Plant Physiol. (1984) 75, 90-940032-0889/84/75/0090/05/$01.00/0

Rhizobium Infection and Nodule Development in Soybean AreAffected by Exposure of the Cotyledons to Light'

Received for publication November 7, 1983 and in revised form January 20, 1984

NASIR S. A. MALIK, MARK K. PENCE, HARRY E. CALVERT, AND WOLFGANG D. BAUER*C. F. Kettering Research Laboratory, Yellow Springs, Ohio 45387

ABSTRACr

The initiation of Rhizobium infections and the development of noduleson the primary root of soybean Glycine max L. Meff cv Williamsseedlings are strongly affected by exposure of the cotyledons/hypocotylsto light. Seedlings in plastic growth pouches were inoculated with R.japonicum in dim light and the position of the root tip of each seedlingwas marked on the face of the pouch. The pouches were covered andkept in the dark for various times before exposing the upper portions ofthe plants (cotyledons and hypocotyls) to light. Maximum nodulationoccurred if the plants were kept in the dark until 1 day after inoculation.The exposure of plants to light 2 days before inoculation reduced thenumber of nodules by 50% while the number of nodules was reduced by70% if the plants were kept in the dark until 7 days after. inoculation.Anatomical studies revealed that exposure to light prior to inoculationreduced both the number of infection centers with visible infection threadsand the number of infections which developed nodule meristems. Plantskept in the dark for 7 days after inoculation formed a normal number ofinfection threads above the root tip mark, but very few of these infectionsdeveloped a nodule meristem. It appears that light stimulates soybean toproduce substances which can both inhibit the formation of infectionthreads and enhance the development of nodules from established infec-tion threads. The effects of light on nodulation appear to be expressedindependently of the Rhizobium-induced suppression of nodule formationin younger regions of the root.

The establishment of a symbiotic association between Rhizo-bium species and legumes involves infection of the root and thedevelopment of organized nodular growths containing bacteria.Nodule formation is a complex process requiring the completionof many steps between the initial contact of rhizobia with theroot surface and the final differentiation of host and bacterialcells to form a mature nodule (1, 10, 12). Previous studies haveindicated that light can have significant, nonphotosynthetic ef-fects on the establishment of the symbiosis (5, 7). Studies byGrobbelaar et al. (5) indicated that exposure of excised beanroots to light prior to inoculation stimulated Rhizobium infec-tion, whereas exposure after inoculation inhibited nodule devel-opment. It has also been reported that nodulation is enhancedin various leguminous plants if they are grown under long daysas compared to short days (1 1), and that nodulation of pea isinhibited upon exposure of roots to far-red light (7). Duringpreliminary studies to examine other regulatory phenomena, weobserved that seedlings inoculated 1 d after imbibition and then

I Supported in part by grant 82-CRGO-1-1041 from the CompetitiveResearch Grants Office of the United States Department of Agriculture.This paper is contribution 814 ofthe C.F. Kettering Research Laboratory.

exposed to light developed 30% fewer nodules on the primaryroot than similar seedlings kept in the dark until 3 d afterimbibition. These observations have led us to investigate moresystematically the effects of light on the initiation of infectionsand the development of nodules in soybean.

MATERIALS AND METHODSBacterial culture. Rhizobium japonicum strain USDA-I- 110-

ARS (resistant to azide, rifampicin, and streptomycin) was ob-tained from Dr. D. Kuykendall, USDA Beltsville, and was usedin all of the studies described here. Freeze-dried ampules of thestock culture of this strain were used to initiate starter culturesin a yeast extract-mannitol-gluconate medium as described pre-viously (2, 3). Subcultures ofthe bacterium in this medium wereused for the inoculation of seedlings after dilution ofa culture inearly exponential growth phase (A at 620 nm = 0.1-0.3) withsterile distilled H20 to obtain 105 cells/ml (108 cells/ml = 0.05A at 620 nm). Viable cell counts ofdiluted inoculum suspensionswere recorded for each experiment to determine the actualnumber of bacteria applied.

Plant Growth. Soybean seeds (Glycine max [L.] Merr. cvWilliams) were purchased from Dewine and Hamma Seed Co.,Yellow Springs, OH. Seeds were surface sterilized with NaOCl,washed, and germinated on agar plates as described previously(2). These plates were kept either in the dark (wrapped in Al foil)or in the light (wrapped in Saran Wrap) at 28°C depending onthe particular experimental protocol. After 24 h, the seedlingswere transferred to disposable plastic growth pouches (NorthrupKing Seed Co, Minneapolis, MN) and kept either in the light orin the dark as indicated below. For those experiments where asecond inoculation was required 48 h after the first inoculation,double length pouches were constructed by joining two standardsized pouches top to bottom. The paper towel wick of the lowerpouch was unfolded and placed underneath the wick of the toppouch.The roots of the two seedlings growing in each pouch were

individually inoculated with 250 Ml of a bacterial suspension inwater (105 cells/ml). The inoculum was added dropwise onto theroot surface from the root tip towards the shoot for a distance of2 to 4 cm. At the time of inoculation, a mark was made withwaterproof pen on the clear plastic face of the growth pouch inorder to indicate the position ofthe RT2 at that time, as describedpreviously (2, 3).

In all experiments described below, the pouching and inocula-tion of seedlings was performed in dim light (<1 ft-c). To ensurethat the effect of light was on the cotyledons or hypocotyls andnot due to diffusion of light onto the root system, the clear plasticfaces of the pouches were covered with black polyethylene. Thisopaque cover was taped at the top of the pouch so that it couldbe flipped over for marking the position ofthe RT on the growth

2Abbreviations: RT, root tip.90

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EFFECTS OF LIGHT ON NODULATION IN SOYBEAN

pouch face. The growth pouches were maintained in an uprightposition between spacers in a foil-covered box.Pouched seedlings were grown in an environmentally con-

trolled (Conviron model EF 7) chamber with 14-h days at 28°Cand 1200 ft-c and 10-h nights at 26°C. RH was maintained at70%. Pouches were watered daily to saturation ofthe paper towelwicks with sterile, N-free, half-strength Jensen's medium (2). Thenumber and positions of nodules on the primary root relative tothe RT mark made at the time of inoculation were normallyscored 7 to 8 d after inoculation as previously described (2, 3, 9).If the seedlings were kept in the dark for some period afterinoculation, then scoring was performed 7 to 8 d after theseedlings were first exposed to light. Uninoculated seedlings didnot develop nodules.

Fiber optic illumination was used to expose seedlings to lightof low (2 ft-c) intensity. In these experiments, seeds were germi-nated on agar plates in the dark and were pouched and inoculated48 h after imbibition in dim light (<1 ft-c) as before. The plantswere kept in the dark for 24 h after inoculation and thentransferred to specially made plastic boxes (1 x 1.5 x 2 feet)where they were kept either in the dark, in full light, or in low (2ft-c) light. The boxes were made ofopaque plastic, excepting onebox which had a clear plastic top and was used for the full lighttreatment. Light of low intensity was provided by a fiber opticlight source with a flexible arm that entered the box and pointedtoward the ceiling to produce a uniform reflected light over theseedlings. The boxes were lined inside with white paper.Anatomical Studies. Nodulation on 30 to 40 plants from a set

of 100 was first scored as described above. Subsequently, fivereplicate roots, each with an average number of nodules aboveand below the RT mark, were selected for anatomical studies.Sample root segments were excised 20 mm above the RT markand 50 mm below the RT mark. A nick was made on the rootto indicate the position of the RT mark made on the growthpouch at the time of inoculation. Freshly cut root samples werefixed in FAA solution (40% formaldehyde:glacial acetic acid:50%ethanol, v/v, 5:5:90). The fixed root samples were dehydrated inan ethanol series, embedded in paraffin, serially sectioned lon-gitudinally at 10 gm thickness, stained with tannic acid-ferricchloride-saffranin-fast green, and examined with the light micro-scope for infections (Calvert et al, manuscripts submitted).

RESULTS

Exposure of Cotyledons to Light Prior to Inoculation. Prelim-inary experiments had indicated that exposure of seedlings tolight prior to inoculation with R. japonicum resulted in reducednodulation. These experiments were repeated. As shown in Table

Table I. The Effect ofExposing Cotyledons/Hypocotyls to Lightfor 72Hours Prior to Inoculation

Seeds were germinated either (1) in full light (1200 ft-c); (2) in lowlight(35 ft-c); or (3) in the dark for 24 h. Seedlings were then transferredto growth pouches, in dim light for treatment 3. Pouches were then keptin the dark or light of the indicated intensity (14-h photoperiod) for 48h prior to inoculation. After inoculation all seedlings were exposed tofull intensity light of a 14-h photoperiod. A set of 100 seedlings was usedfor each treatment.

Avg. No. of Nodules on the Primary RootTreatment Nodules above % of Total nodules % of

the RT mark control' on tap root control'1. Full light 3.7 ± 0.2b 57 5.3 ± 0.3 512. Low light 3.4 ± 0.3 52 5.9 ± 0.4 573. Dark 6.5 ±0.3 100 10.3 ±0.4 100' Nodulation relative to plants kept in the dark (treatment 3).b Mean + SE.

I, seedlings exposed to light during the 72-h period prior toinoculation had only about 50% as many nodules on the primaryroot as seedlings kept in dark until inoculation. Plants exposedto light of reduced intensity prior to inoculation had 57% asmany nodules as plants kept in the dark (Table I).

In the above experiments, it was not clear whether light wasaffecting nodulation through its effect on the cotyledons aftertransfer from the germination plates or its effect on emergingradicals during the first 24 h ofgrowth after imbibition. To checkthis point, seeds were germinated in the dark for 24 h and thentransferred in dim light to pouches covered with black polyeth-ylene. Seedlings exposed to light 48 h before inoculation, withroots protected from light, developed 60% fewer nodules thanseedlings kept in the dark (Table II).

Effects of Extended Periods of Darkness after Inoculation.Individual sets of seedlings were kept in the dark for varioustimes after imbibition in order to determine the optimum timeof first exposure of the cotyledons/hypocotyls to light. Theseedlings were inoculated 48 h rather than 72 h after imbibitionin order to permit nodules to develop over a greater length ofthe primary root. Maximum nodulation on the primary roots ofsoybean seedlings occurred if the seedlings were first exposed tolight 1 d after inoculation (3 d after imbibition) (Fig. 1). Nodu-lation decreased considerably if the seedlings were kept in thedark for more than 1 d after inoculation (Fig. 1). Thus, whilelight had an inhibitory effect on nodulation if provided to thecotyledons/hypocotyls before inoculation, it also appears thatexposure of these organs to light after inoculation is very impor-tant or perhaps required for nodule formation.

Nutritional Effects. The reduced number of nodules on theprimary roots of plants kept in the dark for 7 d after inoculation(Fig. 1) could be interpreted in terms of nutrient deprivationeffects due to lack of photosynthesis. Plants were therefore ex-posed to low light intensities (2 ft-c) in order to reduce photosyn-thesis. As shown in Table III, seedlings kept under 2 ft-c lightformed almost as many nodules above the RT mark as plantskept in full light (1200 ft-c), whereas plants kept in the dark for7 d after inoculation formed substantially fewer nodules abovethe RT mark. Thus, light of low intensity appears to stimulatenodule formation by some means other than generation ofnutrients. On the other hand, plants exposed to 2 ft-c light formedsubstantially fewer nodules on younger regions of the primaryroots, below the RT mark, than plants exposed to full light (TableIII). Moreover, nodules on the plants exposed to 2 ft-c light weregenerally smaller in size than the nodules in plants kept in fulllight. These results indicate the involvement of nutritive factorsas well as regulatory substances in the nodulation process.Double Inoculation. The relationship between the Rhizobium-

induced suppression of nodulation in younger regions ofsoybeanroots and the light effects described above was investigated bythe double inoculation technique (9). Double inoculation exper-

Table II. The Effect ofExposing Cotyledons/Hypocotyls to Lightfor 48Hours Prior to Inoculation

Seeds were germinated for 24 h in the dark then transferred to growthpouches in dim light. One set of 100 seedlings was exposed to 14-hphotoperiod light with roots protected from light while the other set of100 seedlings was kept in the dark. After 48 h, both sets of seedlings wereinoculated and exposed to a 14-h photoperiod.

Avg. No. of Nodules on the Primary RootTreatment Nodules above % of Total nodules % of

RT Mark controla on tap root control

Light 1.6± 0.2b 42 2.9 ± 0.3 40Dark 3.8±0.2 100 7.3±0.3 100

a Nodulation relative to dark control.b Mean ± SE.

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Plant Physiol. Vol. 75, 1984

16 / \a.0~~~~

1 120

z

o 0

ct 8_\LLI\2 Nodules above RT

NOCULATION oAND MARKING * -

0 1 2 3 4 5 9FIRST EXPOSURE of COTYLEDONS to LIGHT

(DAYS AFTER IMBIBITION)FIG. 1. Changes in nodulation resulting from exposure of soybean

cotyledons to light at different time intervals before and after inoculation.Open circles (0) represent the total number of nodules on the primaryroot and closed circles (0) represent the number ofnodules formed abovethe RT mark. Seedlings were marked (RT) and inoculated with 2.5 xI0 rhizobia/plant 48 h after imbibition. The cotyledons were exposedto light (14-h photoperiod) either from the time of imbibition or atvarious time intervals postimbibition. The arrow represents the time ofinoculation and marking of the root tip position. Seedlings were scoredfor nodulation either 7 d after inoculation or 7 d after first exposure tolight. Sets of 100 seedlings were used for each exposure.

Table III. The Effect ofExposing Soybean Seedlings to 2ft-c Light 1

Day after InoculationSeeds were germinated in the dark for 48 h and transferred to growth

pouches, and the seedlings were inoculated with rhizobia under dim light(<I ft-c). The inoculated seedlings were kept in the dark for 24 h andthen exposed to either: (1) full light (1200 ft-c); (2) low light (2 ft-c); or(3) kept in darkness. Seven d after inoculation, all plants were exposedto a 14-h photoperiod for another 7 d prior to scoring. Each data pointis an average from at least 50 plants.

Avg. No. of Nodules on the Primary RootTreatment Above % of Total nodules % of

RT mark control' on tap root controla

1. Full light 2.6 ± 0.2b 100 15.4 ± 0.7 1002. Low light 2.4 ± 0.3 92 10.5 ± 0.4 663. Dark 1.0 ±0.1 38 5.9 ±0.4 36a Nodulation relative to plants kept in the light (treatment 1).b Mean ± SE.

iments were also performed in order to determine the effects ofbrief exposure to light on nodulation. Plants were inoculated atthe time of marking RT1, exposed to light for either 0, 1, or 6 himmediately following inoculation and then kept in the darkuntil 2 d after the second inoculation. Control plants wereexposed to full light (14-h photoperiod) from the time of inoc-ulation at RTI. All plants were reinoculated at the time ofmarking RT2, 48 h after the first inoculation.As indicated in Table IV, plants which were exposed to light

for either 1 h or 6 h immediately after the first inoculationdeveloped significantly more nodules in the vicinity ofRTl thanthe dark controls, although not as many as plants exposed to thefull light regime. Plants exposed to the full light regime showed

Table IV. Effects ofLight and Prior Exposure ofthe Root to Rhizobiaon Nodulation

Seeds were germinated for 48 h in the dark and then transferred tolong growth pouches in dim light. Each set of seedlings was inoculated(RTI) with rhizobia and then reinoculated (RT2) a second time withrhizobia 48 h after the first inoculation. Sets of 80 plants were either: (1)kept in the dark continuously until 48 h after the second inoculation; (2)exposed to light for I h immediately after the first inoculation, then keptin the dark until 48 h after the second inoculation; (3) exposed to lightfor 6 h immediately after the first inoculation, then kept in the dark until48 h after the second inoculation; or (4) exposed to light continously (14-h photoperiod) for 7 d after the first inoculation. Nodulation on theprimary root was scored 9 d after the second inoculation.

Avg. No. of Nodules/PlantTreatment

Near RTlb Near RT2b1. Dark control 7.3 ± 0.2c 8.0 ± 0.2c2. 1-h exposure 11.0± 0.3d 8.1 ±0.4c3. 6-h exposure 9.6 ± 0.4d 8.0 ± 0.5c4. Lightcontrol 14.7 ±0.8e 1.7±O.If

a Mean ± SE.b Near RTI = nodules present on the primary root in the region more

than 0.2 relative distance units (RDU) above the mark made for eachplant at the time of the second inoculation (RT2). One RDU in theseexperiments is the distance between RTI and RT2 measured for eachplant. Near RT2 = nodules on the primary root in regions younger thanthe point 0.2 RDU above the RT2 mark made for each plant. Theposition at 0.2 RDU above RT2 was chosen because at this pointnodulation peaks in the vicinity of RTI and RT2 were most fullyseparated, as seen on profiles of total nodulation on the primary roots(profiles not shown).

c.d.e.fNodulation averages designated by different superscript lettersare significantly different from each other by the t test at P < 0.01.

strongly suppressed nodulation in the vicinity ofRT2 comparedto the density of nodulation near RT1. On the other hand, plantskept in the dark or exposed briefly to light at the time ofinoculation showed approximately equal densities of nodulationnear RTl and RT2 (Table IV). The total number of noduleswhich formed on the primary roots was approximately the sameregardless of the time and duration of first exposure to light(Table IV).

Anatomical Studies. Seedlings taken for anatomical analysiswere first exposed to light either: (a) 2 d before inoculation (i.e.from the time of imbibition); (b) 1 d after inoculation; or (c) 7 dafter inoculation. Roots of representative seedlings were takenfor microscopic analysis 7 to 8 d after the initial exposure of theseedlings to normal light in the growth chamber.

Plants exposed to light 2 d before inoculation formed fewerinfection threads in the region above the RT mark than plantskept in the dark until 1 d after inoculation (Table V). Thenumber of infection threads was not appreciably affected bykeeping the plants in the dark for 7 d instead of 1 d afterinoculation (Table V). The different light regimes did not affectthe total number of infection events (Table V).

Exposure to light affected the maturation of those infectionswith identifiable infection threads (Table V). Infection matura-tion was evaluated according to a classification scheme whereindevelopment is divided into recognizable stages based primarilyon the extent of cell divisions evident in serial longitudinalsections of the root. Infections which have progressed to stage 1in this classification scheme are those which show evidence ofonly one or two rounds of host cell division. These are restrictedto hypodermal and subadjacent cortical cells. By stage 3, celldivisions are evident over the width of the cortex from thehypodermis to the stele. Individual cortical cells next to thehypodermis are generally subdivided into four to six daughter

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EFFECTS OF LIGHT ON NODULATION IN SOYBEAN

Table V. The Efkct.s ofJ Light on the Initiation and Development ofInfections

Seedlings taken for anatomical analysis were inoculated 48 h afterimbibition and first exposed to light either: (1) 2 d before inoculation(i.e. from the time of imbibition); (2) 1 d after inoculation; or (3) 7 dafter inoculation. The number, the location, and the stage ofdevelopmentof each infection was determined as described in "Materials and Meth-ods." Each data point is an average from at least three replicate roots.The population average for nodule number is an average from 100replicate plants.

Avg. No. of Events Above the RT Mark

Stage of infectionTotala th developmentc Pop. Avthreadeb nodules

1 2 3 4 5 6+

1. 2 d before 39 20 0.8 4.5 9.8 2.7 0.8 1.8 2.6 ± 0.2d2. 1 d after 45 32 0.8 7.0 11.2 4.0 3.5 5.8 5.4 ± 0.23. 7 dafter 45 31 2.0 11.5 13.5 1.5 0.5 1.5 1.4 ±0.1

a Total = all induced centers of anticlinal cell division with or withoutinfection threads.

b Threads = all infection events with identifiable infection threads.I Stage of infection development: numbers from 1 to 20 indicate the

stage of development of infections from the initial anticlinal division inthe hypodermis (stage 1) through formation of mature 10-day-old nod-ules (stage 20). Stages 1 to 3 are characterized by a progression of celldivisions in the hypodermis and root cortex prior to meristem formation.Stage 4 is characterized by formation of a nodule meristem. Nodulesfirst emerge above the root surface at stage 6. The averages given referonly to development of infections with identifiable infection threads.

d Mean ± SE.

cells. A nodule meristem is evident in infections which haveprogressed to stage 4 and beyond. Nodules have emerged abovethe root surface by stage 6. Plants exposed to light 1 d afterinoculation had an average of 13.3 nodule meristems above theRT mark (Table V). However, an average of only 3.5 nodulemeristems developed above the RT mark in plants kept in thedark for 9 d, and only 5.3 nodule meristems developed in plantsexposed to the light from the time of imbibition (Table V).

DISCUSSION

The results described above indicate that the exposure ofsoybean cotyledons or hypocotyls to light can have two distincteffects on nodulation. The first effect of light is a substantialinhibition of nodulation on the primary root. This inhibition isseen if the cotyledons/hypocotyls are first exposed to light priorto inoculation rather than 24 h after inoculation (Tables I andII; Fig. 1). Analysis of serial sections through representative rootsrevealed that the exposure of cotyledons to light prior to inocula-tion caused an appreciable reduction in the number of infectioncenters with visible infection threads (Table V). This indicatesthat early exposure of soybean cotyledons to light blocks theRhizobium infection process at some stage prior to infectionthread formation. The reduction in the number of infectionthreads could account for some of the 50% decrease in nodula-tion resulting from early exposure to light (Tables I, II, and V;Fig. 1). It should be noted, however, that many more infectionthreads are formed than nodules (see Table V). Thus, the rela-tionship between reduced numbers of infection threads andreduced numbers of nodules may not be a simple one. The earlyexposure of cotyledons to light also resulted in a considerabledecrease in the number ofnodule meristems formed in the regionabove the RT mark (Table V). It is not clear at this time whetherdiminished meristem formation is a consequence of diminishedinfection thread formation or represents a subsequent and rela-

tively independent point of inhibition.The second distinct effect of light revealed by these studies is

that exposure of soybean cotyledons/hypocotyls to light afterinoculation can greatly enhance nodulation. Seedlings kept inthe dark for 7 d after inoculation developed only about 30% asmany primary root nodules above the RT mark as seedlingsexposed to light 1 d after inoculation (Fig. 1). Microscopicexamination revealed that only about 25% as many nodulemeristems developed if the plants were kept in the dark for 7 dafter inoculation instead of for 1 d (Table V). It thus appearsthat the maturation of Rhizobium infections is blocked beforenodule meristem formation unless the cotyledons are exposed tolight within a few days after inoculation. The inhibition ofnodulemeristem formation in these seedlings probably accounts for theobserved reduction in nodulation.These results offer an interesting contrast to the findings of

Grobbelaar et al. (5). Grobbelaar et al. observed that the exposureof Phaseolus roots to light prior to inoculation stimulated theinfection process, whereas light applied to the roots after inoc-ulation was inhibitory to the later stages of infection develop-ment.The differences between our results and those of Grobbelaar

et al. might be due to differences in either the plant species usedor in the plant tissue which was exposed to light. In the experi-ments of Grobbelaar et aL, excised bean roots growing on agarin Petri dishes were directly exposed to light (5), whereas in ourexperiments the cotyledons and hypocotyls of intact soybeanseedlings were exposed. It may be that light triggers differentresponses depending on the type of tissue exposed (e.g. rootversus cotyledons). Light could also elicit different responses inthe same type of tissue if the tissue was derived from differentplant species. Exposure of pea roots, for example, to red lighthad only a slight effect on nodulation (<6% inhibition) comparedto dark controls (7), but in beans there was at least a 50%reduction in the number of nodules produced on roots exposedto red light (5). In preliminary experiments, we have observedthat exposure of soybean cotyledons to red light before inocula-tion reduces nodulation in a manner similar to the effect ofwhitelight (unpublished data).

Plants exposed continuously to 14-h photoperiod light afterinoculation (Table IV) developed very few nodules near RT2.Since soybean roots grow at the rate of 2 to 2.5 mm/h in thegrowth pouches (2), it appears that the Rhizobium-induced sup-pression of nodulation in soybean (2, 9) is expressed at distancesof at least 9 to 12 cm below the upper regions of the primaryroot. This is consistent with the diminished frequency of nodu-lation observed in the field at distances of more than 15 cmbelow the soil surface (6). It also provides support for the earliersuggestion that the suppressive response in soybean may contrib-ute importantly to the commonly observed clustering of nodulesnear the crown of the root system (9).

It is apparent from Table IV that even brief exposure to lightsignificantly enhances nodulation near RT1 relative to the darkcontrol. Since these effects on nodulation are evident with lightexposures of just 1 h, and since the nodulation response doesnot seem to be proportional to the length of exposure, it seemsunlikely that the effects of brief light exposure on nodulation aredependent on photosynthesis. Earlier results (Table I and III)indicated that exposure to light of low light intensity (2 or 35 ft-c) had significant effects on nodulation. This also supports theconclusion that the effects of light on nodulation observed inthese studies are not simply the result of a decrease in photosyn-thesis or the supply of nutrients. We note in this regard thatremoval of one cotyledon at the time of inoculation did notsignificantly reduce the number of nodules produced on theprimary roots and did not reduce the average rate of rootelongation (unpublished). It remains to be determined whether

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Plant Physiol. Vol. 75, 1984

brief exposures to light overcome the blockage of infections atthe meristem formation stage (Table V). It also remains to bedetermined whether the failure to achieve full nodulation afterbrief light exposure is a consequence of inadequate nutnentsupply or of some other factor.

It is of interest that the total number of nodules to develop onthe primary roots was approximately the same regardless ofwhether the seedlings were exposed to brief light, sustained light,or no light immediately following the first inoculation (TableIV). A similar equalization of total nodulation on the primaryroot was observed earlier when various sets of seedlings wereexposed to different numbers of rhizobia at RT 1 and to the samenumber of rhizobia 15 h later at the time of marking RT2 (9).These results suggest that the plant has an effective compensatorymechanism that regulates the extent of nodule formation inyounger regions of the root depending on the extent of infectionor nodule formation in the more mature region of the root. Aregulatory mechanism of this sort, having its effect days prior toany measurable N fixation, could serve to optimize noduleformation for the plant under various circumstances.

It seems that the exposure of soybean cotyledons to light maystimulate the production of both regulatory substances and nu-trients in the cotyledons, and that these are transmitted downthe root where they influence the infection and nodulationprocesses. Elegant studies by Phillips (8) have shown that thecotyledons of pea seedlings produce one or more substanceswhich inhibit the development of nodules, but which do notaffect the formation of infection threads. In soybean, on theother hand, it appears that light stimulates the production ofoneor more regulatory substances which inhibit the formation ofinfection threads and which may also inhibit the formation ofnodule meristems. In addition, it appears that light stimulatessoybean to produce one or more regulatory substances whichenhance, rather than inhibit, the further development ofinfection

centers with threads. The nature of such regulatory substances isnot known. Further studies are required to identify these sub-stances and to characterize the light receptors in soybean whichare coupled to the infection and nodulation responses.

Acknowledgments-We thank Kevin Hemenger, Kim Mills, Padma Pyati, andSusan Parker for excellent technical assistance.

LITERATURE CITED

1. BAUER WD 1981 Infection of legumes by rhizobia. Annu Rev Plant Physiol32: 407-449

2. BHUVANESWARI TV, BG TURGEON, WD BAUER 1980 Early events in theinfection of soybean (Glycine max [L.] Merr) by Rhizobium japonicum. I.Localization of infectible root cells. Plant Physiol 66: 1027-1031

3. BHUVANESWARI TV, KK MILLS, DK CRIST, WR EVANS, WD BAUER 1983Effect of culture age on symbiotic infectivity of Rhizobium japonicum JBacteriol 153: 443-451

4. Deleted in proof.5. GROBBELAAR N, B CLARK, MC HOUGH 1971 The nodulation and nitrogen

fixation of isolated roots of Phaseolus vulgaris L. II. The influence of lighton nodulation. Plant Soil (special volume) 203-204

6. GRUBINGER V, R ZOBEL, J VEDELAND, P CORTES 1982 Nodule distribution onroots of field-grown soybeans in subsurface soil horizons. Crop Sci 22: 153-155

7. LIE TA 1969 Non-photosynthetic effects of red and far-red light on root noduleformation by leguminous plants. Plant Soil 30:391-404

8. PHILLIPs DA 1971 A cotyledonary inhibitor of root nodulation in Pisumsativum L. Physiol Plant 25: 482-487

9. PIERCE M, WD BAUER 1983 A rapid regulatory response governing nodulationin soybean. Plant Physiol 73: 286-290

10. ROLFE BG, M DJORDJEVIC, KF ScoTT, JE HUGHES, J BADENOCH JONES, PMGRESSHOFF, Y CEN, WF DUDMAN, W ZURKOWSKI, J SHINE 1980 Analysisof the nodule forming ability of fast-growing Rhizobium strains. In AHGibson, WE Newton, eds, Proceedings of the Fourth International Sympo-sium on Nitrogen Fixation, Australian Academy of Science, Canberra City,pp 142-145

11. SIRONVAL C, CH BONNIER, J-P VERLINDEN 1957 Action of day-length onnodule formation and chlorophyll content in soybean. Plant Physiol 10:697-707

12. VINCENT JM 1980 Factors controlling the legume-Rhizobium symbiosis. InWE Newton, WH Orme-Johnson, eds, Nitrogen Fixation, Vol. II. UniversityPark Press, Baltimore, pp 103-129

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