re-sponse series and the results are in agreement withthose of experiment I, showing that a direct func-tional relationiship is maintained between the flower-ing response an(l the number of cycles applied. Theresults of this experimnent indicate that a relationshipexists wherely a particular treatment may be assessedquantitatively and compared to a base control andthat the summary effects of all modifications givenin an experimeental cycle may be evaluated anid ex-pressed either in terms of the nulmiber of flowers pro-duced or translated into units of a standard treat-menit cycle. Although this will not be elaboratedhere, suffice it to say for illustration that the floralincrease caused by 4 hours of light given at the 24-hour point in 7 consecutive tridiurnal cycles is equalto the floral increase caused by 2 hours of supple-mentary light given at the 48-hour point for 7 cycles,and that these increases are each equivalent to thatwhich would be lproduced by the addition of 2 cyclesof a basic tridiurlnal treatment. Allowing for thres-hold in(luction, any of these 3 treatments would in-crease the floral response by approximately 34flowers.

Inhibitory Inifluience of Light Given near thelliddle of the Dark Period in a 72-Houtr Cy7cle. Ex-perimenit II gave a quanititative measure of the de-gree of inhibition obtained due to light breaks in thefirst photopholbe zone of a tridiurnal cycle, and in-dicated the supplementary treatments that best over-come the inhibition effects. AAn experiment was de-signied to study the second inhibitory zone in the mid-Cle of the dark period in a similar fashion, as well asthe possible effects of light interaction. In the fol-lowing experiment a 2-hour inhibitory perturbationinitiate(l at the 36-hour point was applied with vari-ous combinations of 2 and 4 hours of light given atthe 24-, 48- and 56-hour points.

There are 2 methods that have been used to scorethe flowering responses for the experiments that arebeing presented here. In general, the relationshipsfound in the 2 scoring techniques are the same, andfor simplicity the re.sults presented for each experi-mleiit in this paper represent only I set of values.\N'hen B3iloxi soybeain is subjected to tridiurnal treat-ments as previously described, the first bud toinitiate floral differenitiationi occurs at approxinmatelythe tenth or twelfth node. The inductioni of receptivebuds procee(ls ui)pward from that point in accordanlceto the amiiounit of floral stimulation effected. WVhenthe floral responise of a plant exceeds 6 or 7 flowers,floral differen,tiation of receptive buds below t'hefirst induced one begins to take place, and floweringprogresses dowlnward as well as upward in accord-ance with the degree of induction effected. Sincemost workers with Biloxi soybean have l)een con-cerned xvith floral inhibition rather than stimulation,3 to 5 flowers per plant would normally constitute a

maxiIm1uIm1 respoInse in their experimients and(l the up-ward induction trend wouldl be the only index conl-sidered. In experiments such as those presented here,however, it became apparent that there was a (lualstandard for recording very high floral responses.Scoring oily, nodes above the first induced bud ismlore equivalent to the standardl methodI and resultedin lower variability for higher levels of induction.On the other hand, an inclusion of downnward as wellas upIward induction, as has been done in experimiientsI an(l II, increases the floral differential betweenmoderate and high levels of flowering and canl beusefuil in many cases. A direct linear relationship ismaintained with both systems of scoring, but the re-gression slopes, or coefficients, are obviously differ-ent. For sake of comparison, the conventional orstandar(d scoring system has been used to present thefloral responses in experiment III, so that the flower-ing xvhich occurs beloN the first induced bud is notincluded.

Experinent III. Plants were moved from theloing day conditions of the greenhouse for experi-mental tridiurnal treatments. One hundred andseventy-five pots were separated into groups of 5which wvere labeled 1 through 35, respectively. These

P,og,a- o f Tr--tm-ts

Tr--tm-ntNo.

234

5

67a910

1112131415

1617181920

2122232425

2627282930

3132333435

Cycle Time in Fmours

0 8 16 24 32 40 48 56 64 7245

- 76

- 68

53-_ 74

_ ~~~~~~~~~~~~~44_________________- ~ 100

82

74

- 101

m 27

- - - 12_in2 2

31

- 22_ ~~~~~~~~~~~~~~~9

- - - 16- - 32

]22540

~ - 102_

_ ~~~~~~~~~~~~~~~~81- 96

_____________________ - 65

m m - 30-_-m 13i_m 65

93

3

43551 1

- 6_~ _____________________ 0

O.- CSJc | of T, Int --t

FIG. 4. Treatment diagram of cycles used in ex-periment III. Various combiniations of 2- and 4-hourlight breaks were given at the 24-, 36- 48- and 56-hourpoints in a 72-hour cycle. Treatments 31 to 35 belowrepresent cycle duration controls. Each treatment cyclewas repeated 7 times.

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878PLANT P1YSIOLOG(;Y

were fturther separated ilto /7 lots conitaininig S treat-ment groups in each lot. rhe treatments and theresponses are given in figure 4. The other 50 potswere dividle(d into 10 groups anid use(d to provide only5 cycles of exposure to treatments 2, 6. 7, 12, 16, 17,18, 19, 20 and 35. The results of this latter seriesare not presente(l but are referre(d to. where al)l)rol)ri-ate, in the text.

Treatments 1 through 10 an(d 21 through 25 rep-resent treatments given without light at the 36-hourpoint. Treatments 11 through 20 and 26 through 30in the same figure are identical, except 2 hours oflight were given at the 36-hotur point. In table ITNpairs of treatments are compared where the 2 treat-miients (liffer fronm onie another only by the presenceor abFence of a 2-hour light break at the 36-hourpoint. In every case the 2-hour light break causecla decrease in flowering, andcl the last colummn of thetable gives the amotunit of re(luction in flowerinigcause(l by this 36-hour poinit light break. Thle aver-age floral inhibition caused is 52.1 -+ 6.3 flowers per10l)plants. There is considerable variationi in the re-sults, and( while some of these variations are justexperimental error, some are doubtless relate(d to (lif-ferenices in the associated treatmetnts.

In each set of treatmlenit comparisonis shown iiitable IV it might be niote(d that a light interrulptionat the 36-houir p)oint is inhibitory to the niormiial floral

responise wshich would l)e pro(luce(l by that treatmenit.The most severe inhibition due to a 36-hour point per-turbation is accomplished when multiple light breaksare given, and particularly when light breaks aregiveni at b)oth the 24-and 48-hour points of the cycle.It also appears that light perturbations at the 36-hour point are more inhibitory when given withsup)plementary light interruptionis at the 48-hour pointthan with similar supplementary interruptions ateither the 56- or the 24-hour poinlts. There is I11clear explanation for this unless the inhibitory per-tnrbation effects a phase shift or perhaps a secondrhytlhmii which conflicts with and damiipens the miiainrhythmii initiated wvith the imain )photoperiod in eachcvcle.

Tlshe lack of concordanice of certain treatments, asl)ointe(d ouit above, in(licates the value calculate(d intable IV is not niecessarily ani accurate one, an(l veryprobably represeilts an overestimate for the degreeof inihibition that w\,ould be effecte(d (Ite to a 36-houir point light break given in ani otherwise t11-mo(dified tridiurnal cycle.

Fuirther comparisonis of the effects of light at the24, 48, andl 56-hour points may be imiade, as well asthe effect of such stpl)lemiientary treatmiients when aninhibitory ilnterruptioln at the 36-hotur ploint is giveni.Treatmiientis 1 andl 6 do not demionlstrate any differ-enice ini the floral resl)oiise dute to anl increase in the

Table IV. Iiihibitorv Influtecicc of a 2-flour Light Initerrutptioni Giveiz (It the 36-Houtr Point

Cycle time24-hr poinlt 36-hr poinlt 48-hr poinlt

2 hir

2 hr0

0

0

0

4 hir

4 hir

0

0

0

2 lhr2 hir

2 hr

2 hr

4 hir

4 hir

4 hir

4 hir

0

2 lhr4 hr

4 hr

2 hr

2 hr

0

2 hr

0

2 hr

0

2 hr0

2 hr0

2 hr

0

2 hr

0

2 hr

2 hir

2 hir

0

2 hir

2 hr

02 hr0

2 lhr2 hr

0

0

0

0

2 lhr2 lr

0

()

4 hr4 hr

2 hr

2 hr

0

4 hr

4 hr

0

0

4 hr2 hr2 hr0

0

56-hr point

002 hr2 hr

0

4 hr4 hr

0

00

004 hr4 hr

0)0)0)

4 hr

Floral response

4527533174

44

7432

1012570)1 2(821

1 (H)9

8216403

1023081139665

Avg floral decrease = 52.1 + 6.3

Treatmenit110.

114

1414

156169

1'31020

123

137178182130222623272428

Difference

18

74

22

42

76-

64

47

91

66

37

72

68

31

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COULTER AND HAMINER-QUANTITATIVE ASSAY OF FLORAL INHIBITION8

durationl of light given at the 24-hour point. Itshould be pointed out, however, that in 5 other experi-ments in which identical treatments were used, 4hours of light was in all cases highly stimulatoryand produced an average of 78 flowers (an averageincrease of 38 flowers above the control base line).Also, a 5 cycle control in this experiment of treat-ment 6 produced 37 flowers, indicating the normiial re-sponse for a standard 7 cycles of treatment would be71 flowers. Treatments 2 and 7 show the effects of2 and 4 hours of light when given at both the 24-and 48-hour points. It is clear that the longer dur-ations of light at these poilnts are more stimulatorywhen no inhibitory interruption is imposed, but asshowni in treatments 12 and 17. 2 hours of light im-posed at the 36-hour point reverses the correlation.It is probable that the reduced flowering responsewith the longer light durations when a 36-hour pointperturbation is imposed. is due to the effects of lightinteractionl. Treatments 11 and 16 tend to confirmthis with respect to the 24-hour point. In all othercases where a 36-hour point interruption is not given(treatments 7-10 vs. 2-5), an increase in the durationof light in photophil zones causes an increased floralresponse. For cases in which 2 stimulatory lightperturbations are given in the cycle, the influence ofan increased light duration at either or both of thestimulation poillts is not only cancelled, but reversedwhen a light initerruption at the 36-hour point is im-posed. Such a reversal again seems to indicate ef-fects due to light interaction. It is apparent. how-ever, that we mlay be dealing with more than one typeof interactioln here.

Discussion

Experiment I demonstrates that there is a func-tional, or linear, relationship between the number ofinductive tridiurnal cycles and the number of result-ant flowers for Biloxi soybean. This linear relation-ship is maiintained in modified tridiurnal cycles inwhich stimulatory and/or inhibitory light treatmentsare incorporated. These results presented the possi-bility of using supplementary light treatments to par-tially overcome the effects of inhibitory treatmentsto the extent that flowering would result. Thiswould allow an analysis of the degree of inhibitionimposed by treatments which might otherwise repressflowering below the inductive level. In experimentsII and III this principle has been utilized, and thecollective analysis of various combinations of lighttreatments has proved to provide an extremely sen-sitive quantitative measure for either floral inhibitionor stimulation.

In Biloxi soybean there are now 4. or perhaps 5.techniques which may be used to measure the photo-periodic flowering response quantitatively. Thenormal measure of flowering after 7 cycles of treat-ment is truly quantitative in that the number offlowers per plant in a population (usuallv 10 plants)

is recorded and the variance of the response may betreated statistically. For most experiments thismethod is completely adequate, but it is limited bothin its range and its sensitivity. Two methods havebeen presented in an earlier paper (2) which willallow increased resolution for highly stimulatorytreatments. Since there is a direct functional rela-tionship between the numlber of inductive cycles andthe flowering responise, a reduction in the number oftreatment cycles may place the flowering in a re-sponse range which is less variable, and thereby moreaccurate in its representation. The normal 7 cycleresponse pattern, of course, may be obtained by directextrapolationi. In the second method described byCoulter and Hamniier (2). a further increase inresolution can be obtained by applying a regressionanalysis to a 3-point treatment assay.The technique described in this paper allows ana-

lvsis of both stimulatory and inhibitory treatments.The inhibition of normally noniniductive treatmentsmay be measured by the application of compensatingfloral stimulation. Furthermore, the response maybe measured at various levels of flowering by coun-terbalancing the inhibitory treatment with variousdegrees of floral stimiiulatioln. By pooling all of theinhibition values calculated from these various treat-ments, the resulting average minimizes the effectsof chance variation as well as minor variations whichmight be peculiar to a particular treatment regime.This formi of analysis is satisfying nlot only in termsof its accuracy, but also in its ability to detect ab-nornmalities which miiight otherwise be overlooked orunexpectedl. This has indee(d beeni true for certaincases of light interaction pointed out in experimenitIII. The apparenit problems of light interactionii(lenitified in experiment III also led us to seriouslyquestion the accuracv of some of our results in ex-periment II. Most of the treatments used for analysisof inhibitioln at the 16-hour point involve a supple-mentary light treatment at the 24-hour poinlt. Anyeffects of interaction between light applied at the16- anid 24-hour poilnts would cause a partial nulli-fication of the stimulation being applied at the 24-hour point. The occurence of interaction here, there-fore, woul(d result in an overestimate of the inhibitionfound at the 16-hour point. By referring to treat-ments A-4 and B-4 in which no perturbation is givenat the 24-hour point, it may be seen that this was notthe case, for the floral differential (31 flowers) isno greater than the calculated value using other treat-ments. It is conceivable that the inhibition at the 16-hour point caused by an interaction between the per-turbation and the main light period was sufficient topreempt anv secondary interaction with light appliedat the 24-hour point.

There is a fifth way by which floral stimulationand inhibitioln may be evaluated (4, 2), but thus farits application has been rather restricte(d. By appli-cation of various photoperiods as intervening treat-nments in bidiurnal or tridiurnal cvcles. floral re-

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sponses above or below the donor conitrol level de-note the stimulatory or inhibitory+ nature of thetreatmenit. It is possible therefore, to assess the in-fluence of treatments which mlight otherwise be non-indtictive. The inhibition of some treatmlents, how-ever, appears to be too great to be completely coun-terbalance(l by (lonor treatments, and cannlot beevcalluated quantitatively by this technique. This ap-proach is inlmany ways similar to that used bySchwabe (3) in which the inhibitory effects of longdays were stu(lie(l by alterinatinig them w\ith variouscomlbinations of long and short days.

The techniques presented in this paper have manyadvanitages. Correspondingly they have shortcom-ings. Aside from their predictive value in the designof other experiments, one of the major contributionsof such quantitative measures is found in the de-termination of maximum inhibition and stimulationoccurring due to an endogenous rhythmi. This de-term-ination would show the amplitude of the oscilla-tion associated with the rhythm, and, given the per-iod, it is possible that a courier or other types ofmiiathemiiatical analyses could be made in an attempt tomiiore clearlyv assess the nature of the rhythm. Quan-titative analy-sis of a single poiint does not detect apoint of miiaximum inhibition. This determinationshould re(luire analytical scan of the entire photo-phobe range. A second shortcoming of the tech-niques presented is associated with the number oftreatmelnts used to evaluate a single point: Manyof the treatmiients used were not useful for analvsis.It is hoped, however, that the results presenited wvilleliminiate the selection of less favorable treatmentcombiniations in ftiture experiments and will serveas a useful index for predicting the best analyticalcombinations for each experimental application. Aftirther variationimight be employed by assessingprogressive points rather than a silngle point. Thisapproach would offer a decided advantage in that itwvould not olnly increase the reliabilitv of the evalu-ation, but would also indicate the slopes of the in-hibition aui(I stimlulation responses as well as theirmaxima.

In the light break response curve of Coulter andHamner (2)t analysis of progressive points hasalready Ieen use(l for zones of stimuilatioln andcl re-*ponises above the inductive base line. By a com-p)arison with other cuirves resultilng fromii the en-dogenous rhythm,,, the details of this curve slhould beextremiiely useful in evaluating the imiechanisml of thebiological clock. It is important, however, that aneintire analysis be comlpleted before a detailed inter-pretation is given for this curve. Oni the basis of theresults obtained in the preceding experiments, how--ever. tllere are a few gelneralizationis that may beoffered.

Tt is clear thlalt light applied in the first photo-phobe zone at the 16-hcour point is approxi.matelyt\-ice as inhibitorv as-light apl)lied in the niiddle of

the secoindI photophobe zone. Since a light breakinthe middle of a tridiurnial cycle should be free froniany influenice of light interaction associated with aspecific dark requiremiienit, it is logical to assume thatthe inhibitioln imposed wvas a direct reflection of thatassociated w-ith the photophobe phase of the endogeni-ous rhvthm. The additionial inhibitioin produced bylight applied at the 16-lhour poinit might be due tolight interactioni wvith the maini light period. How-ever, 2 hours of light applied at the 16-hour point ismore inhibitorv than 30 miniutes. In this case, 2hours of high intenisity light not only may disrupt therequirement for a niidiniutii duratioii of darkiiessdlirectly following the miiain light period, but alsoset tup an oscillation of its own out of phase with thefundamental. The (liscord from this secondary oscil-lation appears sufficielnt to damp the rhythm to theextent that an additional net inhibition of 17 to 18flowers results.

Reference to light interaction in photoperiodiccvcles w-ould normally suggest the type of phenome-non which has been miientiolne(d bv Claes and Lancg(1). This effect miight easily be explained in Biloxisoybean as due to the restriction of a minimlum dark(Itirationi w-hich nmav be requiredI following an ade-qtlate photoperiod. In B3iloxi soybean the miniimumil(lark requiiremiienlt for floral inductionl x\ ould be ap-proxinliately 10 hotirs. Any light treatmiienit whichwx-ould decrease the dark l)eriod followinig a photo-period to less than 12 holurs, therefore, \vould causea noticeable decrease in floral stimulation, and if the(lark period is restricted to less thani 10 hours, floralildtictioin wN-ould be completely canicelled. Silnce man+of the light breaks used inl the experimeints reportedhere are sufficiently long to colnstittute indepenidelntphotoperiods, this rtile xx ould apply sinilarly to suchlight breaks in relation- to any other light treat-nments. Several exanmples of this tvpe may be seeniin table I V. Treatmiients in x\-hich the duration ofdarkniess between light applications has been limitedto less thani 12 hours showv greater floral inhibitionthani xN-ould otherwise be expected. In treatments 11ai(l 16, for example, the 4-hour initerruptionl at the24-hour poilnt is follox-\ed by a subsequent dark periodof only 8 hours which maya explain the greater re-duction in floral stimulationi for these treatments.In treatments 13 and 18 as -,ell as 14 and 19, a de-crease from 14 to 12 hours of darkness followinig lightgi-ven at the 56-hour point seemiis to cause a corres-ponding decrease in flowvering, but treatnments 3 aind8. and 4 and 9, demlonistrate that the reduction is notdue to a restricted dark period. The results of treat-ments 3 anld 4, as wxell as 8 and 9, demonstrate thesupplementarv treatmiielnts at the 24-hour point affectadditional floral stiiumllatioln. Treatnments 13 anid14, as well as 18 and 19, inidicate that the effects on1floweriing dIue to anl ad(lditional 24-hour poinlt perturb-ation are reversed w-heni a ligIht break is a(l(de( at the36-hotir point. Stuch a reversal caninlot be explaitne(lby light interaction as a fuinction of-a dark require-

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ment alone. Treatments 13 and 18 should indicatethe effects of light interaction due to a limitation ofa continuous dark period. These results, therefore.can only be explained by an effect imposed on thebasic endogenous rhythm.

There are, therefore, 2 types of light initeractionwhich must be considered. The first has been re-ferred to as being associated with a dark require-ment. It should be pointed out that this is not a darkrequirement in the normal sense of that term, fordark durations shorter than the critical may showdifferential responses to a light interruption. Thesecond type of light interactioni, as suggested previ-ously, may be due to a discord between a secondaryoscillation and the fundamental, and may have nloassociation with a specific dark requirement. Thisinterpretation is in good agreement with the resultsobtained with Pharbitis by Takimoto and Hamner(5).

Unfortunately, interexperimental variation pre-vents a direct comparison between the inhibitory ef-fects studied in experiments II and III. It shouldalso be remembered that the data presented for these2 experiments were derived using different scoringtechniques so the similar treatments of stimulationfrom experiments II and III should lnot be compareddirectly. Treatmenits responding at levels below thecontrols, however, should be comparable since thereis no difference in the scoring for that responserange. Comparable controls in each experiment alsogive some index of the expected variance between ex-periments.

It is clear that effects of light interruptiolns givenin a tridiurnal cycle are multiple and make the re-sponse difficult to interpret. Some of these effectsin soybean appear to be similar to those being foundin Pharbitis and Xan,thi-iin, but others are distinctlydifferent. Efforts are now being made, however.to assess such diverse organismiis in hope that thesimilarities and differences in their control of photo-periodic responses may help clarify the mechanismsassociated with the biological clock.

Summary

Techniquies are described by which quantitativemeasurements may be made of inhibition and stimu-

lation imposed on the photoperiodic flowering re-sponse in Biloxi soybean. To assess highly inhibi-tory treatments which might otherwise prevent flow-ering, the inhibition may be counterbalanced by spe-cific applications of treatments causing floral stimu-lation. The degree of inhibition in such cases canlbe determined by measuring the amount of floralstimulation necessary to overcome the inhibition.The results indicate that light applied during thefirst photophobe phase of a tridiurnal cycle is ap-proximately twice as inhibitory as light applied dur-ing the second photophobe phase, and that lightgiven in the second photophil phase may be twice asstimulatory as light given in the first photophilphase. The ability to accurately measure specificinhibitory and stimulatory effects has assisted in anassessment of the number of factors which might beinvolved and the nature of their action in photo-periodic responses.

Acknowledgments

The authors would like to acknowledge the assistanceof Mr. John J. O'Connor with the photography and green-house maintenance. Thanks are also due Dr. WilliamH. Shumate and Mr. Paul H. Moore for correcting themanuscript.

Literature Cited

1. CLAES, H. AND A. LANG. 1947. Die Blutenbildungvon Hyoscyamus niger in 48-stundigen Licht-Dunkel-Zyklen und in Zyklen mit auf geteiltenLicht-phasen. Z. Naturforsch. 2b: 5663.

2. COULTER, M. W. AND K. C. HAMNER. 1964. Photo-periodic flowering response of Biloxi soybean in72-hour cycles. Plant Physiol. 39: 848-56.

3. SCHAWABE, W. W. 1959. Studies of long-day in-hibition in short-day plants. J. Exptl. Botany 10:317-29.

4. SIROHI, G. S. AND K. C. HAMNER. 1962. Floralinhibition in relation to photoperiodism in Biloxisoybean. Plant Physiol. 37: 785-90.

5. TAKIMOTO, A. AND K. C. HAMNER. 1964. Effect oftemperature and preconditioning on photoperiodicresponse in Pharbitis nil. Plant Physiol. 39:1024-30.

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