(Fabaceae)' - Plant Physiology

Plant Physiol.(1987) 84, 318-3230032-0889/87/84/0318/06/$01 .00/0

Photonastic Control of Leaflet Orientation in Melilotus indicus(Fabaceae)'

Received for publication July 15, 1986 and in revised form January 27, 1987

AMNON SCHWARTZ*, SARAH GILBOA, AND DoV KOLLERThe Hebrew University ofJerusalem, Department ofAgricultural Botany, Rehovot 76100, Israel

ABSTRACI

Leaflet orientation in Melilot,,s indicus (L.) All. Is under photonasticcontrol during the day and nyctinastic control during the night, but alsoexhibits a diaphototropic (solar-tracking) response. Detached leaves withthe two lateral leaflets excised were used to study the solar-trackingcapability of the terminal leaflet. Perception ofthe photonastic excitationis located in the pulvinule. The lower (abaxial) and upper (adaxial)surfaces perceive photonastic excitation, which results in concomitantcontraction of the side exposed to light and/or expansion of the oppositeside. Steady state laminar elevation is determined by the fluence rates ofthe light incident simultaneously on the opposite sides. Light sensitivityof the lower side exceeds that of the upper. Response to photonasticexcitation of either side is affected by angle of incidence of the light, butangular dependence is restricted to a limited range of angle of incidence,which differs for the two sides. This may be accounted for by the differenttopography of the two pulvinar surfaces and the localization in them ofthe light-sensitive tissues.

Nyctinastic leaves are characterized by the folding movementof their leat(let) lamina(s) upon transfer from light to darkness.These leaves unfold into a more or less co-planar configurationupon transfer from darkness to light. Most, if not all of theseleaves also exhibit autonomous movements that take place withcircadian rhythmicity. The physiology of these leaves has beenunder intensive study during the past three decades, especiallyby R Satter, AW Galston, and co-workers, and the results havebeen recently reviewed (16, 17). The movement is brought aboutby bending of the pulvinus (at the base of the petiole, lamina,leaflet, pinna, pinnule) caused by differential, turgor-driven ex-pansion/contraction in opposite sectors of motor tissue of thepulvinus. Phytochrome is involved in the control of the nycti-nastic closing response, which is promoted by Pfr and delayedby Pr. On the other hand, the action spectrum for the light-driven opening response exhibits a major peak in the blue spectralregion, between 400 and 480 nm, generally a minor peak around720 nm, according to the species, and certain characteristics ofthe high irradiance response system, that is also attributed tophytochrome. However, it is virtually certain that the blue-absorbing 'cryptochrome' plays the dominant role in the photo-nastic opening response ( 1, 4, 5, 7-9, 16, 20, 23, 26). The adaptivevalue of the nyctinastic response is not clear (16). On the otherhand, the capacity for solar tracking (diaphototropism) maxi-mizes the interception ofphotosynthetically active radiation, and

'This study was supported by a grant to D. K. and A. S. from theSchonbrunn Foundation, through the Research Committee of the He-brew University.

thus has self-evident adaptive value (2, 6, 29). A large numberof nycti- (photo-) nastic leaves were also reported to exhibit amarked capability for solar tracking, by continuously reorientingthe laminar surfaces approximately normal to the sun throughoutmost of each day, with different degrees of fidelity (2, 10, 11,19-24, 28).The light-driven reorientation was studied with leaves of Mel-

ilotus indicus, in view of the detailed observations made on thesolar tracking exhibited by its close relative, M. alba (28).

MATERIAILS AND METHODS

Seeds of Melilotus indicus (L.) All. were collected on thegrounds of the Faculty of Agriculture, and sown in a mixconsisting of equal portions (v/v) of basalt gravel, vermiculite,and peat moss. The plants were grown in the Phytotron of theFaculty of Agriculture (12), at a day temperature of 22°C (0800-1600) and a night temperature of 17°C (1600-0800) and an 8-hphotoperiod ofnatural light. When the plants were about 5 weeksold, they were used to provide the experimental (excised) leaves.Only the four youngest fully expanded leaves were used. Thesewere excised as close as possible to their bases, using a new, sharprazor blade. Immediately upon excision, the petioles were im-mersed in tap water in 25 ml individual scintillation vials andfastened to the rim of the vial at an angle of elevation of 50' to60' (from the horizontal) by means of plasticine. The vials werethen placed in diffuse natural light in the laboratory and theleaves allowed to recover during 24 to 36 h, before the experi-ments. The two lateral leaflets were then excised, leaving onlythe terminal one. The experiments were conducted in a darkroom at 22 ± 1'C. They were all started at approximately thesame time of day, to avoid circadian complications.Two light sources were used. A 15W Daylight fluorescent tube

(Sylvania, GTE), provided diffuse light at a fluence rate of 10 to20 Mmol m2 s' (400-700 nm) at leaf level. The other was amicroscope illuminator (Nikon, Kogaku, Japan), equipped witha 30 W incandescent filament lamp and a lens system, by meansof which a light spot (100 umol m-2 S-l, 400-700 nm) could bedirected at the leaflet at desired angles. Fluence rate was measuredwith a Quantum-Radiometer-Photometer (model 1 85B, Licor,Lincoln, NE), with the sensor normal to the light source. Redlight (Xmax= 654 nm; 30 ,gmol m-2 s-') was obtained by meansof interference filters (Balzers, Liechtenstein, 10 nm half-maxi-mal bandwidth). Blue light (Xm.. = 470 nm; 30 Mmol m-2 S- )was obtained by filtering through blue Plexiglas (No. 2424, Rohmand Haas, Hayward, CA, 100 nm half-maximal bandwidth). Thespotlight was directed onto the entire leaflet in the plane ofsymmetry of its midvein. A12 ofthe beam is stated with reference

2Abbreviations: Al, angle of incidence (degrees) of light-beam onleaflet ; LE, laminar elevation (degrees); both with reference to thehorizontal, with positive values above and negative ones below horizon[Al = O when beam is directed toward base of leaflet, 180' when directedtowards its tip]); w, angular velocity of laminar reorientation in verticalplane.

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FRUCTAN METABOLISM IN BARLEY

Table III. Fructan Content and SSTActivity in Excised Primary LeafBlades ofH. vulgare

The leaf blades were standing in 0.5 M solutions of different sugars for16 h in the dark.

Sugar SST Activity Fructan Contentnkat/ml mg/gfresh weight

None 0.08 ± 0.01 0.01Sucrose 0.64 ± 0.17 1.48Maltose 0.99 ± 0.19 2.07Melezitose 0.87 ± 0.19 -'Maltotriose 0.50 ± 0.07 0.38Trehalose 0.48 ± 0.08 0.01Fructose 0.47 ± 0.21 1.08Raffinose 0.42 ± 0.07 a

Cellobiose 0.33 ± 0.08 0.43Lactose 0.31 ± 0.07 0.13Lactulose 0.23 ± 0.10 a

Glucose 0.22 ± 0.15 0.31Melibiose 0.11 ± 0.05 0.11

Sucrose + 5 gg/ml CHI 0.08 ± 0.01 0.01a Determination of fructan content impossible because of interference

with the sugar tested.

the activity of SST, a possible key enzyme of fructan synthesis.Accumulation of fructan was found to be strictly preceded by anincrease in the activity of SST (Fig. 3). If this increase wasinhibited by CHI, no fructan was synthesized (Table I). Thisfinding is in agreement with previous reports showing that inhi-bition of cytoplasmatic protein synthesis also inhibits the accu-mulation of fructan in certain Asteraceae (4, 14).

Besides the induction of SST activity a surplus of photosyn-thates, most probably of sucrose, is necessary to allow accumu-lation of fructan in the leaves (Table II). Photosynthates are notonly the substrates for fructan synthesis but they appear to bealso effectors for the induction of SST activity as the experimentwith exogenously supplied sucrose demonstrates. Curiously, notonly sucrose but also a number of other sugars induce SSTactivity in the leaves, whereby, as the result with trehalose shows,it is not necessary that the sugar can actually be transformed tofructan (Table III). It will be an interesting task to study themechanism of SST induction by these sugars.Another question concerns the control of fructan degradation.

The activity of fructan hydrolase changed only little and slowlyupon transfer of the leaves to the dark which induced thedegradation of fructan (Fig. 2). A further difficulty in the under-standing of the control of fructan hydrolysis emerges from therecent finding that the hydrolase is located in the vacuoles (19),i.e. the same subcellular compartment in which fructan synthesisand accumulation takes place (18). The question arises, therefore,whether the depletion of fructan in the dark is initiated by adecrease of the rate of synthesis only, caused by inactivation ofSST, in the presence of an unchanged rate of degradation. Thispossibility could be tested by measuring turnover rates offructan.

In any case, it will be interesting to see whether or how the cellsprevent a futile cycle of continuous fructan synthesis and degra-dation. It may be asked also how SST, a vacuolar enzyme shownto be quite stable in vitro (unpublished results), can suddenlylose its activity so rapidly. It is feasible that SST is unstable inthe absence of its substrate, sucrose, and is consequently digestedby the vacuolar proteinases (6). Another question concerns thepossible regulatory role of oligosaccharide synthetic activity (8).A clear answer to this question must probably await successfulpurification and characterization of all the enzymes involved infructan metabolism. These and many other fundamental prob-lems of fructan metabolism in cereals are unsolved. This issurprising since the vacuolar fructan pool plays such a prominentrole on the partitioning of photosynthates in cereals.

LITERATURE CITED

1. ARCHBOLD KH 1940 Fructosans in the monocotyledones. A review. NewPhytol 39: 185-219

2. BORLAND AM, JF FARRAR 1985 Diel patterns of carbohydrate metabolism inleaf blades and leaf sheaths of Poa annua L. and Poa jemtlandica (Almq.)Richt. New Phytol 100: 519-531

3. BLACKLOW WM, B DARBYSHIRE, P PHELOUNG 1984 Fructans polymerised anddepolymerised in the internodes of winter wheat as grain filling progressed.Plant Sci Lett 36: 213-218

4. CHANDOKAR KR, FW COLLINS 1972 De novo synthesis of fructooligosaccha-rides in leaf disks of certain Asteraceae. Can J Bot 50: 295-303

5. FREHNER M, F KELLER, A WIEMKEN 1984 Localization of fructan metabolismin the vacuoles isolated from protoplasts of Jerusalem artichoke tubers(Helianthus tuberosus L.). J Plant Physiol 116: 197-208

6. HECK U, E MARTINOIA, P MATILE 1981 Subcellular localization of acidproteinase in barley mesophyll protoplasts. Planta 151: 198-200

7. HEGNAUER R 1962-1973 Chemotaxonomie der Pflanzen, Vol 1-6. BirkhauserVerlag, Basel

8. HOUSLEY TL, CJ POLLOCK 1985 Photosynthesis and carbohydrate metabolismin detached leaves of Lolium temulentum L. New Phytol 99: 499-507

9. KAISER G, E MARTINOIA, A WIEMKEN 1982 Rapid appearance of photosyn-thetic products in the vacuoles isolated from barley mesophyli protoplastsby a new fast method. Z Pflanzenphysiol 107: 103-113

10. KUHBAUCHW 1978 Die Nichtstrukturkohlehydrate in Grasern desgemassigtenKlimabereiches, ihre Variationsm6glichkeiten und mikrobielle Verwertung.Landwirtsch Forsch 31: 251-268

11. MEIER H, JSG REID 1982 Reserve polysaccharides other than starch in higherplants. In FA Loewus, W Tanner, eds, Plant Carbohydrates, Encyclopediaof Plant Physiol, New Series, Vol 13A. Springer-Verlag, Heidelberg, pp 418-471

12. POLLOCK CJ 1979 Pathway of fructosan synthesis in leaf bases of Dactylisglomerata. Phytochemistry 18: 777-779

13. POLLOCK CJ 1984 Sucrose accumulation and the initiation of fructan biosyn-thesis in Lolium temulentum L. New Phytol 96: 527-534

14. PoTIms HG 1966 Observations on the de novo synthesis of fructosans in vivo.Arch Biochem Biophys 116: 416-424

15. PONTIS HG, E DELCAMPILLO 1985 Fructans. In PM Dey, RA Dixon, eds,Biochemistry of Storage Carbohydrates in Green Plants. Academic Press,New York, pp 205-227

16. SICHER RC, DF KREMER, WG HARRIS 1984 Diurnal carbohydrate metabolismof barley primary leaves. Plant Physiol 76: 165-169

17. SMITH D 1972 Carbohydrate reserves in grasses. In VB Younger, CM McKell,eds, The Biology and Utilization ofGrasses. Physiological Ecology. AcademicPress, New York, pp 318-333

18. WAGNER W, F KELLER, A WIEMKEN 1983 Fructan metabolism in cereals:induction in leaves and compartmentation in protoplasts and vacuoles. ZPflanzenphysiol 112: 359-372

19. WAGNER W, A WIEMKEN 1986 Propertiesand subcellular localizaton offructanhydrolase in the leaves of barley (Hordeum vulgare L. cv Gerbel). J PlantPhysiol. In press

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Plant Physiol. (1986) 81, 448-4530032-0889/86/8 1/0448/06/$0 1.00/0

Heat Inactivation of Starch Synthase in Wheat EndospermTissue

Received for publication September 30, 1985 and in revised form December 17, 1985

ANTONIUS H. G. C. RIJVENDivision ofPlant Industry, Commonwealth Scientific and Industrial Research Organization, P.O. Box1600, Canberra A.C. T. Australia

ABSTRACI

The effect of temperature on accumulation of starch was studied ingrain slices of wheat (Titicum aestivum cv SUN9E), taken 15 days afteranthesis. As compared with pretreatment of such slices at 25°C, pretreat-ment at 30 or 35C reduced the subsequent conversion of sucrose tostarch. In contrast to rice (Oryza sativa cv Calrose), pretreatment ofwheat soluble starch synthase in vitro at 30°C or higher temperaturesreduced its activity. In zymograms using nondenaturing polyacrylamidegel electrophoresis followed by activity staining, the slowest miratingband represented the most temperature sensitive isozyme. Althoughpreincubation of a soluble enzyme sample in vitro at 25°C did not resultin loss of starch synthase activity, it did result in a gradual shift ofzymogram banding pattern toward faster migrating species. Pretreatmentof isolated starch granules at 40°C increased their bound starch synthaseactivity. Both soluble and bound enzymes in the grains of whole wheatplants lost activity when the plants were held above 30°C for 30 minutesor longer. Both activities lost from the grains after a 1 hour treatment at37°C were restored in 1 to 2 days by a return to 21°C. In slices,inactivation of the soluble starch synthase was increased by incubationwith 2,4-dinitrophenol. It is tentatively suggested that in vivo heatinactivation of soluble starch synthase may be a direct effect of heat onthe enzyme protein and that of bound enzyme an indirect effect involvingmetabolic factors.

In wheat the optimum temperature, of plant culture for dryweight per grain at maturity occurs at a day/night temperatureof 15°C/10°C (4). Increasing the temperature of the ear inde-pendent of the rest of the plant from 15 to 20°C and 25C hasresulted in an increased growth rate of the ear but this wasaccompanied by earlier senescence and a shorter duration ofgrowth (6). Ear warming at 33°C/25°C (day/night) in comparisonwith 2 1C/16°C, resulted in a reduction ofgrain growth rate afterthe 6 d warming period (1). From these data, it would be expectedthat at a suitably high temperature such a reduction in rate couldbe observed earlier and analyzed more directly. Also it has beensuggested that for maize kernels grown in vitro, the adverse effectsof high temperature on starch synthesis during the grain-fillperiod were due to deficiencies in the synthetic process ratherthan in the supply of substrate (8). In the following experimentsgrain slices, incubated in sugar solutions, have been used toinvestigate the temperature sensitivity of starch accumulation indeveloping wheat grains. Further, since starch synthase is thefinal specific link in the conversion of sugar to starch, effects oftemperature on its stability in vivo and in vitro have also beeninvestigated.

MATERIALS AND METHODSThe present procedures are based on those described elsewhere

(8, 9).Plant Material. Plants of Triticum aestivum cv SUN9E (and

of Oryza sativa cv Calrose) were grown at 21C/16°C and 27C/22C, respectively (8 h day/16 h night) in natural daylight in theCSIRO Phytotron (CERES) in Canberra. Ears of wheat weretaken approximately 15 d after anthesis to provide grains in the40 to 60 mg fresh weight range and ears of rice at a green stagewith grains ofapproximately 18 mg fresh weight. In some exper-iments, plants were transferred to temperature controlled growthcabinets, lit artificially and continuously at 550 itmol m-2 s-1.

Preparation and Processing of Wheat Endosperm Slices. Theprocedure was as described in Rijven and Gifford (14). The 0.9mm slices, 10 per replicate, were incubated in scintillation vialswith 500 ul of fresh aqueous medium including [U-14C]sucrose,in a water-bath shaker and thereafter extracted exhaustively (14).

Estimation of Starch Production. Starch was estimated essen-tially as described before (14), incubating each replicate with0.25 mg porcine pancreatic a-amylase and with 1 mg Rhizopusniveus glucoamylase (7) (instead of ,@-amylase) for 0.5 h at 40°Cand 2.5 h at 50°C in an acetate buffer at pH 4.8. The 70%ethanol-soluble fraction formed was deemed to represent starch.As a control, label in starch granules, incorporated in a starchsynthase reaction as described below, was 96 to 100% solubilizedby the above incubation. Label in the insoluble residue wassolubilized by acid hydrolysis (14).

Starch Granule Isolation and Enzyme Preparation. The grainswere cut across at the basal end to remove the embryo. Thestarchy endosperm, uncontaminated by other tissue, wassqueezed out by pressure exerted judiciously by the tip of theindex finger and collected in PEG-1000 buffer (pH 7.4) thatconsisted of 300 mm (30%, w/v) polyethyleneglycol mol wt 1000(PEG-1000), 25 mm Tes-KOH, pH 7.4,50 mM KCl, 1 mm DTT.Starch granules were freed from the endosperm cells by choppingin an ice-cooled container for 1 to 1.5 min with a modifiedelectric knife. The brei was filtered on ice through one layer ofMiracloth with enough PEG-1000 buffer to give 5 endospermequivalents ofgranules per ml. Triton X-100 was added to make1.25% (v/v). The suspension was centrifuged at 4000g for 10min at 2°C and the pellet twice washed, thereby removing aninhibitor and enzyme activity involved in cell wall synthesis (13).The pellet was resuspended in buffer without PEG (designated'No PEG') to give a suspension equivalent to 4 endosperms (or20 slices) ml-'. Subsequent centrifugation separated the boundenzyme in the pellet from the soluble enzyme in the supernatant(13). The pellet was washed in No PEG buffer, and a glutenagglomerate was removed and discarded. The presence of aninhibitor in the crude extract prevented the estimation ofenzymerecovery from the tissue (13).

In the case of rice, whole grains (husks removed) were chopped448

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PHOTONASTIC CONTROL OF LEAFLET ORIENTATION IN MELILOTUS

z 800

' 60-jwZc 40zG,_L-

I D' 20

w1- 0I-LL

0 -20w0n -4

0 20 40 60 80 100FLUENCE RATE (jmoI mr2 seC-1)

FIG. 6. Steady state LE of terminal leaflet ofM. indicus as a functionof fluence rate from spotlight striking its lower surface (Al =-45°).Upper surface simultaneously spot-lit at constant fluence rate (100 Mmolm-2 S-1).

FIG. 7. Steady state LE of light-adapted terminal leaflet ofM. indicuswith its lower surface spot-lit over a range of angles of incidence (Al).

_ I60

z

0

40

w

j-Jw 20

-20

-400 60 120 180

ELAPSED TIME (min)FIG. 8. Time-course of reorientation (change in LE) of light-adapted

terminal leaflet of M. indicus with its lower surface exposed to diffusefluorescent light and its upper surface spot-lit. Angle ofincidence changedfrom +900 to + 135' at arrow.

5, open circles), by expansion of the upper side and concomitantcontraction of the lower side, thereby reducing the curvature ofthe pulvinule. By definition (16, 17), in the pulvinule ofMelilotusindicus the extensor sector of the motor tissue is located on theupper side and the flexor on the lower.

Leaves of M. indicus resemble their relative M. alba (28) intheir capacity to track the sun throughout the day (Fig. 1). Themechanism involved in the diaphototropic response in nyctinas-

- 0 30 60 90 120 150 180ANGLE OF INCIDENCE (degrees)

FIG. 9. Steady state LE of light-adapted terminal leaflet ofM. indicusas a function of angle of incidence (AI) of spotlight striking its uppersurface and its lower surface simultaneously exposed to diffuse fluores-cent illumination.

100_ R

1 800'

60B0z0

-40

w

20-

-4010 60 120 180 240

ELAPSED TIME (min)FIG. 10. Time-course ofreorientation ofdark-adapted terminal leaflet

ofM. indicus with its lower surface exposed to red (0), or blue (0) light(30 umol m-2 s-'). R, Upper surface simultaneously exposed to red light(at 90 min); W, upper surface simultaneously exposed to white light (at180 min).

tic leaves is quite different from the one in malvaceous leavesthat track the sun, because in the latter the site of perception forvectorial photoexcitation is quite separate from the pulvinar siteofresponse (13, 14, 18, 30), while in the former, photoperception,as well as the response to it, are localized in the pulvinus itself(1, 3-5, 15, 16, 20-22, 27). In M. indicus, exposure to light ofthe lower surface ofthe pulvinule of the dark-adapted leaflet wasboth necessary and sufficient to cause negative (downward)curvature ofthe pulvinule and the opening response ofthe leaflet(Fig. 5), as in Mimosa (25). Exposure to light ofthe upper surfaceof the horizontal, light-adapted leaflet resulted in positive (up-ward) curvature, by increasing the curvature of the pulvinule.When a similar light-adapted leaflet was transferred to darkness,it reoriented in the same (upward) direction and at a similarangular velocity, but after a substantial lag-phase (Fig. 5). Thisraises the question whether action of light on the upper surfaceis also photonastic (i.e. light-driven), or merely eliminates thelag-phase of the nyctinastic closure. However, the closure causedby spotlighting the upper surface of the leaflet was maintainedeven when the lower surface was simultaneously spot-lit at afluence rate of about 10 ,umol m-2 s-' (Fig. 6), which is aboutdouble the photonastic excitation required to retain the leaflet inthe open orientation when the upper surface was not illuminated(Fig. 4), and is therefore also light-driven. These results suggestthat the upper, as well as the lower, sides ofthe leaflet are capableof perceiving photonastic excitation. But whereas the former

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3Plant Physiol. Vol. 84, 1987

leads to decreasing pulvinular curvature, the latter leads to in-creasing it. Assuming that the photonastic excitation ofthe uppersurface is also perceived by the pulvinule, it appears that bothsectors of the pulvinus respond to photonastic excitation bycontracting, with concomitant expansion of the opposite, unex-posed sector. Therefore, the opening of the dark-adapted leafletsupon exposure to abaxial illumination (Fig. 5, open circles) maybe a result of contraction of the flexor as a direct response to itsphotonastic excitation, rather than ofexpansion ofthe unexposedextensor. Likewise, the closing of the light-adapted leaflet uponexposure to adaxial illumination (Fig. 5, open triangles) may bea result of shrinkage of the extensor as a direct response to itsphotonastic excitation, rather than expansion of the nonexposedflexor.Two mechanisms have been reported in photonastic leaves,

that may account for their solar-tracking capabilities. One ofthese involves a capacity for photonastic excitation in the upper,as well as in the lower flanks of the pulvinus (3-5, 20). Thelower, as well as the upper side of the pulvinus of the primaryleaf of Phaseolus multiflorus and of the secondary pulvinules ofRobinia pseudacacia and Macroptilium atropurpureum cv Sira-tro are light-sensitive. Exposure of the upper surface to lightcauses 'positive' (upward) curvature, while exposure of the lowersurface causes 'negative' (downward) curvature. In P. multiflorus,the response to simultaneous and equivalent illumination ofboth sides ofthe pulvinus coincided with the calculated differencebetween the opposite responses of the two sides individually. Onthis basis, it was predicted (3) that leaflet elevation could bemodulated according to the fluence rates intercepted simultane-ously by the two opposite surfaces. Furthermore, exposure of thelateral flank of the pulvinule to light causes a torsion movementthat twists the leaflet to face the light source. The other mecha-nism is a function of pulvinular morphology. This has first beendescribed in Phaseolus vulgaris, where the pulvinule is curved,with the upper flank concave and the lower convex. Thus,pulvinular morphology is such that parts of the light-sensitiveupper surface of the pulvinule may become shaded by its laminawhen light falls on the leaf from certain directions, which thenresults in a nyctinastic response (27). Self-shading has also beenreported in M. atropurpureum (20). The pulvinule ofM. indicushas a similar architecture. The combined effects of these twomechanisms make leaflet orientation in light dependent on thedirection (azimuth), as well as on the elevation of the incidentlight, thus transforming the photonastic response into a photo-tropic one.By definition, the extensor is contracted in the dark and

contracts upon transfer from light to darkness. This, in additionto its lesser responsiveness to light, has made it difficult to detectits photonastic contraction. In P. multiflorus, P. vulgaris, and R.pseudacacia, detection was possible because light-adapted leaveswere used (4, 5, 27), in which the nyctinastic upward foldingupon transfer to darkness took place very slowly, presumablybecause their phytochrome was in the Pfr form at time oftransfer.In M. atropurpureum cv Siratro, the greater sensitivity of thelower side of the pulvinule was deduced from equivalent andsimultaneous irradiation of the lower and upper sides over a

range of angles of incidence, which invariably resulted in down-ward reorientation ofthe leaflet (20). The bilateral photosensitiv-ity of the pulvinus of M. indicus is clearly apparent from thequantitative results in Figure 6 that show that (a) when a constantlevel (100 ,umol m-2 s') of photonastic excitation of the upperpulvinular surface of the leaflet was balanced against a variablelevel of excitation of the lower surface, the steady state LE was alinear function of the variable excitation; (b) the photonasticresponsiveness of the upper and lower surfaces differs quantita-tive; (c) when photonastic excitation of the lower side wasbalanced against the nyctinastic response of the upper side, 4.7

,gmol m-2 s-' sufficed to maintain the leaflet in the open orien-tation. However, in making quantitative comparisons, it mustbe remembered that the upper surface ofthe pulvinule is concaveas well as folded transversely, while the lower surface is convex(Fig. 2), which means that their photoreceptive surfaces may beexposed to light to different extents. Furthermore, the angle ofincidence of the light may have different effects on the twosurfaces. The extent to which these surfaces are exposed mayalso change in opposite directions as the pulvinus changes itsshape.

In Macroptilium atropurpureum cv Siratro, the leaflets reorientindependently towards the light source. The tracking fidelity ofthe individual leaflets (taken as their 'steady state' laminar ele-vation as a function of angular elevation of a simulated 'sun'directed at the horizontal leaf in the plane of symmetry of itspetiole) was greatest when the 'solar' angle of incidence was ±30%, and was considerably less at greater angles of incidence.Within that range, the steady-state orientation was within 150 ofthe normal to the light beam. When a light source was moved ina vertical arc along the petiole, displacements <150 caused thelaminae to reorient to approximately the same extent, whiledisplacements >150 resulted in a lesser reorientation (20). In M.indicus, effects of Al on the response to photonastic excitationof the lower surface of the light-adapted leaflet were observedonly over a limited range of AI (0° to -40°), where steady stateLE decreased progressively from -6° to -43° (Fig. 7). This raisestwo questions: (a) What is responsible for the angular dependenceat the lower range ofAI? (b) Why is the response independent ofAl beyond LE = -430? The results suggest that at each AI smallerthan -40°, leaflet reorientation in response to abaxial illumina-tion proceeded until the lamina shaded its own lower pulvinarsurface from the oblique beam. Laminar reorientation at Algreater than -400 did not proceed beyond LE ~ -43°, probablybecause of structural limitations of the pulvinus. Results inFigure 8 suggest that Al also controls the photonastic responseof the upper surface of the light-adapted leaflet. Exposure to aspotlight of AI = +90° resulted in immediate reorientation to asteady state LE of + 12°. Further increase of Al to + 1350 causedthe leaflet to depart from that steady state and increase its LE ata near-constant angular velocity. The steady state LE for Al =

+ 1350 was not reached by the end of this experiment. The uppersurface of the pulvinule, which contains the array of receptorsfor its own photonastic excitation is concave. Exposure of thissurface to direct spotlight (at AI = +90°) may have changed thebalance between the photonastic excitation of the upper andlower pulvinar surfaces that preexisted in the light-adapted leaf,in favor of the upper surface, which resulted in the new steadystate LE (+ 120). Increasing Al to + 1350 may have exposed agreater area of the light-sensitive upper surface to light, whichthus caused an additional response. This explanation is supportedby data in Figure 9, which show that steady state LE was virtuallyindependent of AI between 0° and +100°, beyond which itbecame very strongly affected by Al, with diminishing effective-ness (ALE/AAI) as Al was further increased. Rate of laminarreorientation (Fig. 8) was greater after exposure to AI = +900than after increase to Al = + 135°, despite the fact that a greatersurface was presumably exposured to photonastic excitation. Onepossible explanation is that in the initial, light-adapted orienta-tion the pulvinule was already under considerable mechanicalstrain, and that relaxation of this strain accelerated the light-driven reorientation caused by spotlighting (at Al = +90°).

Finally, results in Figure 10 suggest that the blue spectralregion is more effective than the red in photonastic excitation ofboth surfaces ofthe pulvinule, as is characteristic for photonasticpulvini in general.

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LITERATURE CITED

1. BJORKMAN 0, SB POWLES 1981 Leaf movement in the shade species Oxalisoregana. I. Response to light level and light quality. Carnegie Inst WashYear Book 80: 59-62

2. BONHOMME R, C YARLET ORANCHER, P ARTIS 1974 Utilization de 1'energiesolaire par une culture de Vigna sinensis. II. Assimilation nette et accroise-ment de matiere seche, influence du phototropisme sur la photosynthese despremieres feuilles. Ann Agron 25: 49-60

3. BRAUNER L 1959 Phototropismus und Photonastie der Laubbliatter. In WRuhland, ed, Encyclopedia of Plant Physiology, Vol 17(1). Springer-Verlag,Berlin, pp 472-491

4. BRAUNER L, M BRAUNER 1947 Untersuchungen uber den mechanismus derphototropischen reaktion der blattfiedern von Robinia pseudacacia. Rev FacSci Univ Istanbul 12B: 35-79

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