flavonoid complexes in pisum sativum. ii. effects of red and far

Flavonoid Complexes in Pisum sativum. II. Effects of Red and Far-RedLight on Biosynthesis of Kaempferol Complexes and on

Growth of Etiolated Plumules 1

Masaki Furuya 2 and Roderick G. Thomas 3

Josiah Willard Gibbs Research Laboratories, Department of Biology, Yale University, New Haven, Connecticut

Flavonoicl synthesis is, in many plants, either pro-mloted by light or (lepen(lent upoIn light (3). Thenature of this light re(quirement appears to vary de-pending UpOnl the conmpounds ancl plant miiaterialsbeing studiedl ( 15, 17, 19, 21, 23 ), and little is knownregalrding the relationship between photoinducedsy-nthesis of these compounds and photoinclucedgrowth an(l (levelopmlenit. In etiolated pea plumules,low intensity red light stimulates both leaf growth(20, 25) ancl the procluction of an in(lole-3-aceticatcid oxidcase inhibitor (13). This inhibitor has quiterecently been found to contain kaeempferol-3-triglu-coside (KG) and kaemplpferol-3- (triglucosv-l-p-couma-rate) (KGC) (9,10,18).

The purposes of the present investigation were,firstly, to (letermine the effect of re(d an(d far-re(dirradiation on the biosynthesis of KGC an(d KG, and,seccundly, to dletermine whether their synthesis is re-latecl to photomorphogenic processes. The resultsobtained show that the red/far-red pigment systenm,phytochrome, does in(leed affect both leaf growthatncl KGC biosynthesis sinilarly in peas, although itappears to have little effect on KG biosynthesis. Theresults provi(le no evidlence that the levels of thesekaempferol complexes affect see(dling growth in vivo.

Materials and Methods

Seeds of Pisum sativumll cv. Alaska, obtaine(d fromAssociate(l See(l Growers, Inc., New Haven, Con-necticut, were sown in moist washed vermiculite in10 X 10 X 10 cm polyethylene containers. Eachcontainer held about 30 to 40 seedIs. The seedlingswere growin in darkness at 27 + 1° and were ex-posed to dinm green light in the (lark rooml for occa-sional inspection and again at the tinme of harvesting.

Red light was supplied by redl fluorescent tubes(Sylvania, 15 w). The far-red light source con-sisted of five 300-w internal-reflector incandlescentlamnps, light from whiclh was filteredl through 7 layerseach of redl and blue (lu Pont cellophane an(d about8 cIm1 of water (8). The emission curves for the red

Received revised manuscript Oct. 28, 1963.2 Present address: Biology Department, Brookhaven

National Laboratory, Upton, Long Island, N.Y.31Holder of the Theresa Seessel Postdoctoral Fellow-

ship in the Biological Sciences at Yale University. Pres-ent address: Plant Physiology Division, Department ofScientific and Industrial Research, Palmerston North,New Zealand.

and far-re(d light sources are slhowIn in figure 1.Absolute miieasuremiients of radliation energy Nweremlade with a photomiieter, consisting of an RCA #926vacuuiml photatube aIndaL D.C. aml)lifier. This photo-

500

480 - FR

120_i

KB C applied (1ole.I/s.pt ) K aOpplied (p m..Ie/.Pot)

FIG. 1 ( tpper). Spectral emissioni of the red (R)and far-red (FR) light sources used. R, red fluorescenttubes; FR, incanidescenit lamps plus cellophanie filters.Energy was measured 50 cm from the lamps. The far-redsource emitted a total energy of about 20 kiloergs cm-2sec-I (1).

FIG. 2 (lowzer). Amounts of KGC and KG recoveredby elution following chromatography of known amountsapplied to paper. Amounts recovered are expressed interms of absorbance. - - -, expected recovery (100 %o)*- *, observed recovery ± standard errors.

634

.04 .08 .12

FURUYA AND THOMAS-RED LIGHT EFFECTS ON KAEMPFEROL IN PEAS

meter had been previously calibrated for the wave-lenth band emitted by each light source by means ofan Eppley thermopile (1). The dose of red lightwas altered by changing the period of illumination(from 7 seconds to 2 minutes), or the distance fromthe source to the plants (15 to 40 cm).

The following quantitative method was developedfor determination of the levels of KGC and KG in atissue. Tissue (300-500 mg fr wt) was harvested,weighed, and placed in 10 ml of methanol in the darkroom. Extraction was carried out by refluxing thetissue 3 times for 15 minutes each with 10, 5, and5 ml of methanol in a flask held at 800 in a waterbath.The successive extracts were combined and thenevaporated to dryness under reduced pressure at 400.The residue was redissolved in 1 ml of distilled water,and 40 to 80 ,A samples of this solution were appliedby micro-pipette to disks (5 mm in diameter) ofWhatman No. 1 paper. One such paper disk wasinserted through 2 parallel slits 1.5 mm apart on thestarting line of each chromatogram (Whatman No. 1filter paper) to provide original spots of constant di-ameter. Two-dimensional development of the chro-matograms was carried out by the descendingmethod at about 240, the first solvent being n-butanol:acetic acid: water (4: 1: 2.2 by volume) for 12hours without presaturation of the tank, and the second5 % aqueous acetic acid for 3 hours.

The average RF values (± standard errors) ofKGC and KG were 0.47 ± 0.02 and 0.42 ± 0.02 inthe first solvent, and 0.45 ± 0.01 and 0.59 ± 0.03in the second, respectively. Spots were detected onthe chromatograms by exposure to ultraviolet lightand these were cut from the paper as disks of uniformarea (6 cm in diameter for KGC, and 5 cm for KG).KGC and KG were eluted from the disks by shakingfor 3 hours in 2 ml of 50 % aqueous methanol. Theabsorbance of the eluates was determined at 267.5 m,ufor KGC and at 266 m,i for KG. The percentagerecovery of these 2 compounds using this methodwas determined by applying various known amountsof purified materials to chromatograms (fig 2). Theaverage recovery of KGC from the paper was 69.8 %and that of KG 65.2 %. This percentage recoverywas not increased by continued elution for periods ofup to 2 days. The experiments described were de-signed, on the basis of preliminary experience, suchthat the expected range of concentrations of KGC andKG fell within the limits tested (fig 2). The absorb-ance observed was transformed to concentrations byusing the molecular extinction coefficient of 2.57 X104 at 267.5 m,u for KGC and that of 2.09 X 104for KG at 266 mju (9).

Besides harvesting up to 100 plumules in eachtreatment group for estimation of their KGC and KGcontent, in many experiments parallel studies of leafand stem growth were made on plumules collectedfrom the same containers. Measurements of youngplumular leaves and internodes were made using astereoscopic dissecting microscope. Between 25 and60 plumules were measured in each treatment group

depending on the variability of the seedlings. Wherefresh and dry weight determinations were made inconjunction with linear measurements, 6 groups of10 plumules each were weighed in each treatmentto facilitate statistical analysis.

Results

Distribution of KGC and KG in Totallv EtiolatedPea Seedlings. In previous work (13), apical budswere harvested by pulling them off the stem. Thismethod inevitably led to varying amounts of SO, stem(fig 3) remaining attached to them. The main aimof the present investigation being the quantitativedetermination of the concentration of KGC and KGin the apical region, it was important to know at theoutset whether or not concentrations varied fromone tissue to another.

An experiment was carried out to determine theconcentration of KGC and KG in the plumule andstem of 8-day-old totally etiolated pea seedlings.Three regions of tissue were harvested (fig 3 ):plumules (P), defined for the purposes of this in-vestigation as including all tissue at and above thelowest node bearing a foliage leaf; S, stem segments,5 mm long and excised 1 mm below the apical crook,comparable with those described by Purves and Hill-man (22); and SO stem segments, the young stemregion between the plumule and the S, stem segment.The fresh weight of each tissue sample and the

so

P{Lec

Noc3

S,

3

FIG. 3. Diagram of 6-day old etiolated seedling show-ing regions harvested (see text). Bract 2 is borne atnode 3 and leaf 1 at node 4.

635

PLANT PHYSIOLOGY

Table I. Distributiont of KGC antd KG in 8-Da)y Old,Totally Etiolated Seedlings

Avg Dose given to ConcentrationTissue fr wt each paper (umoles/g fr wt)region* (mg/ (mg fr wt

plant) equivalent) KGC KG

P 5.0 24.5 1.99 1.076.1 21.0 2.08 1.72

S<, 4.1 20.1 ** 2.194.5 14.7 ** 2.31

S1 12.2 66.0 ** 0.2610.9 61.2 ** 0.38

* See text for explanation.** No KGC was detectable in the

to the chromatograms.extract doses applied

amounts of KGC and KG were nmeasuredl in eachregion (table I).

Applying extracts to each paper chromatogram atthe range of doses shown in table I, KG was foundin all regions, but KGC could be detected only in theplumules. The concentration of KG was highest inSO and very much lower in S1. The total kaempferolconcentration was highest in the plumule (about 3 to4 ,umoles/g fr wt) and in this region the ester form,KGC, was found to be at a slightly higher concentra-tion than KG.

Another test showed that irradiation with lowintensity red light did not alter the distribution pat-tern of these flavonoid complexes (qualitatively,although hligh intensity white light is known to changethe overall flavonoid content by promoting the produc-tion of quercetin derivatives (10).

The finding that KGC occurred at detectablelevels in only the plumule led to the use of this regionexclusively in all further studies. Plumules were ex-cised with a scalpel imiimedliately below the fourthnode in all cases, and great care was exercised to

ensure that no steni tissues below this node were in-cluded in the samples.

Synthesis of KGC and KG in Relation to SeedlingAge. Selection of the ideal age at which to treatseellings with light was based on an investigation ofthe changes which occur in growvth rates and bio-synthesis of KGC an(d KG during normlal develop-ment in total (larkness. This investigation alsoassisted the interpretation of the effects of exposureto re(l and far-red light.

The amounts of KGC and KG in plumules oftotally etiolated seedllings were determined dailyfrom the 4th to the 8th (lay after sowing, and growth,in terms of fresh weight and leaf ancl interno(lelengths, was measured simultaneously (table II).Up to and including the 4th day after sowing, it wasdifficult to excise plumules at node 4 in dim greenlight with the result that plumules in the samples forweighing and extraction bore no(le 3 and bract 2 inaddition to the plumular material as (lefined pre-viously. A few plunmules of the 5-day-old seedlingsalso included bract 2, but those of olcder seedlingswere excised strictly at node 4.

During growth from the 4- to the 8-day-old stage,all tissues in the plunmule grew significantly and theratio of stem length to leaf length within the plunmuleincreased more than threefold froml 0.099 at (lay4 to 0.346 at day 8 (table II).

The increase in flavonoid content per plumuleshowed a similar time course to that of growth.The amounts of KGC and KG per plumule increase(din direct proportion to leaf developnment, and con-sequently the concentration of these compounds ap-peared to be rather independent of the seedling age(table II). However, although the concentration ofKG renmained fairly constant (between 1.21 and 1.34,umoles/g fresh weight, that of KGC decreased from3.25 to 2.10 Jmoles/g fresh weight with increasingage. This decrease in KGC concentration might beexplicable on the basis of the conconmitant increase

Table II. Plumnule Size anid Concentrationi of Kaempferol Cornplexres in Relationt to Age in Dark-Grozn Seedlinigs

Age(days) 4 5 6 7

Amount of KGC in plumule(,Amoles/plumule) 0.010 0.011 0.016 0.017 0.

Amount of KG in plumule(,umoles/plumule) 0.094 0.004 0.006 0.009 0.

KGC concentration(,umoles/g fr wt) 3.25 3.04 2.74 2.37 2

KG concentration(,umoles/g fr wt) 1.34 1.21 1.23 1.24 1

Leaf 1 lamina lengths (mm) 3.16 3.75 5.08 6.01 6(0.06) (0.10) (0.16) (0.16) (0

Internode 4 length (mm) 0.31 0.45 0.93 1.41 2(0.02)** (0.02) (0.07) (0.10) (0

I: L* 0.10 0.12 0.18 0.23 0Avg. plumule fr wt (mg) 3.07 3.57 5.65 7.28 8

* Ratio of internode 4 length to leaf 1 lamina length.** Standard error.

8

1.018

1.012

'.10

.33).45).26)'.23).20)).35.72

636


in the stem: leaf ratio in the plumule, as KGC isabsent from, or at a realtively low concentration in,the stem.

On the basis of these observations 6- to 7-day-oldseedlings were chosen for use in subsequent investi-gations. In younger seedlings it is difficult to harvestthe plumules and in older ones the plumules containrelatively large amounts of stem tissue.

Response to Low Intensity Red Light. An exper-

iment was set up to determine: whether low inten-sity red light does affect the synthesis of either KGCor KG in plumules of etiolated seedlings, andwhether seedlings show varying degrees of response

at different ages. Exactly the same dose of redlight (310 ergs cm-2 sec'1 for 2 min) was given tototally etiolated seedlings 4 to 9 days after sowing.These were then returned to darkness until theirplumules were harvested 16 hours after irradiation.The fresh weight of the plumules and the concentra-tion of KGC and KG in them were determined inboth nonirradiated controls and red light pretreatedseedlings of each age. The results obtained are pre-

sentel in table III.

The plants in this experiment were rather variablein size and the same difficulties of harvesting plu-mules from the younger seedlings were experiencedas mentioned earlier. However, the results clearlyshow that similar effects of red light occur in seedlingsof all ages tested. Thus low intensity red lightmarkedly increases the rate of plumule growth andthe concentration of KGC. The effect of red lighton KG concentration is less clearly marked.

An experiment of this type permits a comparison

of the concentrations of KGC and KG in plumuleswhich have received different treatments but whichare of approximately the same weight and dimensions.Reference to table III shows plumules in group 6Rto weigh approximately the same as those in group9D: 5.88 versus 5.55 mg fresh weight. Similarlythere is no significant difference between the KGconcentration in the 2 groups. In contrast, the dif-ference in KGC concentration between these 2 is verymarked: 3.87 ,umoles/g fresh weight in group 6Rcompared with 2.31 ,umoles/g fresh weight in group9D. Thus red light irradiation clearlv not only in-duces an increased growth rate in plumular tissues

but also increases the concentration of KGC to alevel well above that normally occurring in plumulesof a comparable size. That this situation prevailsgenerally in response to red light is shown by figure4 in which the results of all suitable experiments inthe present investigation have been plotted on a singlegraph to show the relationship between KGC andKG concentrations and plumule size. While thereis no doubt that red irradiation increases KGC con-centration almost twofold, any effect there mightbe on KG concentration is relatively slight.

Having shown that plumule growth and KGC con-centration are both increased by low intensity redlight, 3 experiments were set up in an attempt todetermine the relationship between them. Thesewere designed to ascertain the time and rate at whichthe changes occur, and, in addition, whether they arepermanent or short-lived. In each experiment, 6-day-old totally etiolated seedlings were exposed to

4

U:

0

. 2E

I

0

x

-I

00 000o

00 a0

0 0

o0 0

0

00

32 44 5 6 7 a 9 10 I IAVERAGE FRESH WEIGHT(mg/plumule)

4.

310

0 o0 0

2 * .0 ° 0 0

00 0

I 0

0A.a a

2 3 4 5 6 7 a 9 10 1

AVERAGE FRESH WEIGHT (mV plumule)

FIG. 4. Relationship between plumule fresh weightand KGC and KG concentration per plumule. 0, dark-grown; 0, harvested 16 hours after red irradiation.

Table III. Effect of Red Light on Grozewth and Biosynthesis of KGC and KG in Etiolated Pea Plt,nitles of Various Ages

Age Avg fr wt Red light Conc of kaempferol complexes increment of(age Avg fr wt induced (ALmoles/g fr wt) iceeto(adftys Treatment per plumule increment concentration (%)

sowing) (mg) of fr wt (%) KGC KG KGC KG

4 D* 3.00 2.79 1.78 ...R* 5.04 67 3.55 1.99 27 12

6 D 2.63 ... 2.52 1.40 ...R 5.88 123 3.87 1.43 53 2

9 D 5.55 ... 2.31 1.56 ... ...R 9.00 62 4.06 2.13 76 44

* D, totally dark-grown control; R, irradiated for 2 minutes with red light (310 ergs cm2 sec-1) 16 hours prior toharvest.

637

-A-

PLANT PHYSIOLOGY

red light at an intensity oi 33 ergs cm-2 sec-1 for 90seconds then kept in the dark until the plumules wereharvested. The times of harvest differed in each ex-periment but several harvest times were commnon to2 or 3 experiments. In the 3 experimiients conmbined1,measurements of growth and the concentration ofKGC and KG were ma(le immecliately before and4, 8, 16, 24, 32, 48, an(d 64 hours after red lightirradiation (figs 5-7). Changes in dry weight andthe ratio of internode length to leaf length (I: L)were deternmined in 2 experiments (table IV). Al-though there was a marke(d increase in plumule freshweight following irra(liation, the percentage dryweight of each plunmule remained fairly constant dur-ing the first 32 hours. The dry weight changedin direct proportion to the fresh weight, and cal-culations based on one variable are directly compar-able with those base(d on the other. As notice(I ear-lier, the increase in plumule size le(d to an increasedratio of I: L.

As in the previous experiments, exposure to redlight brought about a mlarked increase in growthrate and KGC concentration. The increasecl growthrate was maintained fronm 4 to 24 hours after irradia-tion and then reverted to normal. KGC concentra-tion showe(d a similar pattern of response, reachinga peak after 16 to 24 hours and( tllen gra(lually tle-creasing again. This (lecrease is explicable in termsof the relative growth of stem an(d leaf tissues withinthe plumule. During 64 hours of (levelopment, theratio I: L rose from 0.14 to 0.23 in the dlark con-trols an(d from 0.14 to 0.28 following irradiation(table IV).

The results obtainedl for KG were very variable.The apparent peak in concentration 16 hours afterirradiation cannot be consi(leredl significant and itseenms likely that treatnment once again ha(l littleeffect on KG concentration.

The relationship between the amlount of recl lightenergy given to the plant surface ancl the plant re-sponse, in terms of photoinduced growth an(d bio-synthesis of KGC ancl KG, was investigate clquant-itatively in an attempt to deternline the mlinimumeffective close and the energy level require(d forsaturation.

Total energy levels of irra(liation ranging from0.3 to 1200 kiloergs CmI-2 were provi(led by exposingseedllings to 3 dlifferent light intensities, i.e. 14, 33.

0

6

E

I-

3:0i;j

I/,Lin

crLL

Li-J

ci.

640 4 8 16 24 32 48TIME AFTER IRRADIATION (HOURS)

FIG. 5 to 7. Growth and KGC and KG concenitrationim plumules of 6-day old dark-growni seedlinigs at varioustimes following red irradiation. In figure 7, vertical barsrepresenit + twice the standard error. 0, (lark-growl)controls; 0, red light treated.

Table IV. Effect of Red Light Irradiationt ont Pltum tule Growth duirinig the Suibseqtuentt 64 Houirs

Time after irradiation (hours)0

%drywt D*of plumule R

I: L** DR

4 817.2~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

... ... 1..217.7 17.3 17.4

... ... ~~~0.14

16 24

i... 17.817.0 17.0... 0.16... 0.20

* D, dark-grown control; R, red light treated.** I: L, ratio of internode 4 length to leaf 1 lamina lengtlh.

638

0 4 8 16 24 32 48TIME AFTER IRRADIATION (HOURS)

19.019.00.140.14

32 48 64

. . .

. ..

.. .

0.20

. . .

..

0.230.28

v

4.0 1

3.5 -

3.0ED

2.54

2.0 -

1.5

n I -

64


4.0

a.NU0

to.5

3.5

30

2.5

2.0

0

3.0

&:2.5a,

0S 2.0

co

0

E:k 1.5

0

O0 0.1 1.0 10 100 1000TOTAL IRRADIATION ENERGY

(KILOERGS x cm-2)

0.1 1.0 10 100 1000TOTAL IRRADIATION ENERGY

(KILOERGS x cm-2)FIG. 8 and 9. Effect of red irradiation energy level on

KGC and KG concentrations in the plumules of 6-day oldetiolated seedlings 16 hours later.

and 640 ergs cnm2 sec-1, at a distance of about 40 cmbelow the lamps for periods ranging from 7 secondsto 30 minutes.

Dark-grown seedlings were exposed to red light6 *days after sowing and the plants so treated re-turned to darkness immediately. Plumules were har-vested for extraction and measurement immediatelybefore and 16 hours after red light irradiation. Thegrowth response to treatment and the concentrationof KGC and KG in the plumules 16 hours afterirradiation are shown plotted against light energy infigures 8 to 10.

All levels of red light energy to which plants wereexposed were effective in stimulating growth andincreasing KGC concentration but no irradiationtreatment affected KG concentration. The influenceof irradiation increased with increasing energy up tothe poinlt at which saturation was reached (3 to 10kiloergs cM-2 with respect to plumule growth and

about 100 kiloergs cm-2 with respect to KGC concen-tration). The significance of the difference betweensaturating doses is discussed later. Knowing theamount of KGC synthesized in a plumule during 16hours of darkness without prior red light irradiation,the increased synthesis of KGC resulting from photo-induction can be calculated by subtracting the amountsynthesized in the dark control from that synthesizedafter red light irradiation. Thus, the photoinducedsynthesis of KGC per plumule was 0.005 1imole/plu-mule following 100 ergs cm-2 of red light, 0.01 ,umolefollowing 1 kiloerg cm-2, and 0.015 ,umole following10 kiloergs cm-2. This rate of photoinduced synthesisof KGC is approximately proportional to the logarithmof total energy of red light received by the plantsurface.

Reversibility of Red Light Effects by Far-Red.Six-day-old totally etiolated seedlings were dividedinto 4 groups to receive the following treatments:A) continuous darkness, B) 2 minutes of red light,C) 2 minutes of far-red light, and D) 2 minutes ofred light as in group 2 followed immediately by 2minutes or 30 seconds of far-red light.

The total energy at plant level was 360 ergs cm-2sec-1 from the red light source, and about 20 kilo-ergs cm-2 sec-1 from the far-red source. Duringhandling of the seedlings immediately prior and sub-sequent to treatment, they received a total of about5 minutes of dim green light. Dark controls wereexposed to green light similarly. Samples of plu-mules were harvested from each group 16 hoursafter treatment for determinations of weight andkaempferol content. The effect of treatment on plu-mule fresh weight and the concentration of kaempferol

U

-j.1Z

OL..

a.).zEJ

I I5 - 0o 05 so

4-

3 G

2<

0.1 1.0 10 100 1000TOTAL IRRADIATION ENERGY

(KILOERGSx cmr2)

FIG. 10. Effect of red irradiation dose on the increasein plumule weight during a subsequent period of 16 hoursof darkness. In figures 8 to 10, * and 0 represent theresults of 2 separate experiments in which red lightintensities used were 33 and 640 ergs cm-2 sec-1 respec-tively.

639

Table V. Rev'ersibility of Red Light Iniduiced Effects on Growe,th and KGC anzd KG Conicenitration by Far-Red LightMeasurements were made 16 hours after irradiation.

Increment in Conicentration1xneriment Tre2tment r)iiimiile fr wt (uoe/gf t

D**RFRR/FRD**RFRR/FR

V.UlllUlt 11 W L

(mg)

1.552.071.451.60

1.153.571.252.33

KGC KG

1.772.251.891.61

2.393.282.492.84

1.751.961.451.40

1.982.322.001.91

* A, red irradiation for 2 minutes, far red for 2 minutes.B, red for 2 minutes, far red for 30 seconds.

** D, dark-grown; R, red light treated; FR, far-red lightby far-red.

complexes is shown in table V. As found previously,red light stimulated growth and induced an increasein KGC concentration whereas the effect on KG con-

centration was relatively slight. Far-red light alonehad no apparent influence on growth or the concen-

tration of either kaempferol complex, but 2 minutesof far-red light completely reversed the effect of redlight on growth and KGC concentration. Thirtysecon(ls of far-red light proved sufficient to bringabout only approximately 50 % reversal of theeffect of red light. It is apparent that both KGCsynthesis and plumule growth are affectecl by a phyto-chronme mediated reaction.

Discussion

Phytochrome has been extracted and purifiecd fromtotally etiolated Alaska pea seedlings (4). Whentissues of such plants are irradiated with red or

far-red light, phytochrome is immediately convertedfrom a red light absorbing form (P1t) to a far-redabsorbing one (PFR) and vice versa (6). The pres-

ent results show clearly that recl light stimulates bothleaf growth and KGC biosynthesis, but there is a

lag of about 4 hours between irradiation and detect-able response. It is thus probable that an increasein the level of PFR does not affect KGC synthesisand growth dlirectly, and it seems more likely thatPFIt affects intermedliary reactions whlich in turn con-

trol these stimulated processes.Long lag periods between stimulation and response

in relation to red/far-red light irradiation are com-

mon. The length of lag periods has been variouslyreported to range from about 30 minutes to severalhours (5, 7, 14, 15, 16). In etiolated peas a periodof at least 4 hours was required before significantrises in the rate of leaf growth and the concentra-tion of KGC were detectable.

The final response to red light has been foundto be linearly dependent upon the logarithm of theincident energy in at least 3 instances. Parker et al.

treated; R/FR, red light irradiation immediately folloved

(20) found this to be so with regard to the growthof pea leaves, and Klein et al. ( 15) in the case ofleaf weight andl anthocyanin synthesis in bean seel-lings. A similar linear-logarithnlic relationship hasnow been found in relation to KGC biosynthesis inpea plumlules ancl confirmed with regardl to leafgrowth.

The estimatedl quantum efficiency of KGC syn-

thesis in peas varies mlarke(dly with the incidlentenergy level of red light irradliation. In the plu-mule an average of 0.005 tmnole of KGC was foundto be produced in 16 hours following irradiation with100 ergs cm-2 red light. Assuming the area of theplumule to be 0.02 cmn, the incident energy of red

light at the plumule surface wlas 2 ergs. Since theenergy of a single quantum at 660 mtu is known tobe 3.01 X 10-12 ergs, each plumule received 6.6 X10ll quanta. The number of molecules of KGC syn-

thesizedl in 16 hours in response to red light was

calculated to be 5 X 10-9 X 6.024 X 1023: thatis 3.01 X 1015 molecules. From these calculations,cquantum yield was estimiiate(d to be 3.01 X 1015KGC molecules/6.6 X 1011 (juanta, i.e. about 4600molecules/quantum. By similar calculation, thequantum efficiency was estimated as 910 at a redlight dose of 1 kiloerg cm-2, andl 15 at 10 kiloergscm-2. The decrease in quantum yield with increaseddose is thus consistent with a first order reactionrate in the conversion of the plhotoreceptor to theactive state (B. A. Bonner, personal communication).The high quantum yield supports the previous sug-

gestion that the red light energy absorbed by phyto-chrome does not act (irectly on growth and KGCbiosynthesis. It seems nmore likely that the energyabsorbed acts as a trigger to change the rate andpattern of metabolism.

The complete conversion of PR to PFIR by redlight is not necessarily required to obtain 100 %saturation of photophysiological responses such as

growth and KGC biosynthesis. This is shown clearlyby conmparison of the results of direct observations

F

640 PLANT PHYSIOLOGY

ILIIVIILt11 I I q-atLIIK,lt Lt


of PR to PFR with the results of investigations involv-ing measurements of growth and KGC biosynthesis.

Estimates of the energy required to bring about50 % conversion of PR to PFR in etiolated pea seed-lings have yielded results of 16 kiloergs cm-2 in vivo(11) and 110 kiolergs cm-2 in vitro (B. A. Bonner,personal communication). Studies of phytochrome-mediated physiological responses, however, show sat-uration to be reached well below these levels insome cases. Thus in the present investigation 50 %saturation of photoinduced KGC synthesis occurredat about 2 kiloergs cm-2. Similarly, Bertsch (2),using a single exposure to red light comparable withours showed 60 kiloergs cm-2 to saturate completelythe photoinhibition of etiolated pea stem sectiongrowth. Using a rather different experimental pro-cedure which involved exposing plants to 4 minutesof a much purer red light source than ours daily for4 days, Parker et al. (20) found no sign of satura-tion of the leaf growth response of etiolated peaseedlings up to energy levels of at least 30 kiloergscm-2. Differences between the results of differentinvestigations might be ascribable to differences inexperimental technique, but the conditions underwhich the experimental material is grown might wellbe equally if not more important.

The nature of the relationship between leaf growthand KGC biosynthesis remains uncertain, but thereare 3 main possibilities: firstly, that the increasedsynthesis of KGC is simply dependent on the in-creased growth rate of the plumule; secondly, thatthe increased growth rate is casually dependent onthe increased synthesis of KGC; thirdly, that growthand KGC biosynthesis are not interdependent butthat both are equally affected by a common photo-induced precursor. In the first case KGC concentra-tion would be expected to remain constant, or evento drop initially as the growth rate gradually in-creased, but the opposite is true, and, in addition,saturation of the response to red light is reached atlower energy levels with respect to growth than toKGC synthesis. In the second case the concentrationof KGC would be expected to increase before theinitial growth changes are detectable, but in fact thelag period between irradiation and detectable re-sponse was approximately the same in both instances.The third possibility seems much more feasible:red light might stimulate many processes in the cell,2 of which are growth and KGC biosynthesis.

The present investigation cannot be consideredto clarify the relationship between flavonoid concen-tration and plant morphogenesis. It has provided nofoundation for assuming that there might be a casualrelationship between them. Although the same closerelationship between leaf growth and flavonoid bio-synthesis has been reported in other systems (15),such a relationship cannot be considered general.Thimann (unpublished data) has shown that anthocy-anin synthesis in corn leaf disks and Spirodela cul-tures can be enhanced by light in the absence ofconcomitant growth responses.

The effect of light on flavonoid biosynthesis inpeas differs somewhat from the previously reportedeffects in other plants. Siegelman and Hendricks(23) suggested that 2 different radiation-limited stepswere involved in anthocyanin synthesis in apple skin,and Hendricks (12) further reported that anthocya-nin synthesis in seedlings of Sorghum vulgare re-quires 2 photoreactions. These are an initial reactionin response to high energy irradiation, and a secondreaction involving low intensity red irradiation whichis reversible by far-red. In etiolated peas, however, abrief exposure to low intensity red irradiation is suf-ficient to increase the concentration of KGC. Theapparent lack of effect on KG concentration is in-triguing, but interpretation of this difference mustawait further studies of the biosynthetic pathways ofthe 2 compounds. It is of interest that the synthesisof quercetin apparently does require high intensitylight in peas. Quercetin has never been detected inetiolated seedlings but it is known to be the majorflavonoid in the leaves of pea plants grown underhigh intensity white light (9). Exposure of etio-lated seedlings to low intensity red or far-red lightdoes not lead to quercetin synthesis. The obviousconclusion, that quercetin biosynthesis in pea leavesrequires high intensity light, is contrary to the find-ing (24) that light was not essential for the syn-thesis of quercetin from acetate in buckwheat seed-lings. It is not surprising, though, that different fac-tors should be limiting flavonoid synthesis in differentplants, especially when the differences between flavo-noid patterns in different tissues of the same plant(9) are borne in mind.

Summary

Etiolated pea seedlings contain the flavonoid com-plexes kaempferol-3-triglucoside (KG) and kaemp-ferol-3- (triglucosyl-p-coumarate) (KGC). The ef-fect of low intensity red and far-red light on the bio-synthesis of these compounds in such seedlings wasinvestigated in relation to growth.

Seedlings of Pisum sativum cv. Alaska were grownin total darkness and their plumules harvested at vari-ous times after sowing. Some plumules were har-vested for measurement of their leaves and internodes.KGC and KG were extracted from other weighedsamples in methanol and quantitative estimations oftheir concentrations were made spectrophotometri-cally following elution from two-way paper chro-matograms.KG was found throughout the plumule and stem,

but KGC was detectable only in the plumules.Neither was apparently present in totally dark-grownroot tips.

When 6-day-old seedlings were irradiated withvarious doses of red light, a lag period of about 4hours preceded the onset of any detectable response.At the end of this time the rate of leaf growth andconcentration of KGC began to increase markedly to

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a peak after about 16 to 24 hours. KG concentrationwas apparently unaffectedl.

The effect of red light on KGC concentration andplumule growth was reversed by far-red irra(liation.

Growth and KGC biosynthesis were affecte(d simi-larly by the phytochrome systeml, although 50 %saturation of response occurre(c at 0.2 kiloergs cm-'for growth and 2.0 kiloergs cm-2 for KGC synthesis.It is concluded that recl light stimulates plumulegrowth and biosynthesis of kaempferol complexesindependently and no evi(lence was obtaine(d thatflavonoid complexes play any role in nmorphogenesis.

Acknowledgments

This work was supported by the National ScienceFoundation under a grant to Professor A. W. Galston inwhose laboratories this work was carried out. We aregrateful to Professor Galston for his continued interestand encouragement during this investigation. We thankProfessor K. V. Thimani, Dr. B. A. Bonner, anid Dr.M. B. Wilkins for their helpful discussion during thepreparation of the manuscript.

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