STUDIES IN THE WATER RELATIONS OF

THE COTTON PLANT

I. THE FIELD MEASUREMENT OF WATER
DEFICITS IN LEAVES
BY P . E
Department of Botany, The University, Manchester

{Received i May 1949)

(With 9 figures in the text)

INTRODUCTION
Cotton in Uganda is entirely rain-grown; the growth of the crop is dependent, therefore,
on the intensity and distribution of rainfall, the fluctuations of which are undoubtedly
responsible for much of the year-to-year variation in yield.
Although the effect of rainfall on the crop community is complex, the most obvious
way in which rain affects plants is by its influence on their water balance. The experiments
to be described were designed to find out how far seasonal fluctuations in the water
balance of cotton plants growing in the field are related to changes in those environmental
factors implicit in the term rainfall. The initial step was to devise a suitable method for
assessing the water balance of growing plants. This paper deals with work directed
towards that end. Much of the past work on water relations has been devoted to a study
of the separate processes involved, viz. absorption, translocation and transpiration. Little
attention has been paid to the inter-relationships between these processes. It has long
been recognized that it is the relative rates of transpiration and absorption, and not their
absolute values, which are most relevant in the study of water relations. It is a lack of
balance between absorption and transpiration which causes those fluctuations in turgor
of the cells of importance to the growth and behaviour of plants.
The simultaneous study of absorption and transpiration in rooted plants is fraught
with great technical difliculties, so that it is not surprising that few efforts have been made
to carry it out in the past. The methods used by Livingston & Hawkins (1915) and
Kramer (1937), necessitated the plants being grown in pots under conditions of constant
soil moisture, which would make the experiments irrelevant to the problem under
investigation here. It is evident that there is no method of simultaneously measuring
transpiration and absorption in plants growing under normal field conditions.
An alternative method of attacking the problem lies in the fact that a change in water
content of a whole plant or leaf during a given period reflects the inequality between
absorption and transpiration during that period. Such an investigation of water content
is possible with plants growing in the field. The difliculty with this method lies in deciding
on what basis to express the water content. Most of the past work has been carried out to
* This work was carried out at Serere Experiment Station, Uganda, East Africa, whilst the writer was on
the staff of the Department of Agriculture there.
New Phytol. 49, i 6
82 p. E. WEATHERLEY
investigate diurnal variations in water content, and for this both dry weight and fresh
weight have been extensively used as bases. Many workers (e.g. Runyon, 1936) have
realized the limitations and dangers of assuming that a change in the water: dry-weight
ratio necessarily reflects a change in water content, in the sense of a change in the water
balance. A decrease in the water:dry-weight ratio might equally be caused by a fall in
water or a rise in dry weight. The position is further complicated by the fact that diurnal
changes in moisture and dry weight are often reciprocal, so that a diurnal drop in water
content tends to be exaggerated. In view of this. Mason & Maskell (1928) proposed the
use of 'residual dry weight', the dry weight less the weight of labile carbohydrates. In
this way they tried to obtain a constant weight basis independent of diurnal accumulations
of photosynthetic products. The water: fresh-weight ratio, whilst giving a more accurate
picture of small water fluctuations, is clearly inadequate for expressing fluctuations large
compared with the total water content, for such fluctuations would be minimized.
Little work seems to have been done on the variations in water content of plants over
more extended periods than 24 hr. Brown (1927) reports that the water content (pre-
sumably on a fresh-weight basis) of cotton plants is high at the seedling stage and there-
after decreases. Herrick (1933), Evenari & Richter (1937) and Portsmouth (1937) all
used the dry-weight basis for investigating seasonal changes in the water relations of
various plants. As in the case of diurnal changes, it would seem unjustifiable to draw
conclusions about the water balance from such data, without definite evidence that the
water: dry-weight ratio remained constant under conditions favouring equality of ab-
sorption and transpiration rates.
Lloyd (1913) avoided the use of dry weight by expressing diurnal changes in water
content on the basis of area. A similar method was used by Miller (1917), working on
maize and sorghum. Disks of leaf tissue were punched with a cork-borer from time to
time and the water content of a standard number of disks measured. Also using a disking
technique, Hawkins (1927) was able to demonstrate that the water content of cotton
leaves, expressed on the basis of area, responded to irrigation. Dry weight, however,
fluctuated independently. In this case area seemed to form a more useful basis for
expressing water content than dry weight.
In the present investigations a similar disking technique was used and developed.
Punching leaf disks lent itself to rapid and efl^ective sampling of a large number of plants
growing in the field. The damage to the plants being small, the method permitted periodic
sampling throughout the season. Using this technique it was demonstrated conclusively
that changes in the water: dry-weight ratio were due to changes in dry weight and were
not related to water balance. It was necessary, therefore, to devise a new way of expressing
the water content. This took the form of the ratio between the water content of the leaf
tissue as sampled, and its water content when in a state of full turgidity.

EXPERIMENTAL

(i) Sampling methods

All observations were made on Gossypiiim hirsiitum, Uganda variety BP 50. The plants
were grown according to normal practice in rows 3 ft. apart, spaced at i ft. intervals in
the rows making up plots of about 500 plants. Six such plots were sown at monthly intervals
from April to September, the full range of the planting season for the district. In each
 Studies in the water relations of the cotton plant 83
plot sampling was started about 4 weeks after sowing and continued at weekly or fortnightly
intervals until leaf fall prevented further samples being taken. Thus at each sampling,
plants of several ages were sampled. This was an important feature of the experiments,
for had all the cotton been sown at one time it would not have been possible to distinguish
a seasonal trend from an age effect. However, using several sowings, measurements on
plants of more than one age were made at each sampling date through the season. In this
way trends or fluctuations in water content could be attributed either to seasonal changes or
the effect of age.
The method o^sairipling was to punch disks of leaf tissue with an ordinary cork borer.
The borer wis approximately i cm. in diameter,^sed at the handle end and fitted with
a piston device for ejecting the disks collected inside the borer during sampling. The main
veins of leaves were avoided and the position chosen for punching was restricted to the
angle between the main radiating veins. Twenty-five disks constituted a sample, one disk
only being punched from a chosen leaf, and one leaf only chosen from a selected plant.
Thus the sample was taken from twenty-five plants. The twenty-five plants were selected
at random from the plot being sampled.
The choice of leaves was at random, but only within a certain age category. Choice
was a matter of assessment of morphological characters and not of position on the plant.
Only mature leaves were selected, i.e. those which had ceased rapid expansion and had
lost the sheen characteristic of young leaves, but which showed none ofthe yellowing and
leatheriness associated with old leaves. The shape and texture of leaves selected on these
criteria varied considerably with the age of the plant; in seedlings the leaves were small
and entire or only slightly lobed and soft, whereas in mature plants they were large,
deeply lobed and coarser. Again, in old plants, especially after the onset of the dry
season, they were small, but deeply lobed and somewhat leathery. Yet at any stage in the
growth of the plants the leaves chosen could be described as mature for that stage.
Duplicate samples were taken at 6.30 a.m. on each sampling date. Sunrise was chosen
as probably being the time of maximal water content of the diurnal cycle. In this way it
was possible to follow the course of the diurnal maximum water contents through the
season.
As soon as the twenty-five disks of a sample had been punched they were ejected
directly into tared weighing_bojtles and subsequently weighed in the laboratory. On
many mornings the leaves were wet with dew; in this event the wet disks were not put
into weighing bottles, but taken to the laboratory in airtight containers, surface-dried
with filter-paper and then weighed in weighing bottles. Possible errors arising from the
disks taking up moisture from the dew are considered later. After weighing to the nearest
milligram, the samples were dried to constant weight (24 hr.) in an oven at 92-95° C.
In this way the fresh weights, dry weights and water contents of the samples were found.
Since there was the possibility of seasonal changes or age differences in labile carbo-
hydrates, it was desirable to measure 'residual dry weights' as well as simple dry weights.
The method used followed that of Mason & Maskell (1928); the dried samples were
hydrolysed with dilute acid, and the total reducing sugars estimated and subtracted from
the dry weight.

6-2
84 p. E. WEATHERLEY
(2) Seasonal and age trends in the water.-dry-weight ratio
Values of the water: residual dry-weight ratio plotted against time for three of the six
sowings are shown in Fig. i. It will be seen that in each sowing the ratio decreased as
the plants got older. The ratio was high at the seedling stage and decreased at hrst rapidly,
but less so as the plants aged. It is clear that this trend in the ratio was an age and not an
environmental effect, for high values were associated with young plants and not earliness
in the season. Actually the environment was not without its effect on the form of the
curves, for amongst older plants the ratio was lower at a given age, the later the sowing.
This is brought out in Fig. 2 in w^hich water: residual dry-weight ratios are plotted against
age of plants for the first, fourth and sixth sowings. Nevertheless, it is evident that the
trends in the ratio were inherent rather than environmental.

9r

:; 7

^ 5

May June July Aug. Sepc. Occ, Nov. Dec. Jan. Feb.

Fig. I. Curves showing trends in water:residual dry-weight ratios.

G O April sowing. X X July sowing. A —^ September sowing.

It might be suggested that this decline in water: residual dry-weight ratio with age was
due to a progressive drift in the maturity of the leaves sampled. This could be so if there
was a trend in the ratio passing from young to old leaves on the same plant. In fact, there
was little difference in the water: dry-weight ratios between young and old leaves of a given
plant. This is shown in Fig. 3 in which the ratios for buds, small expanding leaves,
mature leaves and old leathery leaves are plotted against the age of the plants. It will be
seen that the water content expressed on a dry-weight basis declined with increasing age
of plants in all the 'maturity categories' of leaves sampled. Furthermore, the differences
between the water: dry-weight ratios of the leaves of differing maturity on plants of the
same age were small compared with the decline in all leaves with increasing age of plant.
Curves for the water:residual dry-weight ratios for three of the six sowings are shown
in Fig. 4, together with corresponding values for the residual dry weight per sample.
It will be seen that in each case the water; residual dry-weight ratio and the residual
 Studies in the water relations of the cotton plant

2-0
100 150 200
Time from sowing (days)

Fig, 2. Curves showing effect of sowing date on trends in water: residual drj'-weight ratios.
O——O April sowing. X X July sowing. A——A September sowing.

35

30

2-5

2-0

1-5
_L
60 80 100 120 140 160 180 200
Time from sowing (days)

Fig. 3. Curves showing relationship between water: residual dry weight and plant age for
leaves of different degrees of maturity.
^ Z^ Buds. >; .X leaves. O —© Mature leaves. G] H Old leaves
86 P. E. WEATHERLEY
dry weight are strongly negatively correlated. This imphes that the fall in the ratio was
merely a reflexion of the rise in residual dry weight, the water content per sample re-
maining relatively constant. That this was so is demonstrated by the curves m Fig. 5,
170

20 L
20 40 60 100 UO 140 160 180 40 60 80 100 120 140 160 180 40 60 80 100 120 140 160
Time in days from sowing

Fig. 4. Curves showing relationship beUveen water: residual dr^• weight and time (£H — H3 ; and residual
dry weight per sample and tinie B El for three sowings.

170

160

150

140

130

120

110

100

90

80

70

60

50
20 40 60 80 100 120 140 160
Time jn days from sowing

Fig. 5. Cur\es showing trends in residual dry weight per sample, water per sample and water; residual
dry weight, each expressed as a percentage of its mean value. Data from September sowing.
O- O Water-H residual dry weight. ^ ^ Water per sample.
H——H Residual dry weight per sample.

in which the residual dry weight per sample, water per sample, and water per unit
residual dry weight, for a single sowing, are e.xpressed as percentages of the mean value
in each case and plotted against time.
 Studies in the water relatiofis of the cotton plant 87
These data clearly indicate that the observed fall in water content on a residual dry-
weight basis did not represent a change in water balance. The age trend in which the
residual dry weight per sample more than doubled its value masked any relatively small
changes in water content such as might be due to changing water balance.

(3) The water:protein ratio

Phillis & Mason (1943) have indicated that the quantity of protoplasm in the leaf tissue
of G. barbadense is likely to be measured by its protein content. Hence the weight of
protein nitrogen should be a logical basis on which to express the water content of such
tissues. Experiments to test this were of the same form as those described in the previous
section, except that only three sowings were used instead of six. The total protein nitrogen
content of the dried samples was estimated by Barnstein's modification of the Stlitzer

120

110

\ •

100

\
\

80

\ i

70 -

60 1 1 1 1 1 1 ^
June July Aug. Sept. Oa Nov. Dec.

Fig. 6. Curves showing trends in the water:protein nitrogen ratio.

Q Q May sowing, x X J'-'ne sowing. A ^ - ^ ^ July sowing.

process (see Allen's Commerical Organic Analysis, 5th ed., vol. viii, p. 662). The precipita-
tion was carried out on a small scale, and the total nitrogen in the precipitate was estimated
by a micro-Kjeldahl method.
The ratios of water: protein nitrogen plotted against time for the three sowings are
shown in Fig. 6. It will be seen that the general form of the curves is similar to those for
water:residual dry weight. The ratio fell from the seedling stage, the decrease being an
age, rather than environmental effect.
It would appear that the water content cannot be expressed usefully on the basis of
protein nitrogen for the same reasons as those already discussed for the water: residual
dry-weight ratio.
88 P. E. WEATHERLEY
(4) The water:cellulose ratio
It was thought that cellulose merely constituting the skeletal framework of the cells
and presumably not being a food reserve, might give a constant denommator on which
to express the water content. Samples were taken in the manner already described.
'Cellulose' was estimated as the residue after mild acid, followed by alkaline hydrolysis.
The method of Chen & Cameron (1942), suitably modified for small quantities, was
followed.
The results are summarized in Fig. 7, in which values for the water: cellulose ratio are
plotted against time. From these curves it will be seen that the behaviour of this ratio was
105

- V \
95

\
85

75 \ \
\
V X

w
65 -
\ \
55 -

45 1 1 1
July Aug. Sept. Oct. Nov. Dec.
Fig. 7. Curves showing trends in the water:cellulose ratios.
0 0 May sowing. X X June sowing. A A July sowing.

essentially similar to that of the residual dry-weight and protein-nitrogen ratios. Water
content of the leaf tissue, expressed on the basis of its cellulose content, showed a marked
age trend, large variations in the cellulose fraction masking any changes in water content.
It is likely that this increase in cellulose of the leaf tissue as the plants aged was due either
to an increase in cell-wall thickness, or to a decrease in cell size.
From the results set out in the foregoing three sections it was concluded that the
method of expressing the water content of cotton-leaf tissue on its residual dry weight,
protein nitrogen, or cellulose content was not useful as an expression of the water balance
in the plants. It was necessary to turn to criteria of a different sort if this aim was to he
attained.
(5) Relative turgidity
The clue to a more satisfactory expression of water content lay in the fact that cotton-
leaf disks can be made fully turgid by floating on water. Thus the water content at full
turgidity could be used as a basis for expressing the water content found in the field
(field-water content).
 Studies in the water relations of the cotton plant 89
The behaviour of the disks when floated on distilled water is summarized in Fig. 8, in
which the water content of the disks is plotted against time. It will be noticed that there
was a rapid increase in water content during the first few hours, and after about 10 hr.
further increase was slow and steady.
It seems likely that the initial rapid uptake was in response to the water deficit in the
tissues, after which the tissue approached full turgidity. The slow increase in water
content after 10 hr. was probably due to growth of the disks.
For measurement of water content at full turgidity samples were floated for 24 hr. on
distilled water. This period was longer than the minimum necessary, but was chosen for
convenience. The disks were floated in bright diffuse daylight, but during the night no
artificial illumination was used. Such an alternation of light and darkness seemed to have

2 800

720
0 10 20 30 40
Time (tir.)
Fig. 8. Curve shpwing increase in water content of floating leaf disks.

no effect on the water uptake by the disks. After floating for 24 hr. the disks were removed,
rapidly surface-dried with fllter-paper and weighed. Their water content was then found
by oven-drying in the usual way.
Having ascertained the field water content and water content at full turgidity of the
leaf tissue, the former was expressed as a percentage of the latter, the resulting figure
being called the 'Relative Turgidity' of the tissue, thus:
Field water content of tissue
Relative turgidity = i^^^-gy^c^ntenTof^ame quantity of tissue when fully turgid
The relative turgidity had a value of 100 for fully turgid leaves (i.e. no uptake of water
on floating), whilst the lower the value, the greater was the water deficit in the tissues.
It will be noted that the field water content and turgid water content refer to the weights
of water present in the same quantity of tissue in each case. This would have been ensured
had it been possible to measure both the water contents on the same sample of disks.
Unfortunately this could not be done since the process of floating involved a loss in
90 P. E. WEATHERLEY
dry weight. The initial field water content of a floated sample could not be estimated since
its initial dry weight was not known. It was necessary, therefore, to have two samples ot
disks, as nearly as possible identical, for each measurement of relative turgidity.

(6) Procedure for measuring relative turgidity

Twenty-five disk samples were punched at random in the plots in the manner already
described for water: dry-weight measurement. In order to obtain two parallel samples,
two cork-borers were used; after punching one disk from a leaf with the first, another was
punched with the second from the same leaf. The second punch was made in a similar
position to the first, i.e. in the angle between two main veins. Thus the two parallel
samples were taken from the same set of randomly chosen leaves.
The subsequent treatment of the pair of samples so obtained is schematically represented
below:
Pair of twenty-five disk samples

Weighed (F.W.J Weighed

Dried in oven Floated for

for 24 hr. 24 hr. on water
at95°C. (turgid)

Weighed Weighed (F.W.j)

Dried in oven
for 24 hr. at
9S°C.

Weighed

(F.W.,,) and (F.W.J) = fresh weight of samples a and b.

(F.W.() = fresh weight of sample b when fully turgid.
(D.w.n) and (D.w.ft) = dry weight of samples a and b.

The field water content of sample ' a' was found directly by oven-drying (F.W.^ — D.w.^).
Sample '^' was floated on water for 24 hr., weighed, oven-dried, and the dry weight
obtained, and hence the water content at full turgidity (F.W.^-D.W.^). Thus

Relative turgidity = V' '"' x 100.

In practice, the calculation was complicated by the fact that two cutters of identical size
were not obtainable, so that the numerator and denominator of the ratio referred to slightly
 Studies in the tvater relations of the cotton plant 91
different quantities of tissue. In addition to this source of error there were the differences
between the two samples due to chance sampling variation. What was required was some
method of comparison of field and turgid water contents on, as nearly as possible, identical
quantities of tissue. The key to a method for reducing these errors lay in the existence of
a close positive correlation {r= +0-95) between the fresh weights and water contents
within duplicate samples. This meant that an estimate could be made of the field-water
content of sample '6' from the field-water content of sample 'a' and the fresh weights of
samples 'a' and 'b'. Thus:
PFJ = W;(F.W.b - F.W. J ,
where W^ and H4 = the field water contents of samples 'a' and ' 6 ' , //; = regression
coefficient of water on fresh weight, and

T.T , • ... Field water content

iNow relative turgidity = =^-ir. 7-. x 100,
i<ully turgid water content

or ' 5 ^ X IOO = - ^ ^ ^ —-"^ X

( ) ( )

.^ - D.w. J + m(F.w.fc - F.W. J

100.

From an analysis of covariance between fresh weight and water content based on all
available data, the regression coefficient {in) was found to be 0-733; this was used in all
calculations of relative turgidity.
Since sampling was carried out in duplicate, errors of sampling could be estimated.
In a single season duplicate samples were taken on seventy-nine occasions giving the
standard error of a mean of duplicate samples to be o-68. This meant that a difference
between means of duplicates of 1-9 was detectable at the 0-05 level of probability. The
general mean value of relative turgidity for the season was 86-7.

(7) Difficulties in measurement of relative turgidity

{a) Dew absorption. The fact that leaf tissue suffering a water deficit takes up water
rapidly when floated, suggests that disks punched from dew-laden leaves might absorb
water during sampling. Whilst the dew was normally shaken off the leaves by quickly
brushing with the fingers, they still retained some surface moisture. There is no doubt
that some of this moisture was absorbed at the cut edges of the disks, giving too high
values for the relative turgidity. Each leaf could not be surface dried before sampling
since this proved too slow and laborious. In the view of the writer dew absorption
constituted the most serious drawback to this disking technique, especially in a country
where heavy dews are common.
In an experiment to estimate the water taken up from dew by disks during punching,
parallel samples were punched from dew-laden leaves and from the same leaves after
surface drying. A comparison of the water contents of such samples indicated an increase
in water content of between 1-4 and 3-1%. In a second experiment relative turgidity
was measured in each case. The results are given in the table on p. 92.
 p. E. WEATHERLEY
Relative turgidity
Sample
Before surface drying After surface drying

I 92'6 90 2
•2, 92-8 890
Mean 92-7 896 diff. 3-1

Least significant difference = 1-9.

From these results it would seem that there is a significant uptake of water from dew.
Thus any variations in the relative turgidity of the leaves, as found at sunrise from time
to time through the season, might have been related to the fact that dew was a frequent,
though not invariable occurrence. However, the variations in relative turgidity to be
discussed subsequently appear to be larger than could be explained in terms of dew
absorption; furthermore the pattern of the relationship between the relative turgidity at
sunrise and environmental factors did not show any evidence of being affected by the
presence or absence of dew.
(6) Diurnal fluctuations in floating leaf disks. Phillis & Mason (1945) used a floating-
disk technique to investigate the mineral nutrition of cotton leaves. They found well-
marked diurnal fluctuations in the water content of floating leaf disks. Clearly this is of
considerable importance to the present technique, for it was assumed that full turgidity
was attained by the floating disks irrespective of time of day, or external conditions. The
writer (Weatherley, 1947) has provided evidence that these diurnal fluctuations are due
simply to the balance between transpiration from the surfaces and absorption at the cut
edges of the disks, and that using small disks of about i cm. diameter the fluctuations are
negligible.
(c) The injection of intercellular spaces. The floating-disk technique depended on the
assumption that water did not pass into the intercellular spaces. Injected areas of leaf
were easily distinguishable from normal areas by their dark waterlogged appearance.
Observation showed that injection occurred rarely, and where it did occur it was localized
in small spots. Slight waterlogging always occurred at the cut edges of the disks, and
undoubtedly this constituted a small systematic error. It was desirable to get some
measure of the injection error in view of this edge effect, and the fact that 'non-visible'
injection might take place. The evidence for injection being a minor factor in determining
the uptake of water by floating disks is based on two facts. First, whole excised leaves
placed with petioles in water take up similar amounts of water to floating disks. It is
doubtful whether whole leaves under these conditions would become injected. Secondly,
disks punched from leaves rendered fully turgid in the above manner take up little extra
water. These facts were demonstrated in the following experiment.
Two samples of 100 leaf disks were taken from a cotton plot, and at the same time
104 leaves were collected. The disks and excised leaves were immediately placed in sealed
containers. In the laboratory the disks were weighed and floated in the usual way. The
fresh weight of four of the leaves was also found, after which they were placed with their
petioles in water under a bell jar. At the same time the remaining 100 leaves were
similarly placed under a bell jar with their petioles in water. Fresh weights of the disks
and four leaves were determined from time to time. It was assumed that these changes in
fresh weight largely reflected changes in water content. Fresh weights expressed as
percentages of initial values are plotted against time in Fig. 9. It will be seen that the
 Studies in the water relations of the cotton plant 93
courses of water uptake in disks and whole leaves were remarkably similar. The percen-
tage increase in fresh weight after 4 hr. was very similar both for disks and leaves, and
after 17-25 hr., identical. After 24 hr. a sample of 100 disks was punched from the re-
maining IOO leaves which had been standing in water under similar conditions to the four
measured leaves. The fresh weight of these disks was found, after which they were floated
for 24 hr. and the gain in fresh weight measured. At the same time the further increase
in fresh weight of the four whole leaves was followed. It will be seen from the graph that
the disks punched from already turgid leaves increased in water content no more than
whole leaves under similar conditions. It may be concluded that the punching and
floating technique led to no water uptake in itself, and that injection was an unimportant
factor in absorption by floating disks.

-s

0 10 20 30 40
Time (hr.)
Fig. 9. Curves showing the increase in water content of whole leaves placed with their petioles in water,
and floating disks sampled in the field and from turgid leaves.
GH——© Disks. fi\ A Whole leaves. E] H Disks punched from turgid leaves.
^ Disks punched from turgid leaves with 24 hr. value adjusted to that of whole leaves.

It is interesting to note here that there is a marked contrast between floating and sub-
merged disks in this respect. Ashby & Wolf (1947) have demonstrated considerable
injection in submerged disks of Iris leaf in darkness, whilst it was noticed during the
present work that disks which became submerged soon became injected, especially in
the dark.
{d) Changes in osmotic pressure atid cell wall properties. The technique for measuring
relative turgidity was based on the fact that leaf disks punched from a plant took up water
when floated. This uptake was due to the tissue having an appreciable suction pressure.
Variations in this suction pressure from time to time might have been due, not only to
fluctuations in the water balance in the plants, but also to fluctuations in the osmotic
pressure of the cell sap, or the extensibility of the cell walls. Whilst a change in relative
turgidity may be regarded as a change in ' water deflcit' of the tissue whatever the cause,
it is necessary to inquire how far such changes were likely to be due to fluctuations in the
balance between transpiration and absorption or to changes in osmotic and wall properties
of the cells.
94 P. E. WEATHERLEY
It is not easy to predict what ultimate effect a change in osmotic pressure of the leaf
sap would have on the relative turgidity. An increase in solute concentration will cause
an initial increase in suction pressure. This will lead to an absorption of water (mcrease
in volume of the cells) and a decline in suction pressure towards its original value. Now
it seems likely that the relationship between the turgor pressure and cell volume is not
linear. The results of Oppenheimer (1932) indicate that as the volume of the cells increases
there is a progressively greater increase in turgor pressure for each volume increment.
This means that two samples of tissue of equal suction pressure, but unequal osmotic
pressure, will absorb different volumes of water to attain full turgor, the tissue of higher
osmotic pressure absorbing less water. In this way an increase in osmotic pressure in the
leaf cells would lead ultimately to a rise in the relative turgidity. On the other hand, if an
increase in osmotic pressure resulted in a permanent increase in suction pressure slightly
more water would be absorbed to reach full turgor and the relative turgidity would
decrease. In either of these two ways a change in osmotic pressure of the cell sap might
affect the relative turgidity, though in opposite directions, irrespective of the water
balance in the plants.
Whilst no data were obtained on how changes in osmotic pressure of the sap did in
fact alter the relative turgidity, there was evidence, to be described in a subsequent paper,
that it was factors controlling environmental moisture which influenced relative turgidity,
rather than factors calculated to alter the osmotic pressure.
Another factor controlling the uptake of water by floating disks is the extensibility of
the cell walls. The less extensible the walls the less water will the disks take up on floating.
Thus an age trend of increasing cell-wall rigidity might be manifest as an increase in
relative turgidity. The results showed no evidence for such a trend. Plants of different
ages showed similar values of relative turgidity at a given time and in the same environment.
The relative turgidity in the leaves was defined by environmental conditions and not by
the age of the plants. Furthermore, the relative turgidity was found to fluctuate in response
to changes in environmental conditions. It is difficult to see how these fluctuations could
have been caused by changes in the cell walls, unless these were both rapid and reversible.
Whilst it is not suggested that changes in osmotic pressure and cell-wall extensibility
are without significant effect on the value of the relative turgidity of a tissue, it is con-
sidered that such variations in relative turgidity as were observed in the course of these
investigations were due to changes in water balance rather than to the operation of these
factors.
DISCUSSION
The key to the technique described in this paper for measuring relative turgidity was the
fact that leaf disks could be made fully turgid by floating on water. In the past little use
seems to have been made of leaf disks for measuring water deficits, Shreve (1924) measured
the maximal water content in the leaves of Encelia farinosa by immersing disks in water
for 24 hr. He looked upon the uptake of water not simply as the satisfaction of a water
deficit, but as a more obscure imbibitional process. More recently. Mason & Phillis (1942)
floated leaf disks on water and solutions to investigate the effect of mineral nutrition on
leaf hydration of cotton. They indicated that the initial rapid uptake of water on floating
was probably due to the water deficit in the tissues. Following this suggestion, Doggett
(1942) attempted to utilize this method for estimating water deflcits in cacao leaves.
 Studies in the water relations of the cotton plant 95
He was unable to demonstrate much absorption, and suggested that this was probably due
to the mucilaginous nature of the tissues.
A number of other workers have used the uptake of water by tissues as a measure of
water deficit, although in these cases whole shoots were allowed to take up moisture to
saturation. Thoday (1921) measured the water deficit in Passerina spp. by comparing the
ratio of water to fresh or dry weight in shoots taken in the field with that in shoots which
had been placed in water until they showed signs of full turgidity. Maximov & Krasno-
selsky-Maximov (1924) grew plants in pots of soil and measured the water deficit during
the drying out of the soil by expressing the water content of the leaves on a fresh- or dry-
weight basis, and comparing this quotient with that in well-watered control pots, or in
plants recovered by watering.
A similar method was used by Yapp & Mason (1932) for estimating the water deficits in
leaves at different heights on the shoot. Bell jars containing wet sponges were placed over
the plants and the water: fresh-weight quotients in these plants compared with control
plants in the open. Runyon (1936) estimated water deficits in the creosote bush growing
in the desert of Arizona by placing cut shoots in water in a saturated atmosphere. He
found that such shoots absorbed water very rapidly at first, but that the rate of absorption
soon decreased, little further uptake being demonstrated after 24 hr. The curves for the
increase in water content of fioating disks and whole leaves described in the present work
are strikingly similar to those of Runyon for whole shoots. Runyon measured the
water:dry-weight ratio in the 'saturated' shoots and compared this with that of similar
shoots growing in the field.
Less satisfactory methods were used by Evenari & Richter (1937), who followed
seasonal fluctuations in water deficit by comparing the water: dry-weight quotient at any
given time with the maximum value obtained during the season. This ignores the possible
occurrence of large seasonal changes in dry weight discussed above.
More recently, Killian (1947), working in the Sahara, suggested a similar method of
expressing water deficits to that developed here. This technique was similar to that of
Runyon (1936). Shoots were collected in the field, and after weighing were placed in
water in a saturated atmosphere for 48 hr., after which the shoots were reweighed and
oven-dried. Assuming no appreciable fall in dry weight during saturation, Killian was
thus able to obtain the initial water content (field-water content) and the saturation water
content, whence
, - . (water to saturate) - (field water)
water deficit = ^ — x 100,
water to saturate
In all these investigations, for the most part carried out under arid conditions, whole
shoots or plants were used to measure the water absorption under saturation conditions.
The methods used were designed to gain impressions of the magnitudes of water deficits
obtaining in desert plants. It is doubtful whether even the technique of Killian would
permit the estimation of smaller day-to-day variations under less extreme conditions. The
complete surface drying of whole shoots without transpirational loss must be difficult to
carry out. Again, dry-weight changes are very likely to occur during the process of
saturation. The other methods cited above suffer from the disadvantage of expressing
water on a dry- or fresh-weight basis, which detracts from their usefulness in following
seasonal trends. A doubling of the dry weight in the tissues, such as demonstrated in the
96 p. E. WEATHERLEY
present work, would lead to a halving of the estimated water deficit, so that there is
a probability of the water deficits appearing to diminish with advancing age.
Relative turgidity suffered none of these disadvantages, being unaffected by age trends
in dry weight, and the disks being easy of manipulation. Also the errors of samplmg were
such that taking duplicates, any change in relative turgidity greater than about 2 %
attained statistical significance. Thus small changes in water balance over long or short
periods of time, were measurable under field conditions.

SUMMARY
1. Methods of investigating the water relations of cotton plants growing m the field
were studied with a view to discovering how environmental conditions affect the water
balance of the plants.
2. The water content of the leaves was considered to be a guide to the balance between
transpiration and absorption of water at any given time. The initial problem was to find
an adequate basis for expressing the water content of the leaves.
3. The usefulness of various methods of expressing the water content of leaf samples,
taken periodically through the season, was compared. Expressions of water on a dry
weight, protein nitrogen or cellulose basis showed marked age trends unrelated to changes
in environmental conditions. Dry weight and dry-weight components were rejected,
therefore, as inadequate bases for expressing seasonal fiuctuations in the water balance
of the plants.
4. It was found that disks punched from leaves could be made fully turgid by fioating
on the surface of water. In this way the water content of a fully turgid sample of disks
could be measured and compared with that of a similar sample taken directly from the
field. Thus the following ratio was found:
Water content of tissue in the field
Water content of the same quantity of tissue when fully turgid
This ratio was called the 'relative turgidity' of the tissue. In practice, a fixed number of
disks taken at random from a plot consitituted a sample. Samples were taken in pairs;
the water content of one sample was found by oven-drying directly (field-water content),
the other sample was floated for 24 hr. on water after which its water content was also
found (fully turgid water content). A relative turgidity of 100 represented full turgor,
whilst any smaller figure indicated a water deficit in the leaves,
5. Sources of error in measuring relative turgidity are briefiy discussed. It is concluded
that relative turgidity constitutes a useful method of following water deficits in the field.

The writer wishes to thank the Director of Agriculture in Uganda for permission
to publish the data described in this paper, and Prof. E. Ashby, of the University of
Manchester, for his helpful advice on the presentation of the data.

REFERENCES
ASHBY, E . & WOLF, R. (1947). A critical examination of the gravimetric method of determining suction
force. Ann. Bot., Lond. (N.S.), 11, 261.
BROWN, H . B. (1927). Cotton. History, Species, Varieties, Morphology, Breeding, Culture, Diseases, Marketing
and Uses, ist ed. McGraw-Hill.
CHEN, W . W . & CAMERON, F . K. (1942). Cellulose content of cotton and southern woods. Industr. Engng
Chem. 34, 224.
 Studies in the water relations of the cotton plant 97
DoGGETT, H. (1942). Thesis. Imperial College of Tropical Agriculture.
EVENARI, M . & RICHTER, R . (1937). Physiological-ecological investigations in the wilderness of Judaea.
jf. Linn. Soc. {Bot.), 51, 333, 339.
HAWKINS, R. S. (1927). Water and dry-matter in the leaves oi Pima and Acala cotton. Arizona Sta. Tech.
Bull. 17, 417. (Quoted from Exp. Sta. Rec. 59, 35, 1928.)
HERRICK, E . H . (1933). Seasonal and diurnal variations in the osmotic values and suction tension values
in the aerial portions of Ambrosia trifida. Amer. J. Bot. 20, 18.
KILLIAN, C. (1947). Le deficit de saturation hydrique chez les plantes sahariennes. Rev. gen. hot. no. 638, 81.
KRAMER, P. I. (1937). The relation between the rate of transpiration and the rate of absorption of water by
plants. Amer. J. Bot. 24, 10.
LIVINGSTON, B, E, & HAWKINS, L . A. (1915). The water-relation between plant and soil. Publ. Carneg.
Instn, no. 204,
LLOYD, F , E, (1913). Leaf water and stomatal movement in Gossypium and a method of direct visual
observation of stomata. Bull. Torrey Bot. Cl. 40, i.
MASON, T . G . & MASKELL, E . J. (1928). Studies on the transport of carbohydrates in the cotton plant.
(1) A study of diurnal variations in the carbohydrates of leaf,bark and wood and ofthe effects of ringing,
Ann. Bot., Lond., 42, 188.
MASON, T . G . & PHILLIS, E, (1942). Studies on the foliar hydration in the cotton plant, H, Preliminary
observations using the disk-culture method, Ann. Bot., Lond. (N.S.), 6, no. 23, 455,
MAXIMOV, N . A. & KRASNOSELSKY-MAXIMOV, T . A. (1924), The wilting of plants in its connexion with
drought resistance, J. Ecol. 12, 95,
MILLER, E . C . (1917). Daily variation of water and dry matter in the leaves of com and sorghums. J. Agric.
Res. 10, I I ,
OPPENHEIMER, H . R. (1932). Zur Kenntnis der hochsommerlichen Wasserbilanz mediterranen Geholze.
Ber. dtsch. bot. Ges. 50a, 185.
PHILLIS, E . & MASON, T . G . (1943). Studies on the foliar hydration in the cotton plant. III. Preliminary
observations using the pruning method. Ann. Bot., Lond. (N.S.), 7, no. 26, 147.
PHILLIS, E, & MASON, T. G. (1945), Studies on the foliar hydration in the cotton plant, VI. A gel theory of
cell-water relations. Ann. Bot., Lond. (N.S.), 9, no. 36, 297.
PORTSMOUTH, G . B . (1937). Variations in the leaves of cotton plants grown under irrigation in the Sudan
Gezira, Ann. Bot., Lond. (N,S,), i, 277.
RUNYON, E . H . (1936). Ratio of water content to dry weight in leaves of the creosote bush, Bot. Gaz. 97,
S18.
SHREVE, E , B, (1924), Factors governing the seasonal changes in transpiration oi Encelia farinsa. Bot. Gaz.
77. 432-
THODAY, D . (1921). Behaviour during drought of leaves of two Cape species oi Passerina with some notes on
their anatomy, Ann. Bot., Lond., 35, 585.
WEATHERLEY, P. E. (1947), Note on the diurnal fluctuations in water content of floating leaf disks. Nezv
Phytol. 46, 276.
YAPP, R, H , & MASON, U . C . (1932). The distribution of water in the shoots of certain herbaceous plants.
Ann. Bot., Lond., 46, 159.

New Pbytol. 49,