Developmental studies on Euphorbia esula. Evidence of competition for water as a factor in the mechanism of root bud inhibition

GORDON I. MCINTYRE Agric~r11trir.c Ctrrrrrrlrr. Rrsrcrrc.11 Sttrtiotr, 130s 440, Rrgirro, Srrsk., Ctrrrorlrr S4P3A3

Received April 23, 1979

McIN.I.YR~. G. 1. 1979. Developmental studies on G~plrorSitr o.srrltr. Evidence of competition for water as a factor in the mechanism of root bud inhibition. Can. J . Bot. 57: 2572-2581.

Seedlings of leafy spurge (Errrrpl~orl~io rsrrlrr L.) were grown in sand culture under controlled conditions and experiments were designed to test the hypothesis that competition for water between the root buds and the parent shoot is a significant factor in the mechanism of root bud inhibition. It was shown that the watercontent of the root buds increased by approximately 25% within 24 h of the removal of the parent shoot. Root bud length increased significantly between 24 and 48 h from shoot removal but while some of the buds continued to grow others were apparently reinhibited. Shoots with all immature tissue removed continued to exert a high degree of root bud inhibition but this inhibition was markedly reduced by keeping the plants in a high humidity. Increasing the humidity from approximately 50 to 95% also caused a significant increase in the rate of emergence and elongation of shoots produced from root buds following the removal of the parent shoot. This effect on emergence was suppressed by conditions presumed to reduce the degree of root bud aeration. Taken as a whole. the results lend considerable support to the concept of internal competition for water as a factor in the mechanism of root bud inhibition.

Mc I N T Y R ~ , G. 1. 1979. Developmental studies on E~rplrorhirr esrrlrr. Evidence of competition for water as a factor in the mechanism of root bud inhibition. Can. J . Bot. 57: 2572-2581.

Des plantulesd'ErrpIrorhitr esrtlo L. ont etecultiveessursabledans desconditions contr8leeset des experiences ont ete mises au point pour testerl'hypothese que la competition pour I'eau entre les bourgeons racinaires et la pousse parentale est un facteur important dans le mecanisme d'inhibition des bourgeons racinaires. La teneur en eau des bourgeons racinaires augmente d'environ 25% en moins de 24 h apres qu'on les a detaches de la pousse parentale. La longueur des bourgeons racinaires augmente significativement 24 a 48 h apres qu'on les a detaches d e la pousse parentale, mais, alors que certains bourgeons continuent i croitre, d'autres sont apparemment inhibes d e nouveau. Les pousses dont on a enleve tous les tissus immatirres continuent h inhiber fortement les bourgeons racinaires. mais cette inhibition est beaucoup moins forte si les plantes sont gardees a une humidite elevee. Une augmentation de I'humidite d'environ 50 h 95% ppr- voq~le egalement une augmentation significative du taux d'emergence et d'elongation des pousses produites par les bourgeons racinaires apres qu'on a enlev6 les pousses parentales. Cet effet d e I'humidite sur I'emergence est supprime par des conditions qui sont censees diminuer le degre d'aeration des bourgeons racinaires. Dans ['ensemble, les resultats appuient fortement I'hypo- these qu'une competition interne pour I'eau est impliquee dans le mecanisme d'inhibition des bourgeons racinaires.

[Traduit par le journal]

Introduction Previous experiments on the mechanism of root

bud inhibition in seedlings of leafy spurge (Eupho/.- hicr esulrr L.) suggested that, under the experimen- tal conditions, the growth of the root buds was largely determined by their ability to compete for nitrogen with the parent shoot (10). Similar experi- ments with Ci~aiu~n rrrcense (12) and Hieraciunz .flo~.~ntitzu~?z (13) also showed that root bud growth was highly responsive to the external nitrogen sup- ply. In the work with leafy spurge, however (lo), it was noted that, whereas the root buds were re- leased from inhibition at high nitrogen levels, the

symptoms the root buds maintained a slow, con- tinuous growth, some reaching a length of 1-2 cm in agrowing period of 8- 10 weeks. Such behavior is in marked contrast to that observed in the field (G. I. McIntyre, unpublished observations). Al- though some root bud elongation may occur at the end of the growing season, and is associated with the senescence of the parent shoot, the buds rarely exceed afew millimetres in length during the period of active shoot growth and appear to be strongly inhibited.

A possible explanation for the sustained growth of the root buds of the experimental plants was

buds on the roots of low nitrogen plants were not suggested by the fact that they were grown in the completely inhibited. Even at a nitrogen level greenhouse during the winter and received a rela- sufficiently low to produce severe deficiency tively low light intensity, an average temperature of

0008-40261791222572- 10$01 .00/0 @ 1979 National Research Council of CanadalConseil national d e recherches du Canada

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only about 15- 18°C. and an abundant water suppl y. It seemed probable that, under these conditions, the degree of water stress experienced by the plants would be considerably lower than that to which they would normally be subjected during the growing season in the field. The possibility that this postulated difference could account for the appar- ent reduction in the degree of root bud inhibition is well supported by recent studies on the relation of water stress to apical dominance. These investiga- tions (see ref. 1 I), involving a wide range of

M t INTYRE 2573

species, provided considerable evidence that inter- nal competition for water is a major factor in the correlative inhibition of lateral buds. Some evi- dence that this factor may also play a significant role in the mechanism of root bud inhibition is provided by the experiments described in the pres- ent report.

Materials and Methods P/till1 cli111lr~~

The seed source and germination conditions were as previ- ously described (10). Germinating seeds were planted in plastic trays containing 12- to 30-mesh silica sand and were watered with distilled water only until the cotyledons were fully ex- panded. The seedlings were then transplanted into 12.5-cm round plastic pots (except in experiment 2 in which square 10.5-cm pots were used). Three seedlings were planted in each pot and were thinned to one per pot a t the two- to three-leaf stage. Nutrients were supplied as a modified Hoagland's solu- tion (7) in which the nitrogen level was reduced to 105 ppm by the substitutions previously described (10). Previous observa- tions had shown that a t this relatively high nitrogen level the plants developed calcium and potassium deficiency symptoms as they approached the flowering stage. The level of these two nutrients was. therefore, increased by 50% by addition of CaCI, and K,SO,, respectively. This modification did not completely eliminate the symptoms but reduced them sufficiently to permit vigorous growth. Each pot received 200 m L of nutrient solution at 2-day intervals. evaporation losses being replaced with dis- tilled water on alternate days. Experiment 1 was conducted in the greenhouse with only partial control of the environmental conditions. The other experiments were conducted in growth chambers under various well-controlled conditions which are described below.

Callec/iot~ rrtltl A twlysis oJDrr/ti Root buds 5 10 mm long were measured to thenearest 0. I mm

under a binocular microscope at x 16 magnification: those > 10 mm were measured to the nearest millimetre with a ruler. The excised buds were weighed fresh, dried at 80°C for 24 h, and their dry weight and water content determined. Experiment 2 involved daily measurements of the same buds on the intact plant. This was accomplished a s follows: a panel 6 cm x 5 cm was cut from the side of the 10.5-cm square pots and fastened in place with masking tape. When the plants had reached the required stage of development the panel was removed and the sand was washed away from the upper part of the root system. T h e pot was then placed on its side under a binocular micro- scope and suitable root buds were selected for measurement. Diagrams were prepared of the exposed part of the root system showing the position and appearance of the selected buds. Since

the base of the bud was not always well defined, some feature, e.g.. the lower edge of the oldest bud scale, was marked in the diagram a s the basal reference point. Measurements were then made from this point to the clearly defined bud apex at a x 16 magnification and a n estimated accuracy of +_ 0.05 mm. Be- tween daily measurements the exposed portion of the root was covered with absorbent cotton moistened with distilled water.

In determining the carbohydrate content of the excised buds (experiment I) the 25 dried buds comprising each sample were ground up in a glass homogenizer with I mL of 80% ethyl al- cohol. The extract was filtered through a Soxhlet thimble and the residue extracted on the Soxhlet for 18 h with more alcohol. Since the extracts were slightly colored by the ink used to mark the buds they were decolorized by treatment with Norit A char- coal. The extracts were each made up to 100 mL and duplicate 2-mL aliquots were analyzed for total soluble carbohydrate by the anthrone method a s previously described (9).

Data were analyzed statistically by analysis of variance and the significance of the difference between treatment means was determined by the Student-Neuman-Keuls multiple range test.

Experiments and Results EjTc~.t of Cor77pIete or. Pcir.tici1 Remoccil of tlze Pci-

rent shoot E,vpcr.iment I If the inhibited growth of the root buds on the

intact plant is due to their inability to compete for water with the parent shoot it might be expected that the stimulation of bud growth resulting from the removal of the dominant shoot would be closely associated with an increase in the water content of the bud. In an experiment designed to test the effect of shoot removal on the uptake of water by the root buds, seedlings were planted in the greenhouse at the end of March and the experimental treatments were applied 10 weeks later. During this period the temperature ranged from I5 to 30°C and the day- length was extended to 16 h by a bank of 12 very high output (VHO) cool-white fluorescent tubes giving an additional intensity of 180 peinsteins m-' s-I. The temperature and natural light intensity were both considerably higher than in the previous experiments conducted during the winter (10) and this difference was associated with an apparent increase in root bud inhibition. In spite of the rela- tively high nitrogen level most buds were only 1-3 mm in length when the treatments were applied.

Thirty-five plants were selected for the experi- ment. All had just reached the flowering stage and had 60-70 e x ~ a n d e d leaves. The sand was washed away from the upper part of the root system and five buds on each plant, ranging from 2 to 4 mm in length, were selected for sampling. The selected buds were marked with waterproof ink for later identification and were re-covered with sand. Five plants provided the 25 buds for the initial sample.

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2574 CAN. J . BOT. VOL. 57, 1979

TABLE 1. Effect of removal of the parent shoot on the growth, carbohydrate content, and water content of the root buds

Time from Bud growth Water contentc shoot Alcohol-soluble -

removal, h Treatment Length, mm" Dry weight, mg ~a rbohydra t e ,~ mg Milligrams per sample fresh weight

0 Control 2 . 7 5 f 0 . 0 9 ~ 24.4 2.15 73.7 74.1

24 Control 2 . 8 6 k 0 . 1 2 ~ 27.7 2.35 Shoot removed 3.14f 0 . 1 2 ~ 31.1 2.10

72 Control 3 . 2 7 k 0 . 1 8 ~ 35.3 4.35 Shoot removed 4.02f 0.30b 30.1 2.20

120 Control 4.18f0.2Ob 49.6 5.45 165.6 77.0 Shoot removed 5 .02k0.51a 32.1 3.50 170.0 84.1

NOTE: All data based on 25 root buds per treatment, i.e., 5 buds on each of five plants. "Bud lengths are mean values (k SE). Means followed by the same letter are not significantly different at the 5% level, according to Student-Neuman-KeuIs

multiple range tesl. T o t a l soluble carbohydrate per 25 buds, determined with anthrone and expressed as dextrose. ,.Water content is expressed both as milligrams per 25 buds, i.e., per sample, and as percentage of total sample fresh weight.

The remaining 30 plants were divided into two groups. Those in one group had the parent shoot removed at its junction with the root while those in the other group were left intact to serve as controls. Bud samples were taken from five plants in each group at intervals of 24, 72, and 120 h and were measured and analyzed as described above.

The results (Table 1) confirmed previous obser- vations (10) in showing that the buds on the intact plant maintained a slow, continuous growth, as in- dicated by their progressive increase in length and dry weight and the associated increase in their total water and carbohydrate content. All of these parameters were markedly affected by the removal of the parent shoot. Of particular interest was the effect on water content. In the samples taken 24 h after shoot removal, the water content of the buds from the decapitated plants was 25% greater than the controls on a per sample basis and approxi- mately 5% greater when expressed as percentage fresh weight. This effect was correlated with a small increase in mean length, which just failed to reach the 10% level of significance, and a more pronounced increase in dry weight. If, as seems probable, this dry-weight increment was due mainly to the uptake of carbohydrate then the car- bohydrate must have been rapidly metabolized as there was practically no change in total soluble carbohydrate, the level of which was markedly lower than that of the controls. Similar differences in water content and bud length were recorded in subsequent samples, except for the marked reduc- tion in dry-weight increment of buds from the shoot-removed plants as compared with the con- trols, an effect which was reflected in their rela- tively low level of soluble carbohydrate.

Experiment 2 Although the results of the previous experiment

showed that the root buds responded rapidly to the

removal of the parent shoot the data were based on average values and thus provided no information on the behavior of individual buds. A further ex- periment was, therefore, designed to examine the variation in bud response.

The plants were grown in a controlled-envi- ronment room at an alternating temperature of 25 k 1°C (day) and 15 + 1°C (night). Illumination was provided by a bank of 24 cool-white fluorescent tubes plus forty-five 40-W incandescent lamps giv- ing a 16-h photoperiod and an intensity of 550 peinsteins m-' s-I. The relative humidity was 50 + 10%. Under these conditions the degree of root bud inhibition appeared to be somewhat greater than in the previous greenhouse experiment. When the plants were selected for experimental observation after a growing period of 10 weeks none of the root buds exceeded 3 mm in length and their growth appeared to be strongly inhibited. Four plants were selected, each having six root buds, the size and location of which made them suitable for compari- son and accurate measurement. The parent shoot was removed from two of the plants at its junction with the root while the other two were left intact as controls. Using the technique described above, the length of the selected buds was then measured at 24-h intervals for the next 7 days.

The data (Fig. 1) confirmed that the buds on the intact plants were strongly inhibited, their mean length increasing by only about 0.5 mm in 7 days. That this inhibition was mediated by the parent shoot was evident from the rapid response of the buds to shoot removal. No response could be de- tected from a comparison of the 24-h samples but during the following 24-h period the growth rate of the buds from the plants with their shoots removed was significantly greater (P < 0.01) than that of the controls. Measurements recorded after 3 days, however, revealed a marked divergence in the re- sponse of the buds released from inhibition. While

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FIG. I. Effect o f shoot removal on root bud growth. (A) Shoot Experimetzt 4

removed, buds withcontinuedgrowth. (B)Shoot removed. buds The marked degree bud pro- with arrested growth. (C) Intact control plants. Data are mean duced by fully mature tissues of the parent shoot values based on 6 buds in treatments (A) and (B)and on 12 buds was consistent with the hypothesis that transpira- in treatment (C). Standard errors greater than 3 mm are indi- tion from the expanded leaves may be a factor in the cated by vertical lines. mechanism of inhibition. If this inter~retation is

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six of the buds continued to grow rapidly, their mean length increasing exponentially, the growth rate of the other six declined markedly and after 4 days was completely arrested. This experiment was later repeated with the same result. As..in the first experiment, half of the buds initially released from inhibition were apparently reinhibited. Al- though this exact agreement between the two ex- periments was presumably fortuitous, it served to emphasize an important aspect of the root bud re- sponse.

Expc)ritnen t 3 It was postulated that if water competition is a

significant factor in the mechanism of root bud in- hibition then some inhibition should be exerted not only by active meristems but also by expanded leaves which are fully mature. In an experiment designed to test this hypothesis, the plants were grown under the same conditions as in experiment 2 and 30 plants were selected for treatment after 10 weeks growth. The plants ranged from 30 to 40 cm in height and had 65 to 80 expanded leaves. They

were divided into three equal groups. Those in one A group were left intact as controls while those in

another had the shoot removed at its junction with the root. In the third treatment, the plants were decapitated 10cm below the shoot apex, leaving approximately 60 leaves which appeared to be fully mature. All the axillary buds and lateral shoots were also removed. These disbudded plants were examined daily to ensure that there was no re- growth from buds which may not have been com- pletely removed.

It was evident from the results (Table 2 and Fig. 2) that the fully mature tissues of the parent shoot

, strongly inhibited the production of new shoots from buds on the roots. Thus, whereas complete removal of the parent shoot greatly increased the number and length of emergent root shoots ( P < 0.01), removal of the immature parts of the shoot had a relatively small effect which was not significant at the 5% level. There was some evi- dence, however, that the early growth of the root

,,,: ----- buds was promoted by the latter treatment, the

o o - o - 0 c mean number of the unemerged buds being about ------- twice as great as in the other two treatments. The

correct it might be expected that the degree of inhibition would be reduced by conditions which reduce the rate of transpiration. In an experiment designed to test this hypothesis, the plants were grown initially at a light intensity of 450 peinsteins m-2 s - ~ and a temperature alternation of 20 f 1°C (day) and 15 f 1°C (night) with a 16-h photoperiod. After 10 weeks growth, 20 plants had all the imma- ture tissue of the shoot removed as in the previous experiment, leaving 50 fully mature leaves on the decapitated shoot, while the other 20 plants had their shoot cut off at its junction with the root. Half of the plants in each treatment were then trans- ferred to a growth chamber in which the relative humidity ranged between 40 and 50% and the other half to a chamber in which a constantly high humidity of 89-95% was maintained. The light in- tensity in both chambers was 270 peinsteins m-" s-I.

Increasing the humidity produced a marked in- crease in the number ( P < 0.1) and length ( P < 0.05) of root shoots on those plants from

experiment was later repeated under slightly differ- , I I I I ent conditions with essentially the same result.

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TABLE 2. The inhibition of root bud growth by mature tissues of the parent shoot

Root shoots" Root buds 2 5 mm" Treatment of --

oarent shoot Number Mean length, mm Number Mean length, mm

1. Shoot intact (control) 0 .3+0 .3b 0 . 6 2 0 . 6 b 7 . 6 2 2 . 9 a 7 .9 f 1.5a

2. All immature parts of shoot removed 2 . 2 + 1 . l b 12 .2k6 .1b 16.4+5.6a 1 0 . 0 t 0 . 9 a

3. Whole shoot removed 20.4+2.7a 8 3 . 7 f 9 . 6 a 6 . 9 k 1 .5a 11.1+2.0a

NOTE: All data are mean values ( k S E ) based on 10 plants per treatment. Means within the same column followed by the same letter are not significantly different at the 5% level, according to Student-Neuman-Keuls multiple range test; those fol- lowed by different letters are significant at the Iz, level. The treatment period was 12 days.

"The term "root shoot" denotes shoots arising from root buds which have emerged above the surface of the sand, whereas the term "root bud" refers to buds below the snrface.

FIG. 2. Seedlings of leafy spurge illustrating the effect of disbudding and complete removal of the parent shoot on root bud growth. (A) Intact control. (B) All immature tissue removed, including the main apex. growing leaves, and all lateral buds and shoots. (C) Whole shoot removed. Note that in treatment (B) the emergence of root shoots has been inhibited by the mature tissues of the parent shoot whereas numerous root shoots were produced in treatment (C). x i.

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which all immature tissue had been removed (Table 3, treatments A 1 and B1 and Fig. 3). A comparison of these data with those from the treatments in which the whole of the parent shoot had been re- moved showed that this effect of humidity could be mainly attributed to a reduction in the inhibiting influence of the fully mature tissues of the parent shoot. There was evidence, however, from a com- parison of treatments A2 and B2 (Table 3), that increased humidity also had a direct effect on root shoot growth. Complete removal of the parent shoot caused a significant reduction in dry weight of the unemerged root buds at both levels of humidity, and at the higher humidity, the increase in root shoot growth was associated with a marked reduc- tion in root bud number (P< 0.05) and mean length.

Expcrit?lol t 5 A further experiment was conducted to examine

more critically the effect of humidity on the emergence and growth of root shoots following the removal of the parent shoot. The plants were grown under the same conditions as in the previous ex- periment. After 8 weeks growth, the sand was washed from the upper part of the root system and I5 plants were selected and divided into three sets for treatment. All had a similar number of root buds ranging from approximately 1 to 3 mm in length. Since i t was thought that reduced aeration might limit the growth of the buds when evaporation from the sand was reduced by high humidity, two sets of plants had the sand which had been removed from the upper half of the pot replaced with vermiculite. Each set was then transferred to separate growth chambers. In one chamber the relative humidity varied between 30 and 50% and in the other be- tween 90 and 100%. For comparative purposes the sand from the third set of plants was replaced with sand of the same kind, i.e., 12-30 mesh, and these plants were also placed in the high humidity growth chamber. All plants were kept in the dark except for a brief daily exposure to natural light of low inten- sity during watering and the recording of shoot emergence. The temperature was 20 + 1°C. The pots continued to receive an excess of nutrient solution at 2-day intervals and the substrate was resaturated with distilled water on alternate days. The amount of water required was not recorded but only about 1 or 2 mL was required to resaturate the sand in the high humidity, indicating that there was practically no evaporation under these condi- tions.

Daily observations (Table 4) showed that the rate of shoot emergence was significantly greater

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CAN. J . BOT. VOL. 57. 1979

FIG. 3. Seedlings of leafy spurge illustrating the effect of humidity on the inhibition of root shoot production by mature tissues of the parent shoot. Both plants had all immature tissue removed from the parent shoot and were then exposed to relative humidities of 45 f 5% (A) and 92 f 3% (B) for 21 days. Note the emergence of shoots from root buds at the higher humidity. x 4.

(P< 0.01) at the higher humidity. This response, produced by the plants in the high humidity - ver- however, was restricted to the treatment in which miculite treatment were more than twice as great as the sand had been replaced with vermiculite. There at the lower humidity. There was also an approxi- was no significant effect of the increased humidity mately sixfold increase in their total length, on the rate of shoot emergence in the treatment in whereas their mean length, which provides a better which the root buds were covered with sand. When measure of the effect on elongation, was about 60% the final measurements were recorded after 14 days greater than in either of the other two treatments. (Table 5 ) , the number and dry weight of root shoots Where the roots were covered with sand, the

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TABLE 4. Effect of humidity and nature of substrate on rate of shoot emergence from root buds following renloval of the

parent shoot

Number of emerged shoots"

Days from removal of parent shoot

Treatmentb 5 6 7 8 9

1. Low humidity, vermiculite 0.2b 0.4b 1.0b 1.6b 3.0b

2. High humidity, vermiculite 2.2a 4.4a 9.4a 13.0a 19.6a

3. High humidity, sand O.Ob 0.2b l . 6 b 2.2b 4.2b

"All data are mean values based on five plants per treatment. Means in the same column followed by the same letter are not significantly different at the 5% level, according to Student-Neuman-Keuls multiple range test.

bThe treatments are fully described in the text.

number of root shoots was not significantly differ- ent from the number in the low humidity treatment but there was evidence from their final length and dry weight that, after their emergence, the high humidity had promoted their subsequent growth. Although there were no statistically significant ef- fects on growth of the root buds, the increased emergence and growth of the root shoots at the higher humidity was associated with relatively low values for the number, length, and dry weight of the unemerged buds.

Discussion The results of this investigation lend consider-

able support to the hypothesis that internal com- petition for water plays an important role in the mechanism of root bud inhibition. They also sug- gest that, in the absence of correlative inhibition, the water status of the tissues may frequently be the limiting factor which determines the rate of shoot extension.

Evidence favouring the water competition hy-

pothesis was provided, firstly, by the rapid increase in the water content of the root buds following the removal of the parent shoot. When expressed on a per bud basis, the water content increased by ap- proximately 25% within 24 h of shoot removal. However, since the length of the buds also in- creased during this period, the question arises as to whether the increased water uptake by the buds was the cause or merely one of the effects of the associated increase in growth. As previously re- marked (1 I ) , the need to distinguish between cause and effect is the main difficulty involved in the use of this approach when attempting to assess the role of nutrient competition in the mechanism of apical dominance. In the present experiment, the sus- tained growth of the root buds on the intact control plants was indicative of a relatively low degree of inhibition, as compared, for example, with the buds of experiment 2, the growth of which was almost completely arrested. It might, therefore, be ex- pected that, if water was indeed the limiting factor, the buds would respond very rapidly toany increase in the water supply. In these circumstances, even the most critical kinetic analysis may fail to distin- guish between cause and effect. The rapidity with which bud growth may respond to the release from inhibition was well illustrated by Hall and Hillman (5) who showed, by means of photographic en- largement of the axillary buds of Plzas~ol~ ls C L I I - gnr-is, that bud length increased markedly within 30 min of decapitation of the parent shoot. Al- though these workers did not speculate on the mechanism involved, an increase in the water (pressure) potential would seem to provide the most probable explanation of such a rapid re- sponse.

As in the study of apical dominance, most ex- periments on the mechanism of root bud inhibition have been designed to test the hypothesis that the inhibition is mediated directly by auxin (indoleace-

TABLE 5. Effect of humidity and nature of substrate on growth of root buds following removal of the parent shoot

Root shootsb Root buds r 10 mm

Treatmenta Number Length,' cm Dry weight, mg Number Length,' cm Dry weight, mg - -

1. Low relative humidity, 14 .4k4 .9b 45 .8 f9 .8b 98.8k38b 20 .8k4 .7a 55.4+ 16a 59.0f 18a vermiculite 5 .9k0 .98 2 . 5 k 0 . 2 7

2. High relative humidity, 31 . O f 5. l a 292+66.4a 253 k 46a 9 .8f 2.3a 33.2f9 .8a 28 .8 f9 .2a vermiculite 9 . 1 f 0 . 5 2 .3k0 .31

3. High relative humidity, 12 .6 f3 .6b 96.8f27b 120+30b 14 .2k 4.7a 32 .6 f7 .1a 33.6f11a sand 6 . 3 k 1 . 7 1 .8k0 .07

NOTE: All data are mean values (f SE) based on five plants per.treatment. Means in the same column followed by the same letter are not significantly dif- ferent at the 5% level, according to Student-Neuman-Keuls multlple range test.

OThe treatments are fully described in the text. bSee footnote, Table 2, for definition of "root-shoot." <First value, total length per plant; second value, mean length per plant.

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2580 CAN. J . BOT. VOL. 37. 1979

tic acid) produced in the parent shoot (2 ,4 , 15, 16). In support of this hypothesis it was shown that, when the shoot was removed, the growth of the root buds could be at least partially suppressed by applying auxin to the cut surface of the stem o r to the base of the root. However, similar evidence of the role of auxin in apical dominance has been subject to considerable criticism (14) while re- cent data on endogenous auxin levels in lateral buds have failed to substantiate the direct inhibition hy- pothesis (6). 'The present observations on the in- hibition produced by fully mature tissues of the parent shoot are also inconsistent with the concept of an auxin-induced inhibition. It has been well established that the principal sites of auxin synthe- sis are the young growing leaves a t the shoot apex yet the removal of all immature tissue was much less effective than complete shoot removal in re- leasing the root buds from inhibition. This result appears to be at variance with observations re- ported by Eliasson (3) who found that a similar disbudding treatment of young poplar plants (Populrrs ti.ct77~rlcr L.) induced the production of "suckers" on the roots within 2-4 weeks. Elias- son, however, gives no information on the size o r stage of development of the root buds when the treatment was applied and thus it is not clear whether the treatment was affecting the initiation of buds o r their subsequent elongation. Also, in con- trast to the present observations on leafy spurge, there was evidence that the growth of the root buds was dependent on a continuous supply of fresh assimilate from the parent shoot. Removal of the shoot below the oldest leaf completely prevented root bud development. Thus , the promotion of suckering induced by disbudding may have been due, at least partly, to the elimination of shoot growth and to the consequent increase in the amount of carbohydrate available for translocation to the roots.

In the present investigation, the most convincing evidence in favour of the water competition hy- pothesis was provided by the effect of humidity on root bud growth. It was shown that, under the experimental conditions, increasing the humidity from approximately 50 to 90% released the root buds from the correlative inhibition exerted by mature tissues of the parent shoot and also significantly increased their rate of elongation in plants from which the parent shoot had been re- moved. In the latter treatment, the increased humidity was extremely effective in promoting shoot emergence when the root buds were covered with vermiculite but had no significant effect when the buds were covered with sand. It seems probable

that the wet sand. which was prevented from drying by the high humidity, inhibited root bud growth by reducing the oxygen supply. Further investigations are required to test this hypothesis and to assess the importance of bud aeration as a limiting factor under both growth chamber and field conditions.

The apparent reduction of correlative inhibition by high humidity is in good agreement with results obtained in previous experiments on apical domi- nance which showed that when plants are well supplied with nitrogen and carbohydrate the provi- sion of a high humidity is sufficient to release the lateral buds from inhibition (11). A search of the earlier literature showed that the observed effect of humidity in promoting shoot extension has also been previous1 y recorded (see ref. 8). Of particular interest is the work of Wiesner (17) who showed, in experiments with Azrrlcrr it7riic.rr and various other species. that when the plants were grown a t humidities ranging from approximately 50 to 80% they formed only short shoots. whereas humidities of 90 to 97% induced the formation of long shoots and considerably increased the duration of apical growth. Wiesner (17) also showed that high humid- ity induced internode elongation in vegetative rosettes of Capsellcr b~r r sa pcrstor-is and in several other rosette-forming species. He noted that since the plants were grown at a high light intensity the induction of shoot extension could not be attributed to etiolation. Although the factors controlling cell and shoot elongation have been the subject of nu- merous investigations in recent years, most of these studies have been conducted with isolated stem sections floating in test solutions. This tech- nique is well suited for a critical analysis of the factors regulating cell extension in isolated tissues but would seem to be less reliable as a means of investigating the natural mechanism of contru!. This point was recently emphasized by Cleland ( I ) who concluded, from theoretical considerations, that although auxin, by virtue of its wall-loosening effect, may be the factor which controls the rate of cell extension in floating sections, the water poten- tial of the tissues, a s expressed in the degree of cell turgor, is the factor most likely to limit stem elon- gation in the intact plant. This view is clearly well supported by the present observations.

In conclusion, it would seem that the results of this investigation have provided sufficient evidence to justify a more critical assessment of the role of water competition in the mechanism of root bud inhibition. Further investigations will be designed, firstly, to examine under controlled conditions the quantitative relationship between the degree of root bud inhibition and such factors a s xylem water

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potential and the rate of transpiration; and sec- 8. Jos-r. L. 1907. Lectures on plant physiology. (English

ondly, to test the general validity of the water com- translation - . by R. J . Harvey Gibson.) Clarendon Press. Ox- tord. petition hypothesis by conducting similar investi- 9, M t l N T Y R t _ , G , 1969 Apical dominilnce i n the of

gations with other root bud species. Agropy1.or7 repe~rs. Evidence of competition for carbohy- drate as afactor in the mechanism of inhibition. Can. J. Bot.

Acknowledgements 47: I 189- I 197.

1 wish to thank M ~ ~ . ~ ~ ~ i l ~ ~ mit dl and M ~ . ~~h~ 10. M t l N ' r Y n ~ G. 1. 1972. Developmental studies on Giplror- Bitr ~,.srrltr. The influence of the nitrogen supply on the cor-

Mitton for excellent technical assistance and Dr. relative inhibition of root bud activity. Can. J. Bot. 50: J. R. Hay for his critical readinn of the manuscript. 949-956. - I. CLLLAND. R. E. 1977. The control of cell enlargement.

Symp. Soc. Exp. Biol. 31: 101-1 15. 2. ELIASSON, L. 1961. The influence of growth substances on

the formation of shoots from aspen roots. Physiol. Plant. 14: 150-157.

3. ELIASSON. L. 1972. Growth regulators in Pop~rlus rro~lrrlo. IV. Apical dominance and suckering in young plants. Physiol. Plant. 25: 263-267.

4. F A R M E R . R. E. 1962. Aspen root sucker formation and apical dominance. For. Sci. 8: 403-410.

5. HALL, S. M., and J. R. H I L L M A N . 1975. Corl.elative inhibi- tion of lateral bud growth in Plltr.scol~rs urr1gtrri.s L. Timing of bud growth following decapitation. Planta. 123: 137- 143.

6. H I L L M A N , J. R.. V. B. MATH. and G. C. MEDLOW. 1977. Apical dominance and the levels of indole acetic acid in P1lrrseol~r.s lateral buds. Planta, 134: 191-193.

7. HOAGLAND, D. R. , and D. I . ARNON. 1939. The water culture method for growing plants without soil. Univ. CA. Agric. Exp. Stn. Circ. No. 347.

11. M C I N - T Y R ~ , G. I. 1977. The role of nutrition in apical domi- nance. Symp. Soc. Exp. Biol. 31: 251-273.

12. MCINTYRE. G. I., and J. R. HUNTER. 1975. Some effects of the nitrogen supply on growth and development of Ci~:rirrr,r rrrce~zse. Can. J . Bot. 53: 3012-3021.

13. PETERSON, R. L. 1975. The initiation and development of root buds. It7 The development and functions of roots. Etliretl hy J. G. Torrey and D. T . Clarkson. Academic Press, London. pp. 125-161.

14. PHILLIPS, I. D. J. 1969. Apical dominance. It1 The physiol- ogy of plant growth and development. Edirctl By M. B. Wilkins. McGraw-Hill, London. pp. 165-202.

15. STERRETT, J. P.. and W. E. CHAPPELL. 1967. The effect of auxin on suckering in black locust. Weeds. 15: 323-326.

16. VOGT. A. R.. and G. S . Cox. 1970. Evidence for the hor- monal control of stump sprouting by oak. For. Sci. 16: 165-17 1.

17. WIESNER. J. 1888. Der absteigende Wasserstrom und des- sen physiologische. Bedeutung. Ber. Deutsch. Bot. Ges. 5: 24-30.

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