studies in the morphogenesis of leaves. ii. the area, cell size and cell number of leaves of ipomoea...

Studies in the Morphogenesis of Leaves. II. The Area, Cell Size and Cell Number of Leaves ofIpomoea in Relation to their Position on the ShootAuthor(s): Eric AShbySource: New Phytologist, Vol. 47, No. 2 (Oct., 1948), pp. 177-195Published by: Wiley on behalf of the New Phytologist TrustStable URL: http://www.jstor.org/stable/2429073 .

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[ I77 I

STUDIES IN THE MORPHOGENESIS OF LEAVES

II. THE AREA, CELL SIZE AND CELL NUMBER OF LEAVES OF IPOMOEA IN RELATION TO

THEIR POSITION ON THE SHOOT

BY ERIC ASHBY Department of Botany, The University, Manchester

(With I3 figures in the text)

CONTENTS PAGE

i. Introduction . . . . . . 177 2. Summary of experimental procedure . . I79 3. Details of experimental procedure . . I80

Material . . . . . . . i8o Cultivation and treatments . . .I8I

Collection of leaves . . . . . I8I Examination of leaves . . . . I8I

4. Summary of data . . . . . . I83 Morphology of leaves on control plants . I83 Morphology of leaves from treated plants . I85

5. Discussion . . . . . . .. I88 6. Summary . . . . . . . I9I References . . . . . . . 192

Appendices . . . . . . . 192

I. INTRODUCTION

In Maximov's book, The Plant in Relation to Water (I929), there is an important chapter on the effects of water supply on leaf structure. Two views set out in this chapter are still generally accepted, namely (i) that some xeromorphic characters, especially those depending on cell size in leaves, can be induced by subjecting plants to water shortage; and (ii) that 'the anatomical structure of individual leaves on one and the same shoot is, so to speak, a function of their distance from the root system' (Maximov, 1929, p. 332). This second view follows from the work of Zalenski (I904), who found. on shoots of Nicotiana rustica, Dactylis glomerata, and other plants a consistent decrease in the size of epidermal cells from the lower to the upper leaves, accompanied- by other signs of increasing xeromorphy. This increasing xeromorphy from the lower to the upper leaves of a shoot might be explained on either of two simple hypotheses: the upper leaves may be produced under comparatively drier climatic conditions than the lower leaves; or the upper leaves may be deprived of water during their growth by the lower leaves. Zalenski favoured the second of these two hypotheses and it is now widely believed that the gradient of cell size found in succeeding leaves on a shoot is the result of competition for water among the leaves, whereby the upper leaves are deprived of some water by the lower leaves. Such observations as there are on the water content, osmotic pressure, and



I78 ERIC ASHBY suction force of leaves at different levels of insertion on the shoot are consistent with this hypothesis; but the most convincing evidence comes from a well-known experiment by Alexandrov, Alexandrov & Timofeev (I92I). These authors grew plants of Bryonia dioica and Ipomoea purpurea as single long shoots, by removing lateral buds. In one particular example, when forty leaves had developed, they removed all the leaves. Ex- amination of these leaves revealed the expected trend from leaf to leaf in cell size (measured in this instance as stomatal frequency). Subsequently the authors examined, from the same shoot, succeeding leaves (leaf 4I, 42, etc.) which had developed in the absence of the first forty leaves. These leaves had cells no smaller than the cells in the twentieth leaf.

450

400

V 350

E 300

0

E EE 250

'4200

150

100 I- l l l 1g 0 10 20 30 40 50 60 70 80

Leaves, numbered from base

Fig. i. Stomata per mm.2 on the abaxial surface of Ipomoea purpurea (ordinates), and leaves numbered from the base (abscissae). Leaves i-4o were removed. Data from Alexandrov et al. (I92I), table 3.

The data are illustrated in Fig. i. The authors concluded that the sudden change in cell size of upper leaves is due simply to the greater quantity of water available when the lower leaves have been removed, and they added that the change is less marked when the plants are grown in shade than when they are grown in sun.

There is no doubt that the facts, as described by Zalenski and many workers after him, are correct; but there is considerable doubt as to the interpretation commonly put upon these facts. In the first place the data of Alexandrov, Alexandrov & Timofeev, which are the mainstay of the interpretation, are misleading. These authors had no controls with which to compare their treated plants, and there is no doubt whatever that the striking change in cell size which follows cutting (e.g. the difference between the fortieth and the forty-first leaf in Fig. i) is due in part to the fact that the upper leaves were cut off before



Studies in the morphogenesis of leaves 179 they were fully expanded: it is leaf 40, not leaf 4I, which is abnormal. In the second place, it has been shown by Goodwin (I937) and others that the growth of a young leaf is inhibited by auxin diffusing from the leaf below it; and if the leaf below is removed, the young leaf is released from this inhibition and grows more rapidly. It is possible, therefore, that the effects of removing lower leaves on the cell size of succeeding leaves above them may be due to interference with auxin gradients, and not to the release of additional water for the upper leaves. In the third place, there are no data from plant physiology to justify the belief that the lower leaves on a shoot do deprive the upper leaves of water.

n n n n

n-I n-I 'U~~~~ n-I n-I

n-2n2

I/ n-2 ~~~~~~~n-2 n-3

4 4

3 4 4 3

2 2

2 21

Treatment :-Control A B C

Fig. 2. Diagram to illustrate the four treatments applied to Ipomoea. For explanation see text, ? 2.

On these grounds the author has re-examined the question of the relation between cell size in leaves and their level of insertion on a shoot. This re-examination is a necessary prelude to the author's studies in morphogenesis of leaves, the purpose of which was fully described in the first paper of this series (Ashby, I948).

2. SUMMARY OF EXPERIMENTAL PROCEDURE

The experiments were carried out to analyse the effects of the lower leaves of a shoot on the morphology of the upper leaves, and in particular to examine the hypothesis that these effects are due to competition for water among the leaves.

Plants consisting of a single shoot were grown under dry and under damp conditions, and were subjected to the following four treatments which are illustrated in Fig. 2:

(i) Control. All leaves left on the shoot (Fig. 2, Control).



i8o ERIC ASHBY

(ii) Treatment A. Leaves i to n were removed. Morphological studies were made on leaves (n + i), (n + 2), (n + 3), etc. (Fig. 2A).

(iii) Treatment B. Leaves i to (n -3) were removed. Morphological studies were made on leaves (n + i), (n + 2), (n + 3), etc. and on leaf (n - 2) (Fig. 2B).

(iv) Treatment C. Leaves (n -2) to n were removed. Morphological studies were made on leaves (n + i), (n + 2), (n + 3), etc. and on leaf (n-3) (Fig. 2C).

These treatments were applied on the hypothesis that: (i) if, on the one hand, water supply is the determining factor in leaf morphology, then leaf (n + i) should respond to treatment B in the same way as to treatment A, and it should respond to treatment C in the same way as to the controls; (ii) if, on the other hand, a leaf hormone, or some similar influence, is the determining factor in leaf morphology, then leaf (n + i) should respond to treatment B in the same way as to the controls, and it should respond to treatment C in the same way as to treatment A. Furthermore, if water supply is the controlling factor, the morphological response should be more pronounced under dry conditions than under wet conditions.

3. DETAILS OF EXPERIMENTAL PROCEDURE

Material Three garden varieties of Ipomoea were used:

(i) Ipomoea caerulea Koen. ex Roxb, carrying normal dominant genes for leaf shape (Imai, I930, fig. i); to be called strain x.

(ii) I. hederacea Jacq. var. grandiflora Hort. carrying normal dominant genes for leaf shape, but differing from strain x for other genes; to be called strain y.

(iii) I. hederacea Jacq. 'purple stripe', carrying the incompletely recessive gene (or genes) 'cordate' (Imai, 1930, fig. 6); to be called strain z.

All three varieties are herbaceous annuals, and are twining plants.

Cultivation and treatments Seed was sown on I4 April I947, in 5 in. pots in a 'John Innes compost'. Eighty plants

of each variety were raised, i.e. 240 plants altogether, one in each pot. Immediately after germination, and without transplanting, forty plants of each variety were put into one glasshouse (house i) and forty into another glasshouse (house 2). The two glasshouses lie parallel to each other and IO ft. apart, and they receive the same amount of light. Both houses received the same amount of heating, though they were not always at the same temperature. House i was kept as dry as possible and the plants in it were watered only sufficiently to maintain normal growth. The leaves frequently wilted for a short time during the day, and once during the experiment, when there were about six leaves on the plants, the soil in the pots in house I became so dry that the pots had to be soaked out in water. House 2, in contrast, was kept continually moist. A hot water-bath maintained the air at a high relative humidity, and the plants were watered every day.

In both houses the potted plants were labelled according to the treatments they were to receive, and were then arranged entirely at random, 9 in. apart, on the east side of each house. After thinning, there was a uniform population of seedlings, and by i i May I947, most of the plants possessed one or two leaves. On i i May, and regularly thereafter, the dimensions of every leaf were measured. The measurements taken are illustrated by the



Studies in the morphogenesis of leaves i8i broken lines in Fig. 3. The purpose of these measurements was to enable any changes in shape during enlargement to be followed, and so that there could be no doubt as to whether any particular leaf was fully expanded or not. All lateral buds were removed as they appeared. For the greater part of the experiment the plants in house 2 had significantly longer internodes, but did not have significantly more nodes, than the plants in house i.

On 6 and 7 June, when plants in both houses had produced about ten leaves, the treatments illustrated in Fig. 2 were applied. The leaves were cut at the base of the petiole with surgical scissors, and preserved for examination. Since, at the time of operation, different plants had different numbers of nodes, the nth leaf referred to in Fig. 2 and ?2 was not always the tenth leaf: it was the topmost leaf which had emerged from the terminal bud, and it varied from the ninth to the eleventh leaf.

After the treatment had been applied, the plants continued to grow and produced seven to eight new leaves on the single shoot. The dimensions of these leaves were regularly measured. On 26 and 27 June these leaves, and the leaves on the control plants, were removed for examination.

Collection of leaves a

When leaves were cut from the plants for treatments A, B and C, the following operations were carried out:

(i) Small cuts were made on the margin of each leaf / according to a code system, so that later on each leaf ' ' ' c could be assigned to its node of insertion on the shoot. / : h , d

(ii) The leaves were then cut from the shoot and immediately laid under a sheet of -glass on ferro-prussiate g blue-print paper, in order to provide a permanent record i of the size and shape of the leaves. f/

(iii) The leaves cut from each shoot were then wrapped l in a roll, bound with soft wire (tin-lead alloy fuse wire was used), labelled with the number of the plant, and preserved in a mixture of alcohol, water, and glycerine in the proportions 2:2:I.

(iv) At the end of the experiment, all the leaves from Fig. 3. Diagram to illustrate control plants, and the leaves which had appeared since the measurements taken on the operations on treated plants, were coded as described growingleaves (broken lines),

and the regions of the leaf in (i) above, cut from the plants, photographed on blue- sampled for cell size (a, b, print paper, and preserved in the alcohol-water-glycerine ..., g, h). mixture for examination.

Examination of leaves From the data collected on growth rates and sizes of leaves while still on the plants, it

was possible to decide which of the preserved leaves were not fully expanded; data for area and cell size of these leaves were rejected. On all fully expanded leaves the following observations were made: (i) leaf area, (ii) number of epidermal cells per mm.2 on the adaxial surface of the leaf blade, (iii) total number of epidermal cells on the adaxial surface of the leaf blade, (iv) leaf shape. The last of these measurements, leaf shape, is not



i82 ERIC ASHBY considered in the present paper; it is to be the subject of the third contribution to this series. Details of the other observations are as follows:

(i) Leaf area. Leaf areas were measured from the blue-print records, with a plani- meter. Each measurement is the mean of two readings.

(ii) Number of epidermal cells per mm.2 Estimates of cell size were made from the adaxial epidermis, because it is easier to remove than the abaxial epidermis, and has fewer stomata. Pieces of leaf about I cm.2 in area were cut from the leaf, washed in 20 % alcohol and then in water, and left in Io % nitric acid for 3-5 min. This treatment softens the tissues so that the epidermis can be stripped off with fine forceps. The strips of epidermis were transferred successively to IO, 30 and 50% alcohol, stained in Ehrlich's haematoxylin for about 3 min., washed in 50 % alcohol, left for 3 min. in a mixture of equal parts of 50 % alcohol and glycerine, and mounted in glycerine jelly.

Cell counts from epidermal cells prepared in this way were compared with counts from cells on the same leaf blade mounted without any treatment, in order to determine whether the treatment led to a measurable distortion in cell size. The value of 't' for a null hypothesis was o0Io2, and of P was o-8. The probability of any error from this source may therefore be neglected.

Cell counts were made with the aid of an Ehrlich eyepiece, with an adjustable square field. A 6 in. objective was used, and the area of the square field was 0o0324 mm.2 Since the epidermal cell walls are convoluted, it is difficult to estimate the number of incomplete cells in the field. Accordingly, only whole cells were counted, and a correction was added for incomplete cells. This correction was made by calculating the regression of half the number of incomplete cells on the square root of the number of whole cells in the field. The reason this form of regression was used is given in the Appendix. The regression is approximately linear, with a coefficient of I*649I + O I005.

Cell counts were made from different regions of the sam'e leaf blade in order to determine the reliability of estimates from small areas of leaf surface. Ten samples were taken from each of eight regions of the leaf blade (the regions marked a, b, c, . . ., h in Fig. 3). The results for one cordate leaf (i) and one lobed leaf (2) are summarized in Table I.

Table I. Variance ratio: between regions/within regions (F), and its significance (S) for epidermal cell counts on eight regions of the leaf blade of Ipomoea; also mean cell counts (m) and semi-fidzucial limits at the 5 % level for means of ten readings (L)

Leaf F S m L

(i) All regions I497 I 7 56 I72

(2) All regions 8-043 S 21-64 IP55 IAll regions except tip, and base of sinus 2-I42 20-58 I.58

It is concluded from this table that (i) apart from small local groups of cells at the tip of the blade and the base of the sinus, there is no significant variance from place to place on the epidermis; and (ii) cell counts in ten fields from one region of the epidermis provide a sufficiently precise estimate of mean cell size over the whole epidermis. The differences actually encountered in the experiment were many times greater than the fiducial limits of the means. Therefore, the final routine adopted for estimates of the number of cells per mm.2 was to sample from each leaf an area corresponding to e in Fig. 3 and to find the mean of ten random counts of cell number, excluding stomata, in this area.



Studies in the morphogenesis of leaves I83 (iii) Total number of epidermal cells. Since there is no significant variation in cell size

from place to place on a fully expanded leaf, except at the extreme tip and edges, the product of area and the number of cells per unit area is an estimate of the total number of cells in the epidermis. Such an estimate is, of course, liable to errors; but the fiducial limits of mean cell counts are not high, and the errors are in any case separated out in the analysis of variance. The data may, therefore, be used with confidence. A significant change in the total number of epidermal cells might be due to a change in the number of cell divisions, or to a change in the size of the leaf primordium; it is not possible to distinguish in these data which of these two causes is the more important.

(iv) Leaf shape. The technique for measuring leaf shape will be discussed in the third contribution to this series.

4. SUMMARY OF DATA

The variables to be considered are: (i) three genotypes of Ipomoea, (ii) growing under two conditions of water supply (dry and wet), (iii) subjected to four operational treatments (control, A, B, C). There were ten plants under each of these twenty-four conditions. For each of the twenty-four conditions there are data for the area, cell size, cell number, and shape of each leaf at each node. Clearly the data cannot be published in full. The present section contains a summary of the data sufficient for a discussion of their bearing on the subject of this paper; more data will be found in the Appendix.

Morphology of leaves on control plants The changes in leaf morphology from node to node on control plants are summarized

in Figs. 4-I 2. All the measurements entered on the graphs are for leaves which were fully expanded. The circles represent data from house i (dry) and the crosses represent data from house 2 (wet). At this stage attention is drawn to the following features of the graphs; other features are discussed later.

Leaf area (Figs. 4-6). In strains x and z the second leaf had the greatest area and thereafter there was a steady fall in area from leaf to leaf up to about the eighth or ninth node. The first leaf was significantly smaller than the second. In strain y there was the same relation between leaf area and node of insertion, except that the second leaf was no larger than the first leaf. For all three strains, leaf areas under dry conditions were strikingly less than leaf areas under wet conditions. The analysis of variance merely confirms these observations (Table 2). In strain x there was a significant rise in leaf area between the sixth and the tenth leaves under dry conditions; strain z showed a similar rise, but it is significant only at the io% level. These rises coincided with the period following heavy watering of the desiccated plants, after the sixth leaf had begun to enlarge (men- tioned on p. i8o).

Table 2. Degrees of freedom (D.F.), variance ratio (F), and significance at the i % level (S) for leaf area of three strains of Ipomoea grown in dry and wet conditions

Strain x Strain y Strain z Cause of variance_

D.F. F S D.F. F S D.F. F S

Node of insertion I2 I 354 S I4 7.2I S I I IO-34 S House (wet or dry) I 63 52 S I I6-23 S I 93 75 S Interaction 12 1:49 14 I-87 - I 4.98 S Error I48 - - 99 _ - I03 --



I84 ERIC ASHBY Number of cells per mm.2 (Figs. 7-9). All three strains showed a marked rise in the

mean number of epidermal cells per mm.2 from leaf to leaf over the first few nodes. In strain z this rise continued up to the eleventh node. In strains x and y there was no significant increase in the number of cells per mm.2 after the fifth or sixth leaf. The

60 Strain z

Strain x 5

50

504

4g Strain y 45 e E

3500 Sta ix 20 E 3000 - 35

I I 30o 1 , .25 X

20 660 20 10~~~~~~~~~~~~~~~~0 15 b, J 10 ' ' ' 1

2 4 6 8 10 12 14 16 2 4 6 8 10 12 14 16 2 4 6 8 10 12 14 16 18 Leaves, numbered from base Leaves, numbered from base Leaves, numbered from base

Fig. 47 Fig. 8. Fig. 6. Figs. 4-6. Mean leaf area on control plants, plotted against position of insertion on the shoot; x

wet conditions; 0 --- 0, dry conditions. Mean leaf area of upper leaves on plants after treatment A; +-- -,- - wet conditions; .@ dry conditions.

Strafn z 1500 A 1400-

1300 Strain yd1300 / c

a 1200- Strain x 1200 E 1200 1

1100- O 1100- 110

E21000- / 1000- d/ ' / CL goo. \18i ~ / 900. 4

90

conditions, nowthtndn th veyhgl+infcn ifeecsi efae.Ti

indication from the0 graphs is confirmed by theanalysis (Table)00 / ~700 I - 700 700 E700 /

600- I-~~~~~o '2 ,~~~~~~~~~~~~~~ ~~600/ 600-

E 500- Soo50 z/

400 400o 400/

300 J__ __ _ _300__ __ _ _ 2 4 6 8 1012 1416 300 '00

Leave, nuberedfrombase2 4 6 8 10 12 14 16 2 4 6 8 10 12 14 16 Leaves, numbered from base ~~Leaves, numbered from base Leaves, numbered from base

Fig. 7. Fig. 8. Fig. 9. Figs. 7-9. Number of epidermnal cells per MM.2 on leaves of control plants, plotted against position of insertion

on the shoot; x ~x, wet conditions; 0 - -- 0, dry conditions. Number of epidermnal cells per MM.2 on upper leaves of plants after treatment A; +.- - -+, wet conditions;

0--,dry conditions.

unexpected and 'important feature of these data is that there is no striking difference in cell size between the leaves grown under dry conditions and those grown under wet conditions, notwithstanding the very highly significant differences in leaf area. This indication from the graphs is confirmed by the analysis (Table 3).



Studies in the morphogenesis of leaves I85

Total number of cells in epidermis (Figs. IO-I2). In all three strains the total number of cells in the adaxial epidermis increased from the first to the fourth or fifth leaf; thereafter there was a gradual decline in cell number from leaf to leaf. There was an evident difference between the cell numbers, in leaves at corresponding nodes, grown in dry and wet conditions; and it is clear that in these varieties of Ipomoea the effect of water supply on leaf area operates through cell number and not through cell size. Under dry conditions strains x and z showed a significant rise of cell number in one or two successive leaves from the eighth leaf; these rises corresponded to the heavy watering of the desiccated plants after the sixth leaf had begun to enlarge. They are accompanied by increases in area, but the increases in area reached significance at an earlier leaf than the increases in cell number. The analysis of variance is summarized in Table 4.

55 x106 Strain z

x10 t 445.

Strain x 4-0 E

E 410 6 8 16 Strain y 3

Fis IO s-I2. s Celnme neiemso evscfcnrlplns lte gis oiino rsrno

E ~ ~~ ~~~ 0 20o - ~ c ~ ~ ~ ~ ~ ~~~~ ~~~~~~~~~~~~~~~~~~~0-

o . - 60 C,- ii.

2 4 6 8 10 12 14 16 2 4 6 8 10 12 14 16 2 4 6 8 10 12 14 16 18 Leaves, nambered from base Leaves, numbered from base Leaves, numbered from base

Fig. io. Fig. i[ . Fig. I 2. Figs. I10- I2. Cell number in epidermis on leaves of control plants, plotted against position of insertion on

the shoot; x x c wet conditions; 0--- O,dry conditions. Cell number in epidermis from upper leaves on plants after treatment A; + - - +, wet conditions; * - -*, dry conditions.

Two conclusions from these measurements on the control plants have a bearing on the problems set out in ?2, p. I80. (i) The differences in water supply to plants in the two glasshouses were associated with significant differences in area and in number of epidermal cells. For the areas of leaves in strain z there was a significant interaction of height of insertion and water supply. But epidermal cell size, although it decreased significantly with node of insertion, was not significantly affected by water supply. This is an unexpected result, especially in view of the fact that in other plants, and under other conditions, the cell size of leaves is sensitive to water supply. (ii) Beside the familiar gradient of decreasing cell size from leaf to leaf, which is significant in all three strains, there is also a gradient from leaf to leaf in total number of cells and in leaf area.

Morphology of leaves from treated plants Treatment A (Fig. 2). Attention is directed again to Figs. 4-I2. The circles and crosses

joined by alternate dots and dashes represent the mean dimensions of leaves (n + i) (n ? 2), (n + 3), etc., on plants which had received treatment A. These leaves developeL with no older leaves below them. The following features are noteworthy:

New Phytol. 47, 2 I 3



i86 ERIC ASHBY

(i) In all three strains the area of leaf (n + i) was considerably larger than the area of the fully expanded leaf (n + i) in the control plants. The effect is significant in both wet and dry conditions, and it is more marked in dry conditions.

(ii) There was a difference in the degree of response to treatment among the strains: strain y responded less than strains x and z.

Table 3. Degrees of freedom (D.F.), variance ratio (F), and significance at the i % level (S) for cells per mm.2 in the adaxial epidermis of three strains of Ipomoea grown in dry and wet conditions.

Owing to a lack of symmetry in the data from the two houses, the interaction variance cannot be interpreted with confidence, and is omitted; but there was no significant interaction when symmetrical sets of data from the two houses were analysed.

Cause of variance Strain x Strain y Strain z


Node of insertion II 5 79 5 14 I7.2I S II 83-68 S House (wet or dry) I 0-20 - 0o-3 - I 380 -

Error 152 - - 104 103

Table 4. Degrees of freedom (D.F.), variance ratio (F), and szgnificance at the i % level (S) for total number of cells in the adaxial epidermis of three strains of Ipomoea grown in dry and wet conditions.

Owing to a lack of symmetry in the data from the two houses, the interaction variance cannot be interpreted with confidence, and is omitted

Strain x Strain y Strain x Cause of variance _


Node of insertion I2 9 34 S 14 5 45 S II io-68 S House (wet or dry) I 72.2 S I 31.7 S I 69-6 S Error* 206 - - 175 151

* Since cell number does not change after a leaf is about 3 cm. long, fewer leaves had to be rejected from this analysis on account of their immaturity; this accounts for the greater number of degrees of freedom in Table 4 compared wvith Table 3.

(iii) The curves for area of leaves (n + i), (n + 2), (n + 3), etc., fall very steeply in all three varieties, much more steeply than the curves of area of the corresponding control leaves in this region of the shoot. As a consequence, leaf (n + 4) in all varieties, although it had only three young leaves beneath it, was not significantly larger than the control leaf, which had about thirteen leaves beneath it. This is not a result which would be expected from the hypothesis that the sole effect of lower leaves is related to competition for water.

(iv) The cell size of leaves (n + i), (n+2), (n + 3), etc., is significantly greater if the leaves below have been cut off; but the increase in cell size is not as great as would be expected on the hypothesis that these leaves have available the total water supply from the roots; and from leaf (n + i) to (n + 3) there is an unexpectedly rapid drop in cell size. Furthermore, the response of cell size in leaf (n + i) is about the same whether conditions are wet or dry.

(v) The removal of leaves i to n had a strikingly significant effect on total cell number in the epidermis of leaves (n + i), (n+2), (n + 3), etc., except in strain y, where the effect was small. Since leaf (n + i) was on an average about o'8 cmn. long when leaves i to n were




removed, this indicates that cell division can be stimulated at a relatively late stage of leaf production. The data for total cell number in leaves (n + i), (n + 2), (n + 3), etc., in common with the data for leaf area and cell size, show a very rapid recovery to the values of the controls; for instance, in strain x, leaf (n + 4) from treated plants, with three leaves below it, has about the same number of cells as leaf (n +4) from control plants, with thirteen leaves below it. In all three strains the response of cell number to treatment was greater under wet than under dry conditions.

Treatments B and C (Fig. 2). These treatments were designed to test the simple hypothesis that the lower leaves affect the morphology of the upper leaves by virtue of their competitive effect on the water supply. The results are set out in Table 5. The predictions made in ? 2 (p. i 8o) are now considered in the light of Table 5. More complete data are given in the Appendix (p. I95).

Table 5. Response of area, cell size, and cell number in leaf (n + i) from three strains of Ipomoea to treatments A, B and C

Response of leaf (n + I) = R, mean difference = D, probability of significance = P. Responses in brackets are significant at the 5 % level only. The analysis does not distinguish between responses under wet and dry conditions. Data in Appendix III.

Strain x Strain y Strain z Treat- -_ _ _ _

ment Area Cells Cell Area Cells Cell Area Cells Cell (cm.2) Area no. (cm.2) Area no. (cm.2) Area no.

A. R More Less More More Less (More) More Less More D 29-7 328 I.7 8-4 259 0-52 28.7 5II I *68 P OOOi O-OOI OOOI OOI 001 < 0.05 O-OOI OOOI 001

B. R None None None None None None None (I8ess) None D - 84 -

P 0-4 04 02 0-4 o8 0O2 o0I < 0.05 0-5

C. R More Less (More) More Less More None Less None D 6-8 I27 0-4 IO?3 I 94 o-68 276 P OOOi < O-O 0I05 s O-OO 0I04 0o03 o06 0o03 o6

In treatment B all but the uppermost leaves (n -2), (n - i), and n, were removed from the shoot (Fig. 2B). Therefore the development of leaves (n + i), (n + 2), etc., took place free from competition from the adult leaves below, but not free from the effects of the three young leaves immediately below. If the morphology of upper leaves is affected principally by the competition for water from leaves below, then the response to treatment B should be similar to (though perhaps less than) the response to treatment A. Table 5 shows that this is not the case. Except for one set of oJbservations (cell size in strain z, which is significantly different at the 5 % level), the morphology of leaves after treatment B is the same as that of leaves on control plants.

In treatment C only the three uppermost leaves (n -2), (n - i), and n, were removed from the shoot (Fig. 2C). Therefore the development of leaves (n + i), (n + 2), etc., took place in the presence of all adult leaves below, and free only from the influence of the three young leaves immediately below. If the morphology of upper leaves is affected principally by the competition for water from leaves below, then there should be no significant response to treatment C. Table 5 shows that this, too, is not the case: there were significant responses to treatment C, except for two sets of observations with

I3-2



i88 ERIC ASHBY

strain z, which showed no response. Apart from this inconsistency in strain z, treatment C produced the same sort of responses as treatment A; indeed in one strain (strain y) even the mean differences between observations on leaf (n + i) from treated and control plants are no less for treatment C than for treatment A. The general conclusion to be drawn from Table 5 is that the area, cell size, and cell number of a developing leaf are greatly modified by the influence of the immature leaves immediately below, but are not significantly modified by the influence of as many as 8-io adult leaves below.

An analysis of the response of the lowest leaf (n -2) left on the plant in treatment B reveals another relevant point. This leaf had already enlarged to about half its normal size when the leaves below it were cut off, yet it responded significantly to the operation. The response was, as would be expected, confined to cell size and area; there was no response in total cell number. The evidence is summarized in Table 6. The result indicates that the final cell size and consequently leaf area, can still be influenced even when the leaf has reached half its normal adult size, while cell number can no longer be influenced at this stage.

Table 6. Response of leaf (n -2) to treatment B (see Fig. 2)

Area Cells Cell Strain (cm.2) Area no.

x Response More Less None Mean difference i6z2 336 P 0-00I 0-001 O*I

y Response None Less None Mean difference 238 P o-o8 o ooI o-6

z Response More Less None Mean difference I5-9 586 P 0I 0-001 0I4

When treatment C was applied, the topmost leaf left on the shoot, (n - 3), was not fully grown. An analysis of its response to the treatment gave inconclusive results. There was no response of leaf (n -3) in strains x or y, and there was a slight response, significant at the 5 % level, in strain z. The data do not merit further discussion at this stage.

5. DISCUSSION The data presented in this paper are relevant to several problems of leaf development; at this stage discussion will be confined to two of these problems, namely: (i) to what extent are leaf area, cell size, and cell number influenced by dry and wet conditions of cultivation? and (ii) to what extent is the decrease in cell size from leaf up the shoot due to competition for water among the leaves? The evidence for answering these questions is as follows:

When groups of Ipomoea plants grown under dry conditions were compared with groups grown under wet conditions (conditions of light apd temperature being similar for the two groups) it was found that corresponding leaves between groups differed significantly in area and in cell number, but not in cell size. In both groups of plants there was a decrease in cell size from leaf to leaf. This gradient was not significantly greater under dry conditions than under wet conditions, and in two of the three strains



Studies in the morphogenesis of leaves I89 it was not maintained after the sixth to eighth leaf. This evidence indicates that, within the limits of the experimental conditions, differences in water supply affect leaf area and cell number in the epidermis, but do not affect cell size; also that the decrease in cell size from lower to upper leaves is independent of the wet and dry conditions of cultivation.

The four treatments illustrated in Fig. 2 support and extend this conclusion. If all leaves on the shoot (i to n) except those in the terminal bud are cut off, the next leaf to develop (n + i) has a larger area, larger cells, and more cells, than the corresponding leaf on control plants. If this response were due simply to the release of water formerly monopolized by the lower leaves, then treatment B (Fig. 2) would influence leaf (n + i) in the same way, if not to the same degree, as treatment A; and treatment C (Fig. 2)

would influence leaf (n + i) in the same way, if not to the same degree, as no treatment at all. The results of the treatments, summarized in Table 5, are quite contrary to these predictions; the data clearly show that it is not the bulk of adult leaves, but the three or four immature leaves immediately below a developing leaf, which affect the area, cell size, and cell number of the developing leaf. The effects are highly significant in both wet and dry environments. Furthermore, in successive leaves (n + i), (n + 2), (n + 3), .. .. there is a rapid recovery from the effects of treatments A, B and C; so that in most cases leaf (n + 4) in treated plants, which has only three young leaves below it on the shoot, does not differ significantly from leaf (n + 4) in untreated plants, which has about thirteen leaves below it.

This evidence is plainly inconsistent with the generally accepted hypothesis that upper leaves have smaller cells (and other xeromorphic characters) because lower leaves deprive the upper leaves of water. The evidence is, however, consistent with another hypothesis: that area, cell size, and cell number in a growing leaf are influenced either by substances which diffuse upwards from immature leaves below, or alternatively, by a deficit of substances which are monopolized by the immature leaves below. This proposed hypothesis accounts for the differences between the responses to treatments B and C, and for the fact that the responses to treatment do not persist after the first two or three leaves (i.e. leaf (n + 4) in treated plants is almost a 'normal' leaf). The proposed hypothesis is consistent also with the fact that a gradient of decreasing cell size exists from leaf to leaf among the first few leaves on the shoot: for there is an increase in the tempo of leaf production, resulting over the first few nodes in an increasingly large number of immature leaves below any developing leaf. In strain x, for instance, the data on leaf dimensions reveal the following information (Table 7): there is a high correlation between the number of immature leaves below an unfolding leaf, and the cells per mm.2 in the unfolding leaf when it is fully grown. In treatment C it was shown that even when a leaf is half grown it will still respond to the removal of leaves immediately below it, provided they are not all quite mature. The response is confined to cell size and (consequentially) area: there is no change in cell number. There was no evidence that treatments A, B or C brought about carbohydrate deficiency in the plants.

It cannot be assumed that the conclusions drawn from this experiment are applicable to other plants under other conditions; there were indeed differences in response to treatments even among the three strains of Ipomoea used. However, the conclusions proposed in this paper do raise general issues of some importance in physiological anatomy and do imply that some current opinions should be revised. Therefore it is necessary to inquire whether the data of other workers are consistent with the conclusions



I90 ERIc ASHBY proposed here. There are already in the literature several facts which throw doubt on the commonly accepted hypothesis of the relation between water supply and leaf structure. Even the data of Alexandrov et al. (I92I), an example of which is reproduced in Fig. i, do not fully support their authors' conclusion: for if cell size in developing leaves is deter- mined by competition for water among the leaves below, then the curve of stomatal froquency should continue to rise as more leaves enter the competition. This is not in fact the case. Apart from spurious rises due to the inclusion of immature leaves in the samples, the stomatal frequency becomes stable in Alexandrov's data at 200-300 per mm.2 from the twentieth to the seventieth leaf. Data of Zalenski (I904), Rippel (I9I9) and others support the view that stomatal frequencies do not rise indefinitely with level of insertion on the shoot. Yapp (I9g2) recorded several observations which are inconsistent with the current hypothesis. He found, for instance, that in successive leaves of a non-flowering shoot of Spiraea Ulmaria there was not a gradient of increasing stomatal frequency from leaf to leaf, but an increase to a maximum in the summer, followed by a decrease in autumn.

Table 7. The state of development of leaves below ayoung leaf, when theyoung leaf is o8 cm. long; together with the number of cells per mm.2 when the leaf is fully grown. Strain x

No. of Cells Adult leaves Immature young leaf mm.2 below leaves below

ISt 385 0 0 2nd 506 0 I 3rd 759 0 2 4th 864 I 2 5th 1027 I 3 6th io82 I 4 7th 1132 2 4 8th 1171 3 4 gth 1103 4 4

ioth IO98 5 4 i ith 1Q05 5 5 I2th ioo8 6 5

He found, too, that there was a marked difference in stomatal frequency between basal and distal pinnae of the same leaf; in one particular plant the first leaf on the shoot was more xeromorphic in this and other respects than the basal (but not the distal) pinna on the sixth leaf. In studying hairiness of leaves, which is highly correlated with cell size, Yapp found less variation between sheltered and exposed habitats than he expected; and his observations led him to the suggestion that the glabrous leaves sometimes induced by removing lower leaves on a shoot, and the glabrous leaves found occasionally above injured parts of the stem, may be caused by the injuries themselves, and not by the consequential disturbances in water supply. These scattered facts are consistent with the conclusions advanced in the present paper.

There is an apparent discrepancy between the independence of cell size and water supply reported in this paper and the close dependence of cell size on water supply reported by some other workers. Many of the data which support the view that dry conditions produce smaller cells are concerned with sun and shade leaves, where not only moisture, but light also, will have affected cell size. However, both Rippel (I9I9) and Tumanov (1927) have observed that plants grown in dry soils have small cells. In Rippel's experiments the 'dry' plants were very dwarfed indeed, and in Tumanov's



Studies in the morphogenesis of leaves 191 experiments the 'dry' plants were repeatedly wilted. Cell size may well be affected by such extreme conditions of drought as these, even though it is not affected by the less extreme conditions provided in the present author's experiments. It is noteworthy that even the 'Trockenzwerge' of Rippel did not have consistently smaller cells than normal plants, and the dwarfness of their leaves was due primarily to their having fewer, and not smaller, cells, than normal plants.

Out of the conclusions presented in this paper, three important questions arise, which cannot-be answered from the data. They are: (i) To what extent is leaf structure deter- mined by the external environment at the time the leaf unfolds? Both Zalenski and Yapp considered that the structure of a leaf is affected more by its position on the shoot than by the time of year it appears; but there are no critical data which enable these two variables to be separated. (ii) What is the relation between the structure and the physical properties of a leaf (e.g. between -cell size and osmotic pressure) suction force, elasticity of cell walls, etc.)? (iii) If the structure of a growing leaf is influenced by the three or four immature leaves below it, what is the nature of this influence? Is the influence due to substances diffusing upwards from the immature leaves, as data of Goodwin (I937) and Harder & von Witsch (I94o) might suggest? Or is it due to interception, by the immature leaves, of substances beneficial to leaf growth?

Experiments designed to answer these questions are in progress in the author's laboratory.

6. SUMMARY I. In this paper the author examines the question of the relation between cell size in

leaves and their level of insertion on the shoot. 2. Experiments with Ipomnoea plants were designed to test the current hypothesis that

the gradient of decreasing cell size from lower to upper leaves on a shoot is due to competition for water among the leaves. Tour treatments were applied, illustrated by the following example:

(i) Control. Single shoots grown to i6-leaf stage. (ii) A. Leaves i-io removed; response studied in leaves ii-i6. (iii) B. Leaves I-7 removed; leaves 8-io left on; response studied in leaves iI-I6. (iv) C. Leaves 8-io removed; leaves I-7 left on; response studied in leaves ii-I6. Three strains of Iponaoea were used. Parallel experiments were conducted under wet

and dry conditions. Observations were made on leaf growth, leaf area, and the size and number of adaxial epidermal cells.

3. The principal conclusions from analyses of the data are: (i) There are significant gradients in area, cell size, and cell number from leaf to leaf up

the shoot. (ii) Leaf area and cell number are significantly affected by water supply, but cell size is

not affected. (iii) In respect of area, cell size, and cell number, leaves II, IX, .. ., respond to treat-

ment B in the same way as to the controls (i.e. to no treatment at all); and they respond to treatment C in the same way as to treatment A. Therefore leaves I I, I 2, ..., do not respond to the removal of seven adult leaves below them, but they do respond to the removal of three young leaves below them.



I92 ERIc ASHBY

4. On this and other evidence the view is put forward that the gradients of leaf area, cell size and cell number between successive leaves on a shoot are not the result of competition for water among adult leaves on the shoot, but are related to the influence of immature leaves upon still younger leaves developing above them. This influence may operate either through the diffusion upwards, from immature leaves, of substances which inhibit cell division and expansion in still younger leaves; or alternatively, it may operate through the interception, by immature leaves, of substances which promote cell division and expansion in still younger leaves.

5. According to this view, the relatively xeromorphic characters of upper leaves compared with lower leaves, in so far as they are dependent on cell size, are attributable principally to the influence of immature leaves immediately below the apex, affecting the morphology of leaves developing at the apex. Any disturbance of water economy which may result from the removal of all adult leaves from a shoot, does not, in the strains of Ipomoea studied, influence the morphology of leaves developing at the apex.

6. The consequences of this view are being examined by a further series of experiments.

I have pleasure in acknowledging technical help from Miss E. Wangermann, B.Sc., Mr D. J. Carr, Mr George Breckon, and the garden staff at the experimental grounds. I am indebted to Mr E. N. Roberts, who prepared the text-figures.

REFERENCES

ALEXANDROV, W., ALEXANDROV, 0. & TIMOFEEV, A. (I92i). The water supply of leaves and their structure. Sci. pap. appl. sect. Tiflis bot. Gdn, 2, 85. (Russian.) See also Maximov (I929), p. 342, and Walter (I926), P. 70.

ASHBY, E. (1948). Studies in the morphogenesis of leaves. I. An essay on leaf shape. New Phytol. 47, I 5 3-76.

FISHER, R. A. & YATES, F. (I943). Statistical Tables. Edinburgh. GOODWIN, R. H. (I937). The role of auxin in leaf development in Solidago. Amer. J7. Bot. 24, 43. HARDER, R. & VON WITSCH, H. (1940). Ober den Einfluss der Tageslinge auf den Habitus, besonders der

Blattsukkulenz und den Wasserhaushalt von Kalanchoe Blossfeldiana. Jb. wiss. Bot. 89, 354. IMAI, Y. (1930). A genetic monograph on the leaf form of Pharbitis Nil. Z. indukt. Abstamm- u. VererbLehre,

55, I. MAxIMov, N. A. (1929). The Plant in Relation to Water, ed. R. A. Yapp. London. RIPPEL, A. (I9I9). Der Einfluss der Bodentrockenheit auf den anatomischen Bau der Pflanzen. Beih. z. bot.

Ges. 36, I87. TuMANov, J. J. (I927). Ungenuigende Wasserversorgung und das Welken der Pflanzen, usw. Planta, 3, 39I. WALTER, H. (I926). Die Anpassungen der Pflanzen an Wassermangel. Naturwissenschaft u. Landwirtschaft,

H. 9. Munchen. YAPP, R. H. (19I2). Spiraea Ulmaria and its bearing on the problem of xeromorphy in marsh plants.

Ann. Bot., Lond., z6, 815. ZALENSKI, V. (I904). Materials for-the study of the quantitative anatomy of different leaves on the same

plant. Mdm. Poly. Inst. Kiev, 4, I (Russian).

APPENDICES

I. Correction for number of itncomplete cells in microscopic fields (? (ii), p. I 82)

When the outlines of cells are irregular it is very laborious to count the number of incomplete cells in a microscopic field. Therefore the author calculated a regression from which a correction equal to half the number of incomplete cells could be estimated from the number of complete cells in a field. He is indebted to Prof. M. S. Bartlett, of the University of Manchester, for suggesting a suitable formula for this regression, derived as follows:




Let the area of the square field= 12 and the mean area of a cell = a. Then if there are n whole cells and m incomplete cells in the field, and if n is large,

12 = (n + m) a. Let h= the average length of a chord of a cell intersected by the edge of the field. Then

mh= 41 and 12 = m2h2/i 6 If h2/a = I/c then

m2- 8mc = i 6nc, m2 - 8mc + i6C2 = i6nc+ i6C2,

(m-4C)2= i 6c(n + c),

m-4c=4V[c(n+c)], and -M = 2{V[c(n + c)] + c}.

This formula will not hold exactly when n is not large, especially as it neglects the effects of the corners of the square field; but it suggests that a linear regression of I m on Vn should be found to be a sufficiently accurate relationship. The goodness of fit of the regression is illustrated by Fig. I 3.

14 Y-101514+1-6491 (X-47811) o Regression coefficient= 1-6491 1001005

13 _0/0

12 0 0~~~~

12 0 ~~~~~00

10 _ ?

o 0 O

( ~~~~~~~~~ 0 ~~~~~~~ 000

0~~~~~~~

8 / 0

00 / 7 0

0 0 00

6

5 _ _ . 2 3 4 5 6

In

Fig. 13. Regression of half the number of incomplete cells in a microscopic field on the square root of the number of complete cells in the field.



I94 ERIc ASHBY

II. Control plants: mean values of leaf area, cell size, and cell number, used in analysis of data

Leaf .. . 2 3 4 5 6 7 8

Area (cm.2) I x Dry 32-2 35 7 28.4 25-0 i8-i i8-o I9-8 20 9 Wet 39 0 46-I 43 6 39-2 312 26-I 24.2 22-1

y Dry 24-5 23-5 234 20-4 i8.5 19-2 19-2 17-8 Wet 37 4 34 2 310 28.5 22.3 2I-2 20 5 I8.3

z Dry 216 30-0 23.6 23-2 17-8 I6-2 150 I8-2 Wet 32-6 51s3 501 47 7 404 334 27-2 25 3

Cells x Dry 365 525 836 1044 1115 1030 IOI9 I007

mm.2 Wet 385 5o6 759 864 1027 I082 1132 1171 y Dry 503 751 854 972 1054 Io82 1026 909

Wet 438 548 779 1014 1125 1213 1241 1112

z Dry 408 517 754 Ioo9 1221 1202 1194 1332 Wet 347 430 532 726 977 i i68 1241 1292

Cell no. x IO16 x Dry 1.17 187 2-38 2 55 20o6 IgI 192 ig98 Wet 1-48 2-27 3 14 3 35 30o6 2 77 2-67 2 57

y Dry 1-24 I 87 20o8 I 99 2-01 I 198 I.99 I .84 Wet i '8o 21i8 2-51 3-00 2-71 2-63 2-31 2-07

z Dry o-88 1-58 178 2-36 2.17 v8i 177 2.03

Wet 1-12 210 2 49 3-19 3-48 3-38 3-01 310

Leaf ... ... 9 10 I I 12 13 14 1 5

Area (cm.2) x Dry 24'3 24-0 218 20-3 I58 - -

Wet 23-1 20-8 24 1 22-4 225 - -

y Dry 20.4 i6-8 14-0 i6-3 i6-4 14'5 154 Wet 17'3 17'3 i8-i i6-3 12-9 134 14.0

z Dry I7T5 i67 i6-o I64 - - -

Wet 22,7 20,4 19-9 17 9 - _

Cells x Dry gI9 I082 IOIg 1042 - _

MMn.2 *Wet 1103 Io98 1005 ioo8 _

y Dry Io56 883 Ioo9 977 1244 ii6i 1041 Wet Io87 1045 976 976 902 998 997

z Dry 1485 1237 1270 1576 - - -

Wet 1315 1365 1249 1440 _

Cell no. x io-6 x Dry 219 2-56 2-24 2-14 i68 - -

Wet 2'59 2'41 2-60 2-24 2-65 - -

y Dry i198 140 136 I55 iv8o 169 i 66 Wet i184 176 1-72 154 135 134 139

z Dry 2 50 2-07 2-28 - - _

Wet 2-64 V257 2-48 - - _




III. Differences between leaf (n + i) on treated plants and the mean for the corresponding leaf on control plants

Treat- ment... A B C

Strain x Y z x y z x y z

Area (cm.2) Dry 38-4 3'9 29-9 -0 4 -4.0 I 0 8.5 7-2 4-2

28-9 4-8 I6-2 4.8 2-4 7-0 6-8 24 7 8-5 20-7 78 -9 45 - 65 137 -

- 08-8---- --

Wet 6-4 91I 33I - 2'2 14.2 8- 5-2 9.8 - o8 38 4 15'9 42-2 -4-8 17 2-3 - 141 123 480o 502 0-3 4,7 5.3 - -6-7 45*4 13*7 26-2 -

Cells per mm.2 Dry 307 197 321 44 - 255 326 119 153 490

305 327 384 120 475 - 6 434 385 289 463 696 -199 -97 27 173 332 336 -1I84 I-84

67

Wet 420 208 637 -210 121 314 152 56 335 445 _Z 671I -1I54 I65 125 287 152 -1I47 5I6 36I 536 -30 -i8i i8 i88 315 224 65 I -59 1I37 11 4 _ I62 35

Total no. of cells x I0-6 Dry 2-42 *0'03 2z36 - o28 -0_03 -0 45 o-86 039 -075

I @48 0I4 057 0-25 - 035 o-63 o-o8 I *46 0-I4 027 o079 -0o28 - o i 8 I ' I9 o-o8 - 025 0-44 -

Wet I 43 I'2I Ig98 Ig98 O77 o-o8 0-23 o-8s 2-23

I'56 OgI 2 53 -0o78 O054 0-15 |O'I2 O093 I62 3 o8 o69 38i - 044 0o72 - 3'50 2'22 -o6i O11 - |



studies in the morphogenesis of leaves. ii. the area, cell size and cell number of leaves of ipomoea...

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