TER VERKRIJGING VAN DEN GRAAD VAN
DOCTOR IN DE WIS- EN NATUURKUNDE AAN
DE RIJKS-UNIVERSITEIT TE UTRECHT, OP GE-
ZAG VAN DEN RECTOR-MAGNIFICUS Dr. J
BOEKE, HOOGLEERAAR IN DE FACULTEIT
DER GENEESKUNDE, VOLGENS BESLUIT VAN
DEN SENAAT DER UNIVERSITEIT TE VER-
DEDIGEN TEGEN DE BEDENKINGEN VAN DE
FACULTEIT DER WIS- EN NATUURKUNDE OP
VRIJDAG 1 JULI 1938 TE 15 UUR

Aan allen, die op eenigerlei wijze hebben bijgedragen tot het
totstandkomen van dit proefschrift betuig ik mijn hartelijken
dank.

In het bijzonder gaat die dank uit naar mijn ouders, die de
studie en de promotie hebben mogelijk gemaakt, en die nu,
naar ik vermoed, een steeds gekoesterden wensch vervuld zien.

Maar niet minder dank ik U, professor Koningsberger, hoog-
geachten promotor, voor de aanmoedigingen, die ik herhaaldelijk
van U mocht ontvangen en voor de aandacht, waarmede gij het
onderzoek en de theoretische consequenties, zoomede de vor-
ming dezer publicatie, gevolgd hebt.

Ik hoop, dat gij, hoogleeraren De Bussy, Honing, Jordan, Pulle
en Westerdijk, door mijn belangstelling bemerkt hebt hoezeer
de wijze waarop gij Uw vak doceert en de persoonlijke invloed,
die daarbij op Uw leerlingen uitgaat, door mij gewaardeerd
worden.

Voor Netty ook hier eenige woorden van bijzonderen dank
voor de moedige wijze waarop ze zoo vaak haar persoonlijke
verlangens op zij heeft weten te zetten voor het doel, dat wij
beiden voor oogen hadden.

I 6. The localization of amylase in the pulvinusnbsp;788
§ 4. Micro-method for the estimation of reducing

§ 5. Methods of extraction and hydrolysis . . . . . . . ..795
§ 6. Amylase content of upper- and lower half of the

Insufficient account of the very divergent points of view in
distinguishing several types of movement gave rise to a serious
coMusion m the nomenclature in the field of plant movements

It we restrict this discussion to movements of members of
plants in the first place a distinction can be made between
growth-movements, in which growth is concerned, and movements
oj variation, due to changes in turgor.

A second distinction can be made between autonomous and
aitiogenous (=paratomc) movements. In the autonomous
movements It is not possible to connect them causally with any
agent outside the plant, while in the pa'ratonic movements this

is the case. The autonomous movements are due to a mechanism
that is entirely determined by the structure of the plant; in
plants showing paratonic movements, such a structure can react
only with a preformed response to an agent outside the plant.
In this latter case, where the agent causes a reaction-type,
which is completely determined by the structure of the plant, one
speaks of a nastic movement. Besides these are those plants, of
which the structure is such, that the direction and the type of
movement entirely is determined by the agent; this is the case
with the tropic movements.

Whenever a movement is replicated consecutively in the same
manner, whether under the influence of an inducing (periodic)
agent or not, one speaks of periodic movements; besides, of
course, theie are purely aperiodic movements.

If one would like to summarize the above mentioned points
of view in one system of classification of plant movements, it
would be rather indifferent which distinction should be chosen
for the main division. A usual system is that which for instance
is reproduced in Meirion Thomas' quot;Plant Physiologyquot;; it is as
follows:

Hitherto the word nasty has been used for several kinds of
movements of a divergent type, as for instance was the case with
nyctinasty. For reasons of this kind I propose the following classi-
fication of the movements of members of plants, based on the
arguments mentioned below, (see next page).

Instead of movements of variation one may also speak of
quot;turgor reactionsquot;, as repeatedly has been done by several
authors; this word, however, suggests that changes in turgor are
not concerned in the case of growth movements, and this cer-
tainly does not hold true.

When parts of plants show movements of variation these move-
ments by the structure are restricted to a defined plane. This
IS not the case in tropisms and perhaps this is the reason why
tropisms are not allied with movements of variation. If an organ
liable to tropisms and to movements of variation both, is
attected by an external agent, first a tropic (growth) reaction
occurs until the agent acts in the plane of the movements of
variation.

The sleeping movements of Phaseolus, Canavalia and other
Leguminosae are movements of variation. Of course, the pulvini
which exclusively are the moving organs, show their movements
of variation long before they are full-grown, but in that first
period the mechanism of these movements may be considered
superimposed upon the growth process.

Bunning (1936) has made a similar distinction. He adds that
the movements sub a. especially are sensitive to the light of

shelter wavelengths, the pulvini relaxing during the sinking
of the leaf, while those sub b. are sensitive to the red part
of the spectrum, the pulvini remaining turgescent during the
movement.

The light-turgor-reactions (movements of variation) with a
short latency are called by Mar. Brauner (1933) quot;phototropicquot;,
if the light falls in the plane of the movement, and quot;photo-
nasticquot;, if the reaction is the result of a symmetrical light
stimulus.

Both types of movements, sub a. and sub b., occur under the
influence of light; these are nastic movements. Moreover, a
mechanism in the plant regulates the movements in constant
darkness; the convincing experiments of Kleinhoonte (1929)
have shown that the plants continue their movements in con-
stant darkness (see also the second half of this paragraph).
Even if the movements in the dark should be regarded as an
after-effect of the preceding light stimulus it carmot be over-
looked, that the period of the dark movements is independent of
the period of the light stimuli which regulated the movement
before. Therefore the movements in constant darkness may be
called endonomous movements; the period of their rhythm is
given by the structure of the plant.

Often the term quot;autonomousquot; has been used to characterize the
dark movements of Phaseolus and Canavalia. However, this
woi d suggests metaphysics and further autonomy in the strict
sense is inconceivable in plants.

For these reasons I prefer the word quot;endonomousquot; to charact-
erize the properties of this kind of movements.

Probably in the plant there are many distinct kinds of pro-
cesses that may lead to endonomous, rhythmic movements. The
periods of such movements vary in different objects from about
1 minute to many hours. As examples of plants with very short
or short endonomous periods may be given:

Desmodium gyrans, period ^ - 1 minute,
Trifolium pratense, period l]/2 - 4 hours,
Oxalis Acetosella, period % - 2 hours.

Their periods are longer at low temperature and in aging
(Kabsch 1861, Pfeffer 1875).

Also in Phaseolus or Canavalia one may find such endonomous,
short periodic movements. Examples may be found in Pfeffer
(1915, p. 93, fig. 28B) and in Kleinhoonte (1929, fig. 37 and
p. 67, fig. 33). These concern plants in which the long periodic

movements have been suppressed. One of the graphs of Klein-
hoonte is reproduced in fig. 1.

The endonomous periodic movements in the dark have caused
the great confusion in hterature and gave rise to the question
at issue: are the movements endonomous or are they aitiogenous?

Sachs in 1857 came to the conclusion that quot;autonomous move-
ments regulated by lightquot; are involved; at least he seemed most-
ly mclined to this conclusion, though in 1863 he also mentioned
an after-effect of the light influence in total darkness. Pfeffer
in 1875 gave this classification:

a. die einfache Receptionsbewegung und deren Nachwirkung,
d. die täglichen periodischen Bewegungen.
The sleeping movements were described as a result of the
after-effects (not called autonomous) and continual rhythmic
stimuli. In 1915 Pfeffer, apart from the autonomous spontaneous

1) In this historical survey I still use the word quot;autonomousquot; when the
authors have done it.

movements, distinguished also autonomous after-effects. Sleeping
movements he too then called: quot;autonomous movements regulated
by lightquot;.

The experiments by Stoppel (1932), Brouwer (1926) and others
gave rise to doubt as to the autonomous character of the sleeping
movements. They supposed the movements to be determined by
external influences. Brouwer thought to make things clearer
by introducing an, unfortunately not further to be defined,
factor X.

Kleinhoonte (1929) and BiInning (1932), at the hand of many
experiments, brought the phenomenon again under the definition
already given by Sachs (1857) and Pfeffer (1915): quot;autonomous
movements regulated by lightquot;.

The endonomous processes determine the length of the period
of the movements at constant external conditions.

If these conditions are not constant, the length of the period
is only influenced by them to a certain extent. For instance, the
endonomous rhythm depends on the rhythm of light and dark
var iation within the boundaries 6 — 6 — 6 — and 24 — 24 ^—^ 24 —
(the figures indicate the length of subsequent light and dark
periods in hours). Shorter alternations than 6 — 6 — 6 —■ and
longer ones than 24 — 24 — 24 — were not followed by the
endonomous processes; in these cases the normal 12 — 12 — 12 —
rhythm of the movements was resumed again (Kleinhoonte 1929).

biinning (1931) has given another convincing proof of the
endonomous character of the vai iation movements. He determined
the length of the period at different temperatures. His results
were that at a temperature of 15° C. the length of the period
was about 30 hours and at 35° C. it was about 20 hours;
intermediate temperatures gave intermediate periods.

Resuming it may be stated that the sleeping movements of
the investigated plants are based upon an endonomous system
which, within certain limits, can he influenced hy external
stimuli, e.g., light, temperature.

I have hitherto exclusively dealt with experiments and problems
concerning the character of the movements, without mentioning
the attempts which have been made to elucidate the internal
mechanism of the processes resulting in moving of the leaves.

Several questions have engaged the investigators on this
subject since Sachs. These questions develop from each other
by logical reasoning, and that is the reason why I have treated

them subsequently in separate paragraphs of chapter IV. The
order chosen by me does not only give a logic survey but also
reflects more or less the chronological sequence in which the
questions have been treated. Moreover, the study of the various
processes gives a good idea of the difficulties to be met with
when studying the mechanism of the endonomous system

In chapter IV in this way the available data on the quot;sub-
sequent questions will be linked up and combined. This led to
investigations on starch and amylase and on several questions
concerning the staich-sugar metabolism in the plant

Properly speaking, hitherto all data on the mechanism of
the sleeping movements were descriptive; none of them gave
rise to a theory on the real prohlem of the sleeping movements:
the endonomous periodicity in constant darkness. It is this pro-
blem which continuously engaged me; it meant that I had to
look for a system in the plant, which in itself could account
ior periodic changes in volume. It will be superfluous to sav
that these changes in volume were supposed to correlate directly
with changes in sugar concentration of the cell sap. From this
only a little step brought me to a possible role of starch and of
an^lase and so the outline of the investigations was traced

The results and the conclusions given in the following pases
are not exhaustive but also may be valuable by stimulating to
detailed investigations on the complicated processes involved

In the course of time mainly two species showing sleeping
movements have been chosen as an object for the study of
n^^tiMsty, I.e., Phaseolus multiflorus L. and Canavalia ensiformis
UU. ±Joth plants show the movements equally well, perhaps
Canavalia more regularly (that means, less disturbed by external
iMluences) than Phaseolus. Especially in the winter season
^^aseoius is more vigorous in growth and less liable to diseases
Although the sensitiveness to external agents is higher in Phaseo-
lus tli^ in Canavalia, they have in common the processes which
result m the endonomous movements in constant darkness (see ge-
neral introduction). Since many of my experiments had to be done
m the winter season, I preferred Phaseolus to Canavalia (for
even artificial day-lengthening by means of neon light could not

The first pair of leaves of Phaseolus shows the movements
most clearly. By continually cutting off the main shoot, the
first leaves develop to a large size and consequently the pulvini
at the base of these grow favourably for a detailed study. The
same pulvini (between petiole and lamina) have served as an
object for the experiments of nearly all the earlier investigators.

Throughout the year the plants were cultivated in the green-
house. In the winter the night period was reduced to 7 hours
by means of neon light. The age at which the plants were used
varied froin about 3 weeks until two months after sowing.
Besides the main shoot also the developing axillary buds must
be continually cut off.

§ 2. The anatomical structure of the pulvinus (fig. 2) may be
characterized by the following features:

Macroscopically the motile organ shows a dorsiventral struc-
ture, the surface of the lower side being cylindrical, that of
the upper one is grooved longitudinally, so that a reniform
transverse section results. The same reniform section is shown
by the central vascular bundles, that are fused to one central
cylinder in the pulvinus, in contrast to the several, peripherically
arranged, strands in the petiole.

the centre consists of merely collenchyma cells. They are sur-
lounded by the vascular strands, cylindrically arranged, except
at the upper side, where the collenchyma of the pith is con-
nected with the collenchyma that surrounds the vascular cylin-
der. Round the collenchyma sheath lies the starch layer which
since long ago drew the attention by its local storage of' starch
exclusively found in the one or two layers of cells, which con-
stitute the most central part of the motile tissue. The remaining
motUe tissue consists of parenchyma cells, almost isodiametiic
(somewhat stretched in a radial direction). Intercellular spaces
cannot be found in this tissue, except in the more central part
where two or three layers of cells show air-filled intercellular
spaces. This was already stated by Sachs (1857). The epidermis
closely adjoins the motile tissue, it is unicellular in thickness
Its cells are smaller than those of the motile tissue, stomata
occur very scarcely. The outer walls of the epidermal cells are
somewhat thickened, the cuticule showing a peculiar wavy
striped, pattern. The surface is covered with hairs, that of the
upper side rather densily, that of the lower side sparsely Active
hydathods occur, also more on the upper side than on the lower

Microscopic reactions: only the walls of the vessels are ligni-
fied (phloroglucin-HCl). All other cell membranes consist of
cellulose: with iodine in sulphuric acid they stain deeply blue
with iodine in zinc chloride violet. Boiling in Sudan-glycerin
merely stains the cuticula (red). Starch is present in the starch
layer m considerable amounts; whether it is present in the
other cells of the motile tissue is difficult to state (perhaps in
minute grains).

I will especially point again to the quot;accordeonquot; structure of
the motile tissue (Zimmermann 1929). Such an accordeonlike
system promotes the changes in volume of the cells in axial
direction.

Measurements of the actual changes in volume of the motile
cells have already been applied in a calculation given in a pre-
vious paper, in collaboration with J. B. Thomas (1938). At that
place we anticipated upon the argumentation which would be
given in the present paper and which follows below.

Some shadowgraphs were made on photographic paper of the
same pulvinus in a high and in a low position (corresponding
to the extreme day- and night positions of the leaf). See fig 3
Of course no exact measurements of the changes in volume of

each cell apart could be made, but measuring the changes in
volume of the whole pulvinus gives an average value for the
voltune changes of all cells together.

It can be measured from the photographs that the increase
in length of the upper side during a sinking of the leaves amounts
to about 50% of the original length, while at the same time the
lower side shows a decrease of about the same value. Assuming

that the changes in volume are simply linearly proportional to
the changes in length (according to the quot;accordeonquot; structure
and neglecting the volume changes in transverse directions), it
may be said that the average changes in volume of the cells
amount at least 1,5 X-

This value has been adopted in the calculation in the previous
paper l.c. and it will be used again in the last paragraph of
chapter VII.

In chapters IV to VII of this paper I shall deal with the sub-
jects that, more or less in chronological order, have engaged
the attention of previous investigators. Where it proved to be
necessary, I added some supplementary experiments of my own,
while the material described in chapters V, VI and VII is new
and led to the hypothesis discussed in chapter VIII.

The data, hitherto gathered by several authors in studying
sleeping movements of plants, mainly have a phenomenological
character. They form the assay to which the explanation of the
mechanism of the movements always should be tested.

When I had the opportunity to state personally, by anatomical
mves igation, to which extent the starch layer in the pulvini
:s filled up with starch, it was very tempting to think of a
possible relation between the storage of this starch and the
processes underlying the movement. The simpliest relation viz
a daily-periodic quantitative alteration in starch content soon
proved not to be observable. However, the starch might have
the junction of a source of energy and for that reason I attempted
to determine the relation between the presence of starch and
the occurrence of movements. This once done, as a logical con-
sequence, I continued the investigation in the direction of in-
fluencing the starch content of the plant, hoping so to influence
the movements. - All the experiments concerning starch are
described in chapter V. —

Once conceiving a possible function of starch, it is a matter
of course that one has to include amylases into the investigation
m order to connect changes in turgor, by way of alterations in
the osmotic value of the vacuole, with the eventual role of a
starch-sugar inversion. Now the most plausible assumption is
that the quantity of amylase(s) in both halves of the pulvinus
would alter reversibly, with the same rhythm as the daily-periodic
movements. Accurate measurements of the amylase quantities in
the upper and lower half of the pulvinus, in the uplifted as well
as in the drooped position of the leaves, led to the conclusion
tiiat such differences, though they were perhaps demonstrated
are inadequate to account for the periodic changes of the osmotic
values. However, it might be possible too that the total amount
ot amylase, present in the cells, is constant in its quantity vet
Its activity being regulated by influences inside the cells —
Measurements of amylase activities are reported m chapter VI —

In view of the endonomous periodic character of the processes
investigated, it seemed attractive to examine changes in the
concentration of the cell sap, due to changes in volume, and, in
connection with the sensitiveness of enzymes especially to hy-
drogen-ion concentrations, it was obvious to focuss particular
attention on the influence of pH on the amylase activity — See
chapter VII. —

It follows from the ever greater complexity of the processes
involved that the conception given on the mechanism of the
movements gradually gets an increasingly hypothetical character.
Ihe elements, however, hitherto are in agreement with the facts

I am fully aware that in fact I have not exhaustively in-
vestigated the several parts of the problem. The system soon

proved to be so complicated that this could not be avoided. It
would have been much easier to lose myself in a detailed study
of a well defined inferior part of the process than to survey the
system as a whole, and yet the latter was my intention. Certainly
I may have overlooked vital processes which also play a more
or less important part besides the phenomena described in the
next pages.

Although, at present, it is beyond doubt that the changes in
volume of the motile cells are due to changes in turgor of these
cells, it is interesting that once this subject has been discussed
in literature. Since in 1857 Sachs had mentioned that it is the
turgor of the cells that changes periodically without thinking
of another possibility, Hofmeister in 1862 suggested that the
phenomenon of imbibition of the cell walls (quot;Zellhäutenquot;) was
much more likely the base of the changes in volume. Hofmeister
was influenced by the data and the theories published in 1861
by Graham on the properties of colloidal and crystalloidal sub-
stances. The imbibition of the cell walls would be followed by
water absorbtion by the cell sap. The hydrating and de-hydrating
power of the colloidal system, conditioned by all kinds of factors,
would offer a suitable means to explain the rapid changes in
volume of the motile cells.

In 1875 Pfeffer could not decide which of these two views
should be preferred, but he felt that some data were in favour
of the opinion of Sachs. In 1909 Lepeschkin published experi-
ments, which served to demonstrate that the mechanical pro-
perties of the cell wall did not alter during the movement. He
measured the flexibility ') of the pulvini, after plasmolysis,
before and after light and daik periods. The flexibility did not
prove to alter and therefore Lepeschkin concluded that only
changes of the turgor occur during the movement. Since that
time the attention entirely has been focussed upon changes in

1) To this purpose he used the method of Brücke: the angle between
lamina and petiole is compared in a normal and in an inverse position of
the plant; the weight of the lamina causes a bending of the pulvinus
proportionate to its flexibility.

the turgescence of the cells, and never since has been spoken
of imbibition or such. Yet it may be useful to consider the
possibility that, to a slight extent, changes in the water content
of the cell walls may play a part in the moving effect.

Another question is the co-ordination of the turgor changes
in the upper- and in the lower half of the pulvinus during the
movements. In 1857 it has been stated by Sachs that the changes
in turgor take place in an opposite sense in the antagonistic
halves. He supported his statement by comparing the flexibility
(measured in the same way as by Lepeschkin) of the pulvinus
in a drooped and in an uplifted position of the leaf. Pfeffer
(1875) has endorsed Sachs' view by experiments with parts of
pulvini. He cut away the upper- or the lower half of the organ
and registered the movements of the remaining halves in con-
stant darkness. I have reproduced his results in fig. 4, for they

convincingly prove the correctness of Sachs' original observation.
It was clearly distinguished by Pfeffer that his statement only
holds true for the daily periodic movements (and for the auto-
nomic movements) and not for the movements due to a singular
reception with after-effect (see the scheme by Pfeffer, given
on p. 763). The latter he attributes to changes in turgor, acting
in the same sense but with a different velocity.

about the beginning of this century the question on the co-
ordmation of the turgor changes in the pulvinus arose anew.
Jost (1898) and Schwendener (1887) undermined the results
and the conclusions of Pfeffer. During several years Schwen-
dener worked with Phaseolus (and Oxalis); all his observations
were made in the greenhouse without any precautions regarding
constancy of temperature, humidity, etc.; the cloudiness influenced
his results, as can be seen from his protocols. So it is not
amazing when he remarks: quot;An widersprechenden Reaktionen
fehlte es zwar auch bei dieser Versuchsreihe nicht!quot; Still, he
came to a conclusion contrary to that of Pfeffer: the rapid
reactions upon light stimuli (quot;Einfache Receptionsbewegungquot;
of Pfeffer) were ascribed to an opposite change of the turgor
in both halves of the pulvinus.

Pantanelli (1901), in experiments with Robinia Pseudacacia
and Porliera hygrometrica, confirmed the results of Pfeffer.
Jost has critisized this work; he called the investigation super-
fluous. — Pantanelli (1901), in a reply to Jost, held the view
that the investigations of Jost and Schwendener were more
likely superfluous than his own, since he worked with unknown
plants and the others used a material that had already exten-
sively and seriously been investigated by Pfeffer.

Wiedersheim (a student of Pfeffer), in 1904 attempted to
show that the controversy between Pfeffer and Pantanelli and
Jost and Schwendener was due to a different way of operating
(halving) the pulvini. He held the depth of the cutting responsible
for the result; Pfeffer should have cut up to the vascular cy-
linder, Schwendener should have halved the vascular cylinder
too. His experiments did not convincingly prove that this view
is correct. — Lepeschkin (1909), however, has investigated the
same subject, and he confirmed the results and the conclusions
of Wiedersheim.

In spite of all the supplementary investigations, we conclude
that still Pfeffer was right when he discriminated between two
types of movements and two types of co-ordination of the changes
in turgor in both pulvinar halves.

a)nbsp;Rapid movements, induced by an external stimulus (light,
temperature) — changes of turgor in the upper- and in the
lower pulvinar half in the same sense but at different rates.

b)nbsp;Daily periodic movements, quot;slow movementsquot;, — changes
of turgor in both pulvinar halves in an opposite sense.

stated that separated upper halves (lower half removed) did
not show movements, while the separated lower halves moved
in a daily periodic rhythm. He cut the pulvini so deep that the
white vascular cylinder just became visible. I do not see how
his differing results can be explained.

Many times it has been attempted to determine the osmotic
value of the cell content and often the results were contradictory.
This must be ascribed to a deficient distinction between the
two types of movements, the rapid and the slow ones. Hilburg
in 1881 attempted to state whether changes in the concentration
of the cell content after a short period of illumination of the
plant could be measured. This failed.

Kerstan (1909) applied the plasmolytic method (de Vries);
he found a difference in the quot;saltpetre valuequot; of the upper-
and the lower pulvinar half, in a day- and in a night position.
He did not account for the changes in volume that had occurred
in the meanwhile.

Lepeschkin (1909) combined the data of Kerstan with the
magnitude of the changes in volume as indicated in 1875 by
Pfeffer (40%) and concluded that no changes in the osmotic
values of the turgescent cells had been proved by Kerstan. He
made no experiments of his own.

Zimmermann (1929) estimated the osmotic value of cells when
just turgid, of both pulvinar halves, during the daily periodic
movements. He found striking differences. The variations of the
osmotic value appeared to occur antagonistically in both halves
and parallel to the changes in volume. When the decrease in
volume of cells just turgid was taken into account (according
to Ursprung, 1930), the differences still remained. Weidlich
(1930), using also the formulae of Ursprung, came to the same
results. Unpublished results of experiments by Miss Vaandrager
(Utrecht 1931) confirmed this too.

Since Lepeschkin (1934) and BUnning (1936) have critisized
the results and the conclusions of Zimmermann and Weidlich,
I decided to examine once more the osmotic conditions in the
upper and the lower pulvinar halves during the daily periodic
movements.

The osmotic value and the suction pressure of the cells of a
tissue can be measured in various ways. Always it has been
considered necessary to reduce the experimental data, with the

aid of more or less complicated formulae (Ursprung 1930, Weid-
lich 1930, Lepeschkin 1933, 1934), to the actual volume of the
cells in the tissue.

Now, in studying the movements of variation, the most import-
ant thing to know is, whether and how the suction pressure of
the tissue as a whole changes relative to the movements. I used
a somewhat simplified method, inasmuch as I compared the
volume (proportionate to the length) of a certain strip of pulvinar
tissue in water and in a hypertonic solution of glucose. The
1 mol. glucose solution caused plasmolysis; in this state the length
of the tissue strip was minimal. The length of the strips in water,
as compared to the minimal length in glucose solution, was taken
as a measure for the suction pressure of the tissue.

The strips were first placed in water, then in the glucose
solution; controls showed that first placing in glucose and then
in water gave almost the same values. The shortening in glucose
solution as compared to the length in water was expressed in per-
centage of the length in the
glucose solution. The strips
were longitudinal (radial)
sections of the pulvinar
motile tissue of the upper
and of the lower half. To
facilitate the measuring,
the outline of the strips,
in water as well as in the
sugar solution, was traced
on paper. An example of
this procedure is given in
fig. 5.

Sometimes an increase of
length in a transverse direc-
tion was noticed to be
combined with a decrease
of length in the longtudinal
direction, when the strips
were placed in the glucose
solution. This may support
the existence of an quot;accor-
deonquot; structure of the mo-
tile parenchyma (Zimmer-
mann 1929, Mar. Brauner
1933).

With the aid of tangential strips I first have determined the
sJiortenmg of more or less peripherically situated parts of the
pulvmar tissue. The results of these experiments are given in
table 1.

To ascertain that endosmosis or exosmosis might not give false
results, I arranged special experiments. In these series I placed
^e strips first in paraffin oil (P), to trace the original outline
l^en in two series I placed them first in water (W), then in
glucose solution (S), in two other series first in glucose solution
(S), then m water (W). In each of the media the outline of the
strips was traced. Changes in length were expressed in percen-
tages of the length in paraffin oil. The results are given in the
following table.

Sometimes even a slight lengthening in sugar occurred, showing
that the osmotic value may exceed that of 1 mol glucose
A comparison of the series P—W—S and P—S—W shows that

the relative length of the strips in water shows differences
between the upper and the lower halves, while the values of
the S-column scarcely differ in both halves. Therefore the length
of the strips m water as compared to that in glucose solution
(and expressed in percentage of the length in glucose) may be
used for comparing the suction pressure (and so the osmotic
values) of the tissue as a whole. No actual values of the osmotic
pressures or suction pressures have been calculated, since I only
intended to investigate whether differences between both halves,
m various positions of the leaf, could be detected.

The results of the experiments, carried out to this purpose,
have been summarized ui table 2. The percentages given in this
table are averages of 3 to 8 measurements each. For each
percentage I have calculated the mean error from the formula
■\l S a}

of the difference between the values for the two antagonistic
halves of one pulvmus calculated (from the formula my = ±

II. Leaves in a drooped position (night).

J	upper	half	7
	lower	half	5
L	upper	half	4
	lower	half	4
M	upper	half	5
	lower	half	3
N	upper	half	7
	lower	half	6

12,9	10,26
9,4	12,9
19,9	6,54
9,5	8,32

TABLE 2. Comparison of the osmotic values in both pulvinar halves.
I. Leaves in an uplifted position (day).nbsp;~

III. For leaves in an intermediate position all differences found between
upper and lower halves were far within the limits of error.

The positions of the leaves were valued from the angle between
lamina and petiole; no registering of the movements took place,
since too many plants were involved in the experiments. There-
fore not all results of the series with strips of uplifted leaves
exceeded the limits of error.

The results of these experiments are in full agreement with
the results of the former investigators. They all lead to the
conclusion, that the osmotic value of the upper half is minimal
in an uplifted position of the leaf, while that of the lower half
is maximal in that case; when the leaf is in a drooped position,
the situation is reverse.

It may be emphasized again that this only bears upon the
slow, daily periodic movements.

It may be useful, but it is adventurous, to express the course
of a certain vital phenomenon in terms of mathematics. For, when
the formulae are erroneous, the conclusions drawn from them
are false.

Lepeschkin in 1933 and 1934 has published papers on the
osmotic processes in cells (cells apart and in a tissue) and on
the mechanism of the periodic movements of variation. He has
given a set of formulae, expressing the relation between turgor
pressure, suction pressure and water withdrawing forces of a cell
in a tissue. He also has taken into accoimt the permeability, but
particularly that part of his calculations has led him to somewhat
strange conclusions on the influence of changes in the permea-
bility of cell membranes on the turgor pressure of the cells.

Before critisizing the work of Lepeschkin I will first define
some symbols used by me and partly also used by him:

T — turgor pressure, the pressure exerted on the cell wall
by the cell content, straining elastically the wall.

Pw — the partial hydrostatic pressure of the water in the
solutions outside the cell.

Po — the partial hydrostatic pressure of the solved substances
in the solutions outside the cell.

a — the total area of the surface of the membrane (assuming
the inner- and the outer surface to be equal).

Permeability (p) is defined: the ratio of the number of mole-
cules passing a membrane to the number of molecules colliding
against it.

(K = a constant, dependent on properties of the cell wall
and of the tissue and on conditional factors).

Now Lepeschkin is right in deriving his formulae until the
point where he introduces the permeability into the problem.
Once assuming that the membrane is permeable to osmotically
active substances no final turgor pressure can be expected in a
cell placed in water.

Lepeschkin (1933) expresses the relation between the suction
pressure (Si) of a solution and its osmotic pressure (P) in the
equation: Si =: P (1—fi), where /x represents the quot;permeability-
factorquot; for the solved substance. Considering the permeability for

represents the quot;permeability-factorquot; for water. Now these formu-
lae cannot be used for describing
the effect of a change of /x or a,
since P and /x or P and a are
not varying independently. This
has not been mentioned by Le-
peschkin. Differential calculus
should have been applied in this
case! The problem is rather
difficult to solve and falls beyond
the scope of this paper, I shall
not treat it here.

I prefer to consider the case
of a eel in a system (a tissue),
where a part of its surface is in contact with a solution 1 and
the other part with a solution 2 Several assumptions must
be made before a final turgor value of the cell can be expected.
The most important assumption is that constantly the loss of
osmotically active substances (caused by permeating) by the cell
is supplied by a mechanism in the cell (viz. photosynthesis or

The symbols regarding solution 1 are marked ' that regarding solution 2
are markedquot;; the symbols regarding the cell content have no indices.

activity of enzymes). In this case O is kept constant. I assume
further thatP;gt;K.Ygt; P;^ andnbsp;P;gt; P.; (see fig. 6),

and that a part m of the cell surface is in contact with solution
1, and that P^, P^, P^' andP^ are constant (viz. continuously

refreshed solution). For the equilibrium to be estabhshed, we
now may write that in a certain period the numbers of molecules
entering and leaving the cell must be equal:

It is clearly expressed by this formula how (when equilibrium
has been reached) the volume of the cell V depends on O and on

influences the value of V is entirely dependent on the mutual
proportion of Pw and Po in the solutions 1 and 2 (i.e. the osmotic
values of the solutions surrounding the cell).

in a different value for V, since the formulae represent the statics
of the processes, i.e. the equilibrium. The factors in the formulae
are not independent of each other and therefore an alteration
of one factor affects the value of others and the result carmot
be predicted.

For these reasons I carmot see how Lepeschkin (1935) and
biinning (1936), from their formulae (or even without describing
the phenomena in formulae), could conclude that changes in
permeability (for water and solved substances) of the cell mem-
branes would result in a definite change of the volume or of
the turgor value.

A study of the anatomy of the pulvinus especially called my
attention to the great quantity of starch that normally fills up
the starch layer. Since it seemed probable that this starch might
play a fundamental part in affecting or in supporting the endo-
nomous processes on which the movements are based, I tried to get
some data on this storage of starch under various circumstances.

In the petiole as well as in the pulvinus the starch is stored
mainly in the starch layer (surrounding the vascular bundles),
in granula of rather large dimensions. In the more peripherical
cells of the pulvinar tissue starch will be probably present, but
in scarcely demonstrable grains (very small ones) or perhaps in
a solved form.

The starch layer consists of two or three layers of parenchyma
cells, adjoining closely the collenchyma that surrounds the central
vascular bundle in the pulvinus. Generally on the upper side
the starch storage is markedly less than on the lower side.

To examine the starch content of the moving pulvinus during
a period of 24 hours, every hour of a series of plants one pulvinus
was isolated and fixed (in alcohol 96%); afterwards these pulvini
were tested on starch content. At the same tune of all plants
the angle between petiole and pulvinus was measured (also every
hour), while temperature and air humidity were continually
noted too.

The estimation of the starch content (here and in other experi-
ments) was made by staining the starch with iodine (in potassium
iodide) in a hand-made transverse section of the pulvinus. Of
course minor differences in starch content could not be noticed
in this way.

This standard only refers to the starch present in the starch
layer. When starch occurs in a considerable amount elsewhere

For the 24-hours experiment a nuniber of plants were chosen
grown from the same sowings and under equal conditions of
light, temperature and air humidity, all having a normal and
healthy appearance. During the experiments the plants were
left in their normal habitat (i.e., in the greenhouse, under natural
day and night succession). With the experimental method just
described a variation in the starch content of the pulvinar starch
layer during 24 subsequent hours could not be detected. Through-
out the 24 hours the starch layer was filled up with starch to
the same extent.

By earlier investigators (e.g. Kleinhoonte) it was noticed repea-
tedly that the sleeping movements still continue in the usual
12-12-12-hours rhythm for one or two weeks in constant darkness
until they finally stop. Now it might be of importance to control
the behaviour of the starch during such a dark period.

An ever felt objection to this kind of experiments is that one
cannot control the movement of the leaves after having done a
starch estimation, for to this purpose the pulvinus is cut off,
fixed and microscopically tested. In consequence, conclusions
must be drawn from experiments with more plants, grown under
the same conditions. This is permissible as is demonstrated by
fig. 7, which shows the result of a registration of the movements
of three plants, placed side by side in the greenhouse. The dull
and rainy days, 3rd and 4th of June, have had a corresponding
effect in all three plants (AA 1, AB 3, and AB 5, of two sowings).

In a dark room, with temperature about 23° C. (not accurately
constant), the movements of the same three plants were registered
from June 11th until June 25th. The results of the last part of
this registration are given in the graph of fig. 8. This figure
shows, that the plants AA 1 and AB 5 after one week had fairly
well stopped their movements, while those of AB 3 still continued
and even had not stopped after 14 days ')■ At that moment the
starch content of the pulvini of this series was estimated and
proved to be of

Thus, in spite of the similar behaviour shown before, in the dark these
plants displayed an individual character; the latter is due to internal
conditions, while the parallelism before was induced by external stimuli.

Of course these prehminary indications ought to be controlled
in more objects. The following table gives some more observations
on this subject:

A great number of plants of the same sowings (CY) and grown
under equal conditions, were placed in a dark room (temp, about
20° C.) at the same time. Each day one pulvinus was cut off
and fixed to be examined on starch. The movements of one of
the pulvini were registered during the whole experiment, this
pulvinus being fixed itself at the end.

The movements of the registered pulvinus continued until
about Februar 25th, with some irregularities perhaps caused by
the insufficient constancy of the temperature in the room. During
this first period the starch content gradually decreased, as
demonstrated by the microphotographs of the starch layer (the
starch stained deeply blue with iodine). See the graph of fig. 9
and Tab. XVIII, A. On Februar 26th, from 9h30 until 20h00, the
plants were illuminated by a bulb of 500 W. half a meter over the
leaves (with a water bath between plant and bulb to eliminate
thermic rays). The reaction to the illumination of the registered
plant was a lifting of the leaf. After some oscillations an arrest
was reached again somewhere near March 1st. I especially call
the attention to the fluctuations of the starch quantity during
the last part of the experiment. At the end of the light period
not only the starch layer had been filled up with starch, but
also the parenchyma cells of the motile tissue were unusually
full of starch, though in grains considerably smaller than those
in the starch layer. As to how this phenomenon is connected
with the illumination and as to how (c.q. whether) movement is
linked up with the high starch content, is not clear. The last
photograph shows that after all with the final arrest of the

Several investigators, a.o. Pfeffer
(1915), Stoppel (1932), Cremer (1923),
have mentioned that the plants stop
their sleeping movements in constant
light. That this does not hold true has
convincingly been demonstrated by Brou-
wer (1926) and by Kleinhoonte (1932).

I decided to examine the progress of
the starch content during a period of
constant illumination of the plants. To
this purpose three plants were placed
in a dark room and illuminated by a
bulb of 200 decalumen, 50 cm over the
leaves. The movements of one of these
leaves (AF 9a) were registered from
Januar 9th until 15th (see fig. 10), while
in that period the starch content of one
pulvinus each day was estimated. The
results were that no fluctuation of the
starch content could be noticed.

It may be concluded that during this
period of constant light the starch con-
tent did not increase nor decrease, nor
did any starch appear outside of the
starch layer.

The nature of the problem prevents
that absolute certainty can be gained as
to the direct relation between the starch
storage and the pulvinar motility, since
for studying the changes in starch con-
tent during a long period it is necessary
to draw more than one plant into the
investigation. Therefore all results must
be regarded as averages of several values.
None the less it may be stated that

Fig. 9. The result of the registration of the movements of one among
IT^'r Pl^^ts placed m a dark room. For further explanation see text -
this tigure should be compared with Tab. XVIII, A.

without doubt starch plays an important part as a storage of
energy for the processes that result in movement.

As long as starch is present movements can occur, though I
want to emphasize that this only holds true for a single type
of processes, all concerned with starch metabolism. Apart from

these, it is conceivable that, e.g., changes in permeability may
cause alterations of volume which have nothing to do with the
starch metabolism. I cannot, however, visualize how such volume
changes could be reversible.

As already indicated on p. 769 the possible part played by
starch in the mechanism of the sleeping movements drew the
attention to enzymatic actions inside the cells of the motile
tissue. In connection with starch, amylase(s) ') should be the
most important enzymes. The methods, described by van Klin-
kenberg (1931), for demonstrating the activity and the compounds
of diastasis (malt-amylases), with slight alterations, proved to
be very suitable for a qualitative proof of the occurrence of
amylase in the pulvinus. A more careful examination of the
amylase activity in different parts of a transverse section through
the pulvinus enabled me to localize roughly the amylase. The
next problem was to measure as accurately as possible the
exact quantity of amylase present in both halves, the upper

1) I further use the term quot;amylasequot; for all amylolytic enzymes, eventually
present in the pulvinar tissue.

and the lower, of the pulvinus. To this purpose I made myself
famdiar with the method described by Linderstr0m-Lang for
quantitative estimation of reducing sugars by means of a micro-
titration. Once acquainted with this micro-method, I needed a
good deal of time for finding the best way to extract the amylase
from the object. Not until these things were settled I could
continue my attempts to establish whether a difference in
amylase quantity between both pulvinar halves could be demon-
strated or not.

The method first used by Wijsman (1889) and recently by
van Klinkenberg (1931) to study the properties of the amylase
system of malt, seemed to me a suitable means to examine
whether amylases could be detected in the pulvinar tissue of
Phaseolus multiflorus L.

In this method a gelatin-starch plate is used to demonstrate
the action of the amylase. The plate is made by casting a gelatin-
starch solution (8% of gelatin,nbsp;of soluble staich) in a Petri

dish. The enzyme is allowed to hydrolyse the starch of the
substrate for a long time (1—3 days) at low temperature in a
refrigerator. — Van Klinkenberg has usually worked with en-
zyme solutions, only a few times he placed parts of a plant
tesue (from barley kernels) on the gelatin-starch substrate. —
The action of amylases is determined by staining the plates by
a dilute iodine solution.

I have applied the same method for examining the amylase
content of the pulvinar tissue. I placed some transverse slices

a gelatin-starch substrate;
after 24-hours incubation in the refrigerator, the plate was
stained with iodine. The tissue proved to contain a clearly
demonstrable quantity of amylase. The influence of alcohol and
ether was then tested; alcohol inactivated the enzyme, particu-
larly when heated up to 70° C. Ether did not inactivate at all
even a slight activation was noticeable, but the latter might be
attributed to a better diffusing of the enzyme from a dead tissue
than from a living one. A difference in amylase content between
the upper and the lower half of the pulvinus could not be
detected. The best period for diffusing of the enzyme from the
tissue appeared to be about 20 hours (at 0° C.).

No further data being obtainable with this crude method I
have tried to apply the same principle in a more detailed in-
vestigation, which will be described in the next paragraph

The experiments, described in § 2, only generally demonstrated
the presence of amylase in the pulvinus (as well as in the
petiole). Once familiar with the method of enzyme testing after
van Klinkenberg, I adapted the method to a more detailed
examination of the localities in the plant (in petiole and in
pulvinus), where the enzyme is present in the greatest quantity,
or at least where it is most active. It needs scarcely to be said,
that it would be difficult to discriminate whether one measures
the quantity or the activity of amylase in the plant.

To apply the method of van Klinkenberg for a minute exa-
mination, I casted the gelatin-starch solution (see foregoing §)
in a thin layer on a slide. The edges of the glass were coated
with paraffin, so that by floating it on a water surface, to get
an exact horizontal position of the slide, the gelatin layer became
as homogenous in thickness as possible. After solidifying of the
gelatin the slides prepared in this way were dried completely
for several hours and an quot;enzymographic platequot; was obtained
that, after quot;developmentquot; in iodine solution, fairly well repro-
duced differences in amylase activity of various parts of plant
tissue brought into contact with it.

Yet there is one point which easily can spoil the results and
give a false idea on the distribution of the amylase in the tissue.
An important condition for a good result is that the surface of
the tested tissue must be entirely in good contact with the
enzymographic plate, for if this is not the case the results, of
course, are misleading. The good contact between plant and plate
can be judged from the mark of the tissue that should be visible
on the plate.

Another, less serious, difficulty is the irregularity in staining
intensity, that inevitably occurs when a iodine solution of too
high a concentration is used. The concentration should be low
enough to permit a slow quot;developmentquot;, which can be stopped
at any moment desired.

After the development the quot;picturequot; of the amylase activity
may be copied on photographic paper and in some cases even
an enlargement can be made. ')

Not too great an accuracy may be expected from this method,
since, during the cutting of the pulvinus (or the petiole) j the
blade unavoidably spreads some of the cellsap (with or without
amylase) over the surface.

tlTf^; ^^ Tf^ concluded from the experiments that the
greatest quantity of amylase is situated near to the vascular
bundles or m some cases in the vascular bundles itself Of
course one is mclined to think of the starch layer as a source
of amylase, but it must be doubted whether this is always the
case Moreover, it is generally known in literature that the
amy ase content of a tissue may vary rather rapidly, as will be
amply discussed on p. 804.
Conclusion:

The motile tissue contains the highest quantity of amylase in
the more central parts, while in some cases the vascular bundle
r!nbsp;J ^°Uenchyma sheath) is very rich of amylase.

How these fac s are to be explained still remains uncertain,
since we do not know where starch in a solved form is present
(If such ever be the case). It is, however, clear that, once starch

already may bring about considerablenbsp;alterations in the s^ar
content of the tissue.

§ 4. Micro-method for the estimationnbsp;of reducing sugars and
amylase quantities. ') y y

by means of the following chemical method. The sugar es
glucose, is oxidized stoechiometrically to gluconic acid by addfi
freshly prepared iodine solution of a suitable alcalinitvquot;
CeH^Oe J^ 3 NaOH CeHi^O^Na 2 NaJ 2 H,0
The excess of lodme is determined by titration with thiosul-
phate m acid solution.

To secure that glucose and maltose are oxidized and sac-
charose, fructose and starch are not, the iodine reaction occurs

Na^COa 1 vol 0,4 N HCl). - Since iodine reacts with a
number of substances, all the material used must be cleaned

With due precautions these conditions are realized in the
^o^od described in detail by Linderstr0m-Lang and

') In the §§ 4, 5 and 6 of this chapter the figures represent the auantitip^
of reducing sugars, formed by hydrolysis, expressed in mma thiosSa ?

From the solution of which the sugar content must be esti-
mated, a certain exact quantity (15 mmquot;) is pipetted with a
half-automatic micro-pipette into a micro test tube. These tubes
are made from Jena glass (they are about 25 mm high, outer
diameter 6 mm) and completely coated with a thin (fully trans-
parent) paraffin layer. As to the preparing and the cleaning of
these tubes I refer to Linderstr0m-Lang I.e.. About 50 mmquot; of
a carbonate buffer of pH 10,5 is added to the sample taken with
the half-automatic pipette, in order to establish the conditions
required for the oxidizing by iodine. Next 11.9 mmquot; of a 0,15 N
iodine solution in potassium iodide is added to the mixture
with the automatic pipette (for a description of the automatic
and the half-automatic pipettes I refer to the paper by the
inventors of these useful instruments). Iodine sinks to the
bottom and sublimation is prevented. To check it entirely,
immediately afterwards a ring of 50 mmquot; 1,2 N sulfuric acid is
laid above the level of the mixture with a micro-pipette. By
capillary forces the ring sticks to the wall of the micro-tube. In
the same way another ring of liquid (consisting of ± 30 mmquot;
0,3% starch solution) is placed above the sulfuric acid, in order
to serve as an indicator at the titration. Then the tube is allowed
to stay for 30 minutes. In this period part of the free iodine is
bound, dependent on the quantity of reducing sugar present in
the mixture; when the 30 minutes are over the two liquid
rings in the tube are added to the reaction mixture by centri-
fuging the tube. The sulfuric acid acidifies the mixture (while
producing COg) and then the micro-burette allows a vary careful
addition of the N/20 thiosulphate. During the titration the
mixture is continuously stirred with the aid of a periodic electro-
magnetic interrupter and minute beads of iron melted in glass.
To estimate the amount of sugar, the result of the sugar titration
is compared with a check experiment, in which exactly the same
volumes of solutions are used, yet water instead of the sugar
containing solution. In this way the amount of sugar is expressed
in mmquot; N/20 thiosulphate. How many mg sugar correspond to
1 mmquot; thiosulphate completely depends on the actual concen-
tration of the solutions used, i.e., the iodine- and the thiosulphate
solution. Generally the concentrations slowly alter in the course
of time, even during one experiment if this lasts for some hours.
Therefore it can be indicated only approximately how much

1) Experiments of Linderst0m-Lang and Holier have shown that this time
is sufficient.

sugar corresponds to 1 mm^ N/20 thiosulphate; for maltose
lÏ'abtuT i ofiquot;nbsp;^^^nbsp;^^^ -«thod

In the experiments of the following paragraphs the amounts
of mahose always are given in mm» N/20 thiosulphate, since
^ly the results of one experunent can be mutually compared
Moreover, it could not always be avoided to break micro-pipettes

and It is difficult to make a new micro-pipette of exactly the
same volume. This is also a reason why only the results of no
more than one experiment can be compared

All the figures representing amounts of maltose estimated
are means of at least two parallel titrations. The accuracy of

To use the above described method for the estimation of
^ylase quantities, it is necessary that the amylase acts on a
so ution of pure starch to form maltose (I used quot;Kahlbaumquot;
soluble starch p.a.). For the sake of comparison the sai^i^s
shall be treated m exactly the same way: all auantities nf
solutions shaU be the same and so the time ofhyTcïy^and
of sugar oxidation and, of course, the temperature must be
kept as constant as possible all the tune

^e amylase is extracted from the tissue (for details see
§ 5), and consequently the amount of amylase depends on the

quantity of tissue extracted. In
comparing the amylase contents
of two pieces of plant tissue it
is therefore necessary to reduce
the quantities of amylase estim-
ated to the same weight of tis-
sue. Yet in this reduction is a
difficulty, due to the nature of
the hydrolysing process. The ge-
neral shape of the curve, repres-
enting the course of the process
of hydrolysis — forming sugars
through the action of amylase
out of starch —, is reproduced
in figure 11 (Exp. 01). It can
be seen that the curve of the

sugar production starts almost linear, then bends slowly to a
certain limit, the level of which is determined by the quantity
of starch available for hydrolysis. One might also say: the
quantity of amylase determines the rate of sugar-forming until
the quantity of available starch becomes the limiting factor
m the process. In most of the graphs on the next pages one
easily recognizes the theoretical shape of the hydrolysis curve.
This shape is the result of the quantity of maltose formed per
tune unit, whilst this quantity continuously depends on the
quantity of the available starch-amylase complex (SE). During
the first part of the process of hydrolysis the concentration of
the starch is very high relative to that of the amylase and the
quantity of SE formed per time unit will be constant. The first
prt of the curve is a straight line. As soon as the starch quantity
has decreased below a certain limit, besides the amount of
amylase also the amount of starch will affect the rate of the
process S E SE; the reaction-velocity slowly decreases un-
til the amount of enzyme has become high relative to that of
the starch and then, at the end of the reaction, the curve will
be almost a straight line again. In several cases I have clearly
noticed such a theoretical course of the hydrolysis curve. —
If hydrolysis lasts for a very long time the amount of sugars
produced decreases again; it is not with certainty known to
what kind of processes this is due.

If the reduced values lie in the sphere of influence where the
available starch quantity is limiting factor, a reduction of the

Fig. 12. The influence of reducing the determined values to the same
weight of tissue on the shape of the curve,
ordinate: sugar in mm'' thio sulphate,
abscissa: time in hours.

values to the same weight of tissue will give a false idea of
the amylase activity. This is demonstrated by the experiment
02, in which two pulvini were cut into thin slices; these were
mixed and divided into two unequal parts, each of which was
weighed and extracted in the usual way.
Exp. 02. Extraction at 0° C. for 24 hours.
2 pulvini sliced and divided in:

b - 11,7 mg tissue, extracted in 170,8 mmquot; buffer pH 5,9.
c - 15,6 mg tissue, extracted in 170,8 mmquot; buffer pH 5,9.
results hydrolysis in table 4 and in fig. 12.

It always has been a puzzle how to express the quantity or
the activity of enzymes. Generally one is accustomed to compare
the quantities of sugar formed by hydrolysis in a definite time.
It will be evident from the last experiment that the application
of this procedure may lead to completely false values on the
relative amylase activities, when not the greatest precaution
is taken. This will be demonstrated in the next experiment:
Exp. 03. Extraction at 0° C. for 24 hours.
2 pulvini extracted in 88,4 mmquot; buffer pH 5,9.
ai — undiluted extractnbsp;— amylase content x

32 — diluted: 3 aq. dest.: 1 a^ — amylase content 1/4 x
ag — diluted: 3 aq. dest.: 1 ag — amylase content 1/16 x.

In the table 5 I have compiled the ratios of amylase
activity as can be read from fig. 13, considering several periods
of hydrolysis.

It appears clearly from this experiment that for com/paring
amylase quantities it might be the best to compare the slope of
the hydrolysis curve in its initial part, viz. expressed in the
tangent of the angle curve/abcissa.

In many of the experiments described in the next paragraphs
it will be seen that the hydrolysis curve at the hour 0 does not

start at zero. This is due to an initial sugar content of the extract
or of the substrate (starch solution), in most cases of the extract.
To eliminate these errors one should always try to estimate two
points of the first straight part of the curve.

For a full application of the procedures explained above may
be referred to the experiments 68 and 69, p. 809.

Since the quantity of amylase extracted from the tissue is
estimated by the quantity of reducing sugars, formed by hydro-
lysis of starch, it is necessary to keep in mind that the quantity
of sugars determined is not a measure for the amylase quantity
without comment.

The quantity of reducing sugars, formed from starch by hydro-
lysis through the action of amylase, depends on:
I. the quantity of amylase,

II. the conditions under which hydrolysis takes place.
I. The quantity of amylase on its turn depends on
the conditions under which the extraction of the tissue happens,,
viz.:

the Uquid in which the amylase is extracted,
the way of fixing the tissue,
the way of crushing the tissue,
the temperature,
the time of extraction,
the activation by various substances).

Of all these factors I had to determine the effect on the
sugar quantities finally measured.

a. As liquids to extract amylase from the plants I compared
distilled water and buffer solutions of various composition.

Exp. 11 gives the result of a comparison between the extraction
m aq. dest. and in a phosphate buffer of pH 5,9.
Extraction at 0° C. for 27 hours,
a — 25,3 mg tissue in 170,8 mm» buffer pH 5,9
b — 19,7 mg tissue in 170,8 mm» aq. dest.
results in table 6 and in fig. 14.

Exp. 12 shows that extraction in buffer pH 5,9 givesnbsp;a higher
yield than that in buffer pH 10,5;
Extraction at 35° C. for 6 hours,
k — 16,9 mg tissue in 170,8 mm» buffer pH 5,9
1 — 23,7 mg tissue in 170,8 mm» buffer pH 10,5.
results hydrolysis in table 7 and in fig. 15.

b. In order to stop as abruptly as possible the metabolic pro-
cesses of the tissue, I looked for a method of fixing the tissue
before or perhaps during the extraction. Preliminary experience
had already tought me, that ether doesnot affect the amylase,
while alcohol does.

The following experiments were made to control these effects*
Exp. 21. Extraction at 0° C. for 4 hours.

from 12^00 — 14^-30 in 170,8 mm» buffer pH 5,9.
results hydrolysis in table 8 and in fig. 16.

c. The common way in which I prepared the pulvini before
extracting them, was to cut them into thin slices. I compared

this way of extraction to a crushing of the shced tissue in the
extraction-vessel (with a glass-stamper). Experiments 31 and 32
show the results of this comparison:

Exp. 31. Extraction at 35° C. for hours,
a — 40,0 mg tissue in 170,8 mmquot; buffer pH 5,9, in slices.
b — 35,9 mg tissue in 170,8 mmquot; buffer pH 5,9, crushed.
results hydrolysis in table 10 and in fig. 18.

The influence of crushing the tissue. See text. Ordinate: sugar in mm'
thiosulphate, abscissa: time in hours.

Exp. 32. Extraction at 0° C. for 16 hours,
k —28,2 mg tissue in 170,8 mm^ buffer pH 5,9, in slices.

1 — 17,8 mg tissue in 170,8 mm-' buffer pHnbsp;5,9, crushed.
results hydrolysis in table 11 and in fig. 19.

d, e. The influence of the temperature on the quantity of active
amylase, extracted from the pulvinus, often was investigated in
combination with the effect of the time of extraction.

Exp. 41. Extraction for 12^/2 hours,
a — 1 pulvinus in 170,8 mm® aq. dest., at 35° C.
b — 1 pulvinus in 170,8 mm® aq. dest., at room-temp. {18° C.)
results hydrolysis in table 12 and in fig. 20.

The experunents 31 and 32 show that the same order of
values is reached with the extraction at 35° C. and that of
0° C., though of course the extraction times are not the same.
Eocp. 42. 2 pulvini in 341,6 mm' buffer pH 5,9, at roomtemp.
The amylase content is estimated after 6, 8 and 113^ hours of
extraction; hydrolysis for Ij/^ hours,
results hydrolysis in table 13 and in fig. 21.

	hours
	/ 8 hours
	/ / 6 hours
r 1	1

0 1
Fig. 21.

Exp. 43. Extraction at 0° C.
31,1 mg tissue in 170,8 mm» buffer pH 5,9.
Extraction-time Ij^, 3 and 4 hours.
Results hydrolysis in table 14 and in fig. 22.

The amylase quantity after these three extraction times may
be compared from the curves representing the rate of hydrolysis
Exp. 44. Extraction at 35quot;= C.nbsp;j' o- •

a — 18,5 mg tissue in 170,8 mm» buffer pH 10 5
b — 23,7 mg tissue in 170,8 mm» buffer pH 10 5
a — extracted for hours, b — for 6 hours.' '
Results hydrolysis in table 15 and in fig. 23.

Fig. 23 -^e influence of the time of extraction. See texl Ordinate: sugar
m mms thiosulphate, abscissa: time in hours.

Fig. 24. The influence of papayotin. See text. Ordmate: sugar in mm»
thiosulphate, abscissa: time in hours.

The injurious effect of the longer extraction time probably
must be due to the high temperature during the extraction.
ƒ. In one experiment only I examined the activating influence
of papayotin; since this aspect of the enzymatic reaction was
of no interest to me, I did not continue my experiments in this
direction. Later on I never have used the stimulating effect (that
will be shown below) of the papayotin.

Exp. 51. Extraction at 35° C. for 23^ hours,
a — 18,5 mg tissue in 170,8 mmquot; buffer pH 10,5.
b — 19,9 mg tissue in 170,8 mm' buffer pH 10,5,
Results hydrolysis in table 16 and in fig. 24.

The temperature determines the rate of starch hydrolysis.
For completeness' sake this may be demonstrated by the results
of the experiment 55, which follow below:

Exp. 55. Extraction at 0° C. for 16 hours.
22,1 mg tissue in 170,8 mmquot; buffer pH 5,9.
Results hydrolysis in table 17 and in fig. 25.

Another factor of importance in hydrolysis is the hydrogen-ion
concentration. This subject will be dealt with in detail in the
next chapter.

§ 6. Amylase content of upper- and lower half of the pulvinus
in various positions of the leaf. ')

amy ase-extraction methods, was that the mechanism of move-
ments of variation possibly might be simply explained if the
amylase content of both halves of the pulvinus would p'rove to
be unequal in various positions of the leaf.

The dependency of the activity of the pulvinar amylases on
several enyiromnental influences (e.g. temperature hydrogen
^n^concentration) urged me to do the experiments in^arLs

~ o170,8 mm® buffer pH 59
m —18,9 mg lower half in 170,8 mm® buffer pH 59
Beginning of the extraction: a and b — 0h30
(extr for 6 hours)nbsp;k and m - 14h00.

There is a striking conformity in this experiment between the
amylase content of the upper and of the lower half, both at
O^'SO and at 14i'00; the smn of the amounts of amylase of both
halves seems to differ to a certain extent at different times.
This, however, can be attributed to the fact, that the pulvini at
O'^SO and at 14''00 were from two different plants and it is a
well-known fact, that two individuals need not contain the same
amount of active amylase').

In this connection it may be emphasized that this divergency
too is another reason (besides that already mentioned in § 4
of this chapter) why the figures, representing the estimated

») I refer, for mstance, to a remark of Oparin (1934): quot;Wir sehen demnach,
dasz die Aktivität irgendeines Ferment ('z.B. der Amylase oder Invertase)
keinen für eine bestimmte Zelle charakteristischen, konstanten Wert vor-
stellt. Die Fermentaktivität erfährt im Gegenteil under dem Einflirsz innerer
und äuszerer Faktoren weitgehende, rasch ablaufende, reversible Verände-
rungenquot;.

quantity of reducing sugars, cannot be mutually compared in
more than m one experiment only.

In this experiment the reduction of the figures to the same
weight of tissue leads to a divergency in the curves (see graph)
which, however, is not essential at all. Only in this case f
have calculated and plotted the reduced values completely to
demonstrate the conformity of these two divergent curves to
that ot exp. 02 (§ 4).

Exp. 63. Extraction at 0° C. for 5 hours, begmning IQhlS.
a — 12,5 mg upper half m 170,8 mm» buffer pH 5 9
b — 12,8 mg lower half in 170,8 mm» buffer pH 5 9
Results hydrolysis in table 20 and in fig 28

Exp. 64. Extraction at 0° C. for 5 hours, beginning S'^OO.
a — 10,8 mg upper half in 170,8 mm® buffer pH 5,9.
b — 10,6 mg lower half in 170,8 mm® buffer pH 5,9.
Results hydrolysis in table 21 and in fig. 29.

In this experiment the mitial sugar lt;
seems to have been rather high (see § 4).

Exp. 65. Extraction at 0° C. for 4 hours, beginning 10^30.
a — 7,9 mg upper half in 170,8 mmquot; buffer pH 5,9.
b — 8,8 mg lower half in 170,8 mmquot; buffer pH 5 9
c — 7,1 mg upper half in 170,8 mmquot; buffer pH 5,9.
d — 7,7 mg lower half in 170,8 mmquot; buffer pH 5 9
Results hydrolysis in table 22 and in fig. 30.

^e apparent difference in amylase content between both
pulvinar halves, at least partly, might be the effect of a differ-
ence m initial sugar content of the extract or of the starch
solution used for hydrolysis.

Exp. 66. Extraction at 0° C. for Sj^ hours, beginning 8'gt;30.
a — 8,8 mg upper half in 170,8 mmquot; buffer pH 5 9
b — 10,4 mg lower half in 170,8 mmquot; buffer pH 5 9
Results hydrolysis in table 23 and in fig 31

Fig. 31. Amylase content of up-
per and lower half. See text.
Ordinate: sugar in mmquot; thio-

Exp. 67. Extraction at 0° C. for 6 hours, beginning 81=15.
a — 12,6 mg upper half m 170,8 mm' buffer pH 5,9.
b — 12,4 mg lower half in 170,8 mm' buffer pH 5,9.
c — 10,3 mg upper half in 170,8 mm' buffer pH 5,9.
d — 10,7 mg lower half in 170,8 mm' buffer pH 5,9.
Results hydrolysis in table 24 and in fig. 32.

(The figures have not been reduced to the same tissue weight, because
the differences in weight between the two halves of one pulvinus are
minimal).

In two experiments the amylase content of upper and lower
halves was measured during 24 hours (every 6 hours an extract-
ion was started). The extraction happened at 0° C. for 2 hours
in 44,2 mm' ether, then for 22 hours in 170,8 rmn' buffer pH 5,9.

The sugar formed by hydrolysis was estimated in each pair of
extracts after 1, 3, 5, and 34 hours, to be sure that the
part of the curves used for comparison still is below the bending
of the hydrolysis curve. Only the curves of one pair of extracts
(e and f) are given as an example of this procedure (fig. 33).

Results of hydrolysis (after 1 and 3 hours, and all reduced to
the same weight of 17,1 mg) in table 25 and in fig. 34.

the tangent of the angle between the hydrolysis curve and the
abscissa in the graphs. In this way a single figure expresses the
differences in amylase activity between the upper and the lower
half of the pulvini. At the same time eventual errors by differ-

Fig. 34. Amylase content during 24 hours. See text.
Ordinate: sugar in mm» thiosulphate,
abscissa: time in hours.

ences in the initial sugar content of the extracts are eliminated.
The slope of the amylase-activity curve is a better standard for
the amylase activity than e.g. the quantity of sugars formed by
hydrolysis in a certain period.

At all four hours chosen the lower half appears to have a
higher amylase content than the upper half, although the differ-
ences were small. For the values see fig. 34. The values calculated
for the upper halves (expressed in the tangents described above)
prove to be strikingly equal in all four cases.
The same procedure was repeated in

a — 11,1 mg upper half
b —13,2 mg lower half
c — 25,6 mg upper half
d — 19,7 mg lower half
e —15,8 mg upper half
f —15,9 mg lower half
g — 18,3 mg upper half
h — 16,9 mg lower half

Results of hydrolysis (all reduced to the same tissue weight of
15,7 mg) in table 26 and in fig. 35.

The values for the amylase activity and for the differences in
amylase activities between the upper and lower halves can be
read from fig. 35.

Fig. 35. Amylase content during 24 hours. See text.
Ordinate: sugar in mm' thiosulphate,
abscissa: time in hours.

Summarizing the results of the experiments in this chapter,
I have shown that amylase is present in the pulvini of Phaseolus',
further where it occurs and how the method of extraction affects
the finally measured quantity of reducing sugars, formed by
hydrolysis from soluble starch.

For extracting, buffer solutions of a certain pH proved to
yield a higher amylase content of the extract than distilled
water. I, therefore, further always used a buffer solution (of
pH 5,9) as extraction liquid.

It has been stated by Oparin and Riskina (1932) that ex-
tractions in buffer (they used a buffer solution of pH 8,0 after
Mc. Ilvaine) show a much higher amylase content than ex-
tractions in water. They suppose, that in the first case the total
quantity of enzyme (also the inactive part) is extracted from
the tissue, while in the latter only the active enzyme compound(s)
enters into the extraction solution. They remark further, that
for this reason the daily changes in amylase activity of leaves
would not be observable when extraction happens in a buffer,
while extracting in water would reveal differences.

I found this information at a time, when all my attempts
to state a difference between the amylase contents of both
pulvinar halves had already been made. Besides, it still remains
open to doubt whether the changes in amylase activity under
the influence of hydrogen-ion concentration in the tissue (the
importance of which will be described in chapter VII and VIII)
ever might be reflected in the amylase content of extracts,
independently of the extraction liquids used.

The reason why I used ether to start the extraction in most
of the experiments, was to kill the tissue without destroying
the amylase from the tissue.

Crushing the tissue instead of only slicing it yielded a
much higher demonstrable quantity of amylase. Yet I have never
used this method in estimations where a high degree of
accuracy was desired, because it proved impossible not to alter
the total quantity of tissue already weighed, in removing from
the liquid the glass-stampei used for crushing.

A perusal of the results of the experiments on the influence
of the temperature and the time of extraction on the amylase
content of the extract will show that the higher the temperature
and the longer the time of extraction, the higher the amylase
content. Still I hesitated to extract at a high temperature and
for a long time, because it is uncertain whether one does
not partially destroy the enzymes (though raising their activity).

It is known from literature that most of the enzymes are
gradually losing their activity, when extracted from the living
tissue, while also many enzymes are known to be very sensitive
to too high or too low temperatures. Therefore I have applied
several temperatures and different times of extraction, when
trying to find a difference in amylase quantity between the
upper and the lower half of the pulvinus.

These latter attempts have not been successful. In some cases
a difference was found but reverse results were so frequent.

that no sufficient proof is present, to maintain the possibility
of differences in amylase quantity between both halves as a
probable means for explaining the mechanism of the movements.

It is a well-known fact that the activity of enzymes is
highly dependent on the concentration of certain ions, among
which H-ions play the most important part.

In literature the influence of H-ion concentiation on amylase
activity has repeatedly been investigated. An extensive review
is given bij Oppenheim (1936). Ahnost all investigators have
described an optimum-curve. The subject has thoroughly been
investigated by Van Klinkenberg (1932), who estimated the
pH optimum-curves for both «- and /S-amylase (from malt).
Of all types of amylases, hitherto examined on their activity
in different H-ion concentrations, the pH optima lay between
pH 4 and pH 6. In some cases the range of optimal activity
is wide, in other cases it is only a minor part of the investigated
H-ion concentration traject.

In view of the dependency of the amylase activity on pH,
I decided to determine the pH activity curve of the pulvinar
amylase, in order to see whether these data somehow might
reveal a relation with the phenomena of reversible volume
changes. Of course I expected to find a pH optimum-curve
with one optimum at about pH 5, and so I wondered, when
finding a two-peaked curve, or, if one prefers to say so, a
curve with a very broad optimum (from pH 4 to pH 7) and
a sinking near pH 5,8 — 6,0 (see fig. 36).

The results of two of the experiments are reproduced ffisquot;
36 and fig. 37).nbsp;i-nbsp;v s-

The hydrogen-ion concentrations were obtained in using
buffer solutions after Mc Ilvaine; the buffering compounds
are 0,1 mol citric acid and 0,2 mol Na2HP04. The pH's obtained
with the aid of these buffer solutions cover a range from
about pH 2 to pH 8. The values of the pH were controlled

The pulvini were extracted first in ether and then in
distilled water. No buffer was used here, to prevent shifting
of the pH of the solutions in which hydrolysis had to occur.
Since rather large quantities of extract were required in these
experiments, in some cases I made the extract from a mixture
of petiolar and pulvinar tissue; no deviations as compared
with plain pulvinar extracts were noticed.

The extracts were filtered through a glass-filter, in order
to avoid inaccurate readings by slight contaminations in the
solutions. — In the experiments described below all quantities
used are exactly mentioned.

in 5 cm» aq. dest. — temp. 35° C.
Extraction-time: ether 30 minutes
aq. dest. 150 minutes

Extraction-time: ether 30 minutes, aq. dest. 150 minutes.
Quantities used in hydrolysis:
58,6 mmquot; extract

') In tables 27, 28 and 29 the figures represent the quantities of reducing
sugars, formed by hydrolysis, expressed in mm« thiosulphate.

Since I did not investigate whether a- or /?-amylase was
present in the pulvinar extract '), I am fully aware that one
may argue that the presence of both amylases may acount for
the two-peaked curve. I believe that this cannot be the case,
since Van Klinkenberg (1931) has shown that, though the
curve of ;8-amylase has its optiminn at pH 4,5—5,2, the optunum
of the a-amylase curve (pH 5.8—^6.0) just falls in the sinking of
teh pH-activity curve of the pulvinar amylase (see figs 36 and 37).

A much more serious objection might arise from the method
used in estimating the sugars formed by hydrolysis. This method
allows the estimation of the quantities of quot;reducing sugarsquot;
and therefore, besides maltose, its cleavage product (glucose)
might also be determined. Now the enzyme maltase, which
splits up the maltose in twice as much glucose, acts optimally
at a pH of about 7-8. Thus, the two-peaked curve might be
caused by the action of maltase, partly hydrolysing the maltose
formed by the amylase from the starch. One molecule maltose
(with one reducing bond) forms two molecules glucose (each
with one reducing bond). At pH 5,5—6,0, where the activity
of the amylase decreases, that of the maltase is increasing.
If the pulvinar extract contains maltase, this fact would
neutralize the effect of the decrease in amylase activity.

To control this posibility, I have examined the extract on its
maltase content. Using extremely pure maltose (quot;Kahlbaumquot; p.a.)
as a substrate, no maltase appeared to be present in the
extract. One of three experiments is reproduced here.
Exp. 73.

1) I was interested only in the activity of the extract as such at various
H-ion concentrations. Whether this activity has to be ascribed to one or
more different amylases is a subject for a special detailed enzymatic study.

126,4 mm» buffer -f 1% maltose (or 1% starch)
TABLE 29. Maltase not present in the extract.

Though, as I have already mentioned, generally only one
single pH-optimum has been described for amylases in literature,
I have found some indications as to the occurrence of two-
peaked pH curves. Orru (1930) described a pH optimum at
5,2—5,4 and a second, though less prominent one, at 6,4—6,6.
Giri and Sreenivasan (1937) reported on the amylase system
of the rice-kernel that the pH-optimum of j8-amylase lays at
4,5—5,1 and that of a-amylase at 6,4—7,1. Bois et Nadeau (1936)
estimated the quot;neutral pointsquot; of amylase and found that these
lay at pH 4,6—4,9 and at 6,5—6,7.

It thus appears that the phenomenon reported by me stands
not alone. It would be useless to try to explain the two-peaked
curve of the amylase activity; to this purpose it would be
necessary to investigate in detail which types of amylase are
present in the extract and how their activity is at various
hydrogen-ion concentrations.

An explanation, however, is at, yet not of special interest to
me; the important thing is that the pulvinar amylase is less
active at pH 5,8—6.0 than at both higher and lower hydrogen-ion
concentrations.

Still there is one thing to be considered: the amylase content
of the tissue must be much higher than that measured in the
extracts, since in extracting one dilutes to a high degree the
original amylase concentration. It seems probable, that the
extract is at least about 5X diluted as compared to the
tissue.

') Viz. in exp.'s 71, 72 and 73 a certain weight of tissue is extracted in
5 x as much water!

This being the case, also the amylase activity of the tissue
at any pH must be about 5X higher, and it will be clear that
the shape of the amylase activity curve, given in fig. 36, is
only a faint reflection of the activities of the actual amylase
concentration in the tissue.

To realize what this means, we want to compare for instance
the amylase activity at pH 5,8 with that at pH 6.5.

The same difference in the amounts of produced sugar, plotted
in figs. 36 and 37, would have been obtained in at least 1/5
of the time at a concentration of amylase as present in the
tissue. Further it also may be probable that the differences would
be much more pronounced.

It cannot be denied that, up to now, it has not been possible
to measure the pH of the cell sap in a tissue. Yet it seemed
necessary to me to try to get some information on the pH
in the pulvinar tissue. Direct measurements, of course, were
out of question.

One of the great difficulties met with, is the small quantity
of juice of which the H-ion concentration is to be determined.

Since I was once accustomed to the use of micro test tubes
and micro-pipettes, I applied these tools for a colorimetrical
method of comparing H-ion concentrations. To this purpose I
made a colour standard for several pH-indicators, viz. methylred,
bromthymolblue and bromcresolpurple. For preparing the scale
I used buffer solutions of citric acid and Na2HP04 (Mc Ilvaine;

was filled with 126,4 mmquot; buffer solution and 9,4 mmquot; mdicatS
solution, the pH of the buffers being controlled Xad S a
chinhydrone-electrode. The series used for the three tyj^ of
indicators are given in full in table 30.
Also some tests are given in that table. Most of the observations

a.nbsp;pulvinar tissue, pressed out in tap water, changed the
colour from purely yellow (tap water) to the blue side.

b.nbsp;pulvinar tissue cut into slices and extracted in tap water
VlTdnbsp;^^^nbsp;-de (Ltween

means of a glass-stamper. The colour shifted still more to
yellow, now being between V and VI.

In the experiments with bromcresolpurple, distilled water
was used to extract the tissue. Distilled L er itself sited
with a nearly yellow colour as soon as water and indicatorhS
been brought together; however, its colour changed rather
rapidly to completely violet. Now, in observating the c^
changes brought about by the tissue in aqua destUlatr^^
s roke me that the colour of the latter did not hife' a

d and e represent the colour given by the juice of the pulvinar
tissue (the pulvmus first being cut up into thin sliLs) S
some cases the colours obtained were between IV and V
(d), but in most of the experiments the pH of the extracted
jmce was between 6,28 and 6,66.

It has been attempted to determine too whether a difference
m pH of the upper- and the lower half might be detected, but

will not be possible to detect any differences in H-ion con-
centration between both sides by means of this crude metS
ConcZus^ns; Generally the value of pH in the extracted eel
sap was found at about pH 6,5. - Since in extracting the
electrolyte solutions of the cell sap have considerltybeen
dduted, the actual value of the pH of the cell sap in the tissS

must be less than 6,5. It depends on the buffering capacity of
the cell sap how much the actual value has changed by diluting.
Therefore we may conclude, that the pH of the cell sop probably
lies between 5,5 and 6,5 (assuming a maximal dilution of 1: 10
in extracting).

No differences in pH of the upper- and lower half of the
pulvinus could be demonstrated with this method.

§ 3. Shifting of H-ion concentration recorded by measurements
of potential differences.

Since more than a year J. B. Thomas is engaged with
measurements of P.D.'s in several plants. When he proposed
me to proceed to measurements of the P.D. of the petiole
and the pulvinus of Phaseolus multiflorus, I have eagerly
accepted that proposal, since no means should be neglected which
perhaps might throw new light on the mechanism of the
movements.

So we started to investigate whether a relation could be
found between the movements of the leaves of Phaseolus
multiflorus and the P.D. variations in the motile pulvini. When
such a relation had been stated, it seemed interesting to
examine to which extent the P.D. variations could be explained
as concentration effects.

The method of measuring potential differences was described
in a previous paper (De Groot and Thomas 1938). I will shortly
report the experiments which are of special interest for an
explanation of the motile mechanism.

In 11 experiments we measured the P.D. in petiole and
pulvinus four times a day. At the same time the movement of
the leaf was registered. The distribution of the potential in the
petiole appeared not to be related with the movement, while just
in the pulvinus such a relation exists. The results of one ex-
periment are reproduced in fig. 38.

In all experiments a shifting of the quot;potential levelquot;,
synchronous with the movement, was found in the pulvinus.
The results of this series of experiments show that a constant
relation exists between the value of the P.D. across the
pulvinus and the state of movement; besides, the potential
level in the petiole remained practically constant during each
experiment.

minute) the shifting of the P.D.
in the pulvinus, synchronously
with the movement. The results
of one of several experiments
have been reproduced in fig.
4 of our previous paper. It
was evident that the variations
of the movement are preceded
by those of the P.D. of the
pulvinus. The space of time
between both reactions amounts
to 20—30 minutes. The lower
side of the pulvinus showed
more intensive changes in its
potential than the upper one.
The potential levels of both
sides in general change in an
opposite direction.

A discussion of the results
yielded data of special interest
in relation to the periodic move-
ments. We have calculated the
potential differences to be ex-
pected when a leaf moves from
a high to a low position, if
one considers the membranes
in the plant impermeable for
anions and assuming that the
changes in volume and in con-
centration of the cell sap
amount 3/2 in the upper half
and 2/3 in the lower half (for
argumentation see § 3 of chapter
II). Substituting these values
in the formula of Nernst for
diffusion potentials, we found:

Fig. 38. The quot;potential patternquot; in petiole and pulvinus, represented in a
3-dimensional graph. The horizontal plane 1-2-3-6-5-4-shows the topographic
situation of points measured on the median section through petiole and
pulvinus: see situation at the top of the graphs. The potential value of
1 each time was chosen as the level of the abscissa plane; the potential of
each point is referred to 1. The upper side of the quot;potential planequot; is
dotted, its under side shaded.

This gave E = 10,45 mV for the upper half and E = —
10,45 mV for the lower half; the difference between the P.D. in
a high and in a low position of the leaf should be 20,90 mV
across the pulvinus. As an average of all the experiments we
found that the difference of the values at a sinking and a rising
state of the leaf amounts to 24,85 mV (theor. 20,90 mV)
across the pulvinus, while for the upper and the lower side apart
(compared to the constant potential level in the petiole) the
corresponding values are resp. 14,21 mV (theor. 10,45 mV)
and —10,03 mV (theor. —10,45 mV). This rather close agreement
made us assume that we were right when considering the mem-
branes practically impermeable for anions, while it also seems
correct to assume the value for n (in the formula) = 1. Since
among the univalent cations the speed of the H-ions is of a
paramount rate, it is plausible that the influence of other cations,
even if they were polyvalent, is only of secondary importance.

In this connection it is important too to know which kind of
electric effects were really measured by means of the gelatin-
ZnS04-electrodes applied to the plants. Some experiments with
models, to be reported by J. B. Thomas in a next paper, pointed
in the direction that we were exclusively measuring the effect
of the shifting of H-ion concentrations.

These results and conclusions considerably will support the
theory, discussed in the next chapter, which attributes a probable
function to alterations in the hydrogen-ion concentration in the
movements. However, no definite method is available to verify
the measured values of the pH-changes and to ascertain which
changes actually occur.

In the preceding chapters I have made an attempt to analyse
some of the complicated processes involved in the motile system
of the pulvini of Phaseolus multiflorus (and alhed species). A
tentative synthesis will be given now.

a hypothesis on the role of the subjects studied. The periodic
movements in the dark can be explained as follows ').

Continuously sugars are removed from the tissue by the
respiration and by diffusion to places of lower concentration The
sugar content of the cells is maintained by the action of amylase
starch into sugar. If the removal by respiration
and diffusion exceeds the supply by hydrolysis the volume will
decrease, it the supply exceeds the removal the volume will
increase, because the sugar content chiefly determines the
osmotic value of the cells. However, changes in volume must be
accompanied by changes in the concentration of the electrolytes
solved in the cell sap. It is highly probable that the ionic equili-
bria in the cell sap will be disturbed when a volume change
occurs. A new equilibrium will be established and, for instance
the hydrogen-ion concentration will have got another value
ihis possibihty has been proved by the measurements of P D's
(chapter VII). The pH of the cell sap was found to be about 6 0
and it is exactly in this region that the amylase activity in a
peculiar way depends on the H-ion concentration. As a matter
of course these facts have led me to the assumption that the
interaction between amylase activity and H-ion concentration
forms a system that in itself may account for the periodic
changes m volume.

Similar thoughts on the influence of ionic equilibria on enzyme
activity I have found in a paper of St. J. von Przylecki (1935)
He says: Angenommen, dasz die Amylase ein amphoterer Stoff
ist, so konnten wir uns die Aktivität folgenderweise vorstellen-
Um die maximale Amylasewirkung zu erreichen ist 1 ein be-
stimmtes Verhältnis zwischen dissoziierten Anionen/Kationen
Gruppen, 2. vielleicht auch ihre Menge (d.i. die Entfernung von
gleichnamig und entgegen geladenen Gruppen u.s.w.) nötig Die
Grosze des Verhältnisses übt vielleicht einen Einflusz auf die
Affinität, besonders aber auf die Stabilität des Komplexes E-S
aus In the same paper he remarks: quot;Ein charakteristisches
Merkmal der lebenden Materie bildet ihre Fähigkeit zu Autore-
gu ationen - quot;Die Regulierung der chemischen Prozesse im
Zellverbande ist innigst mit der Regulation der Enzymreaktionen
verbundenquot;.

fJ^-ta T^T ^^ described with the aid of the scheme in
ng. A closed chain of reactions consists of the following com-
ponents: the amylase activity varies with the pH of the cell sap,

Fig. 39. Scheme of the theoretical relation between amylase activity,
quantity of sugars, volume of the cells and H-ion concentrations. For
reasoning see text.

the pH depends on the volume of the cell, the volume varies
accordmg to the osmotic value of the cell sap and this osmotic
^lue is dependent on the amylase activity. One might also say:
Ihe pH IS closely related to the water content of the cell and
the amylase activity is determined by the pH as well as it deter-
mines Itself the water content and the volume of the cell.

I emphasize again that this system concerns merely the periodic
movements in the dark. The theory, however, can account for
all changes m turgor, also those in the light. For superimposed
on the periodic (endonomous) dark processes, all kinds of

stimuli may influence the various parts of the processes resul-
ting in a certain turgor pressure. To realize how this might
proceed, we have to consider how the three processes determining
the sugar content (and the turgor) of the cells are dependent on
various external and internal agents. The three processes involved
are:

I.nbsp;The intensity of respiration is affected by a) the amount
ot available respirable substrate, b) the concentration of oxygen
in the air, c) the water content, d) the concentration of carbon
dioxide in the air, e) the acidity, f) the salts present, g) the
temperature and h) the light (?).

II.nbsp;As to diffusion of soluble material out of the cells, it must
be remembered that

a)nbsp;the direction of the diffusion is determined by the differ-
ences in concentration of the solutions involved,

b)nbsp;the rate of the diffusion is determined by 1) external
conditions, as light, temperature, etc. and 2) internal conditions,
as acidity concentration of electrolytes, etc., which all effect the
state of the permeable membranes in the tissue.

d)nbsp;internal conditions: pH and all kinds of activating or
paralyzmg influences.

and, if I would be complete, I should have to mention all kinds
of metabolic processes determining the physiological state of the
cells in the pulvinar tissue.

In spite of the great uncertainty as to the actual part of each
process in the resulting value of the turgor of a cell, some indica-
tions may be given to account for phenomena known in literature
and cormected with pulvinar movements.

For instance, it seems highly probable that the rapid changes
in turgor, caused by the light, studied by Mar. Brauner (1933)
and others, have nothing to do with amylase action. They can
be attributed to an influence of the light on the diffusion system
in the tissue. I do not say quot;on the permeability of the cell mem-
branequot;, since I cannot imagine how an increase or a decrease
of the permeability — at a certain concentration gradient —■ ever
might account for a reversible change in the turgor of the cell.
Of course changes in permeability may have something to do
with the influence of light, but certainly they cannot merely
account for the phenomena.

In this paragraph I will discuss how the various processes,
studied by former investigators and by myself, are related to the
system described in the last §.

The endonomous periodic movements in constant darkness can
be explained by the endonomous periodic changes in amylase
activity in the cells of the motile tissue. No changes in the inten-
sity of respiration or in the rate of diffusion need to be
involved in the mechanism of the movements. This does not
mean that, if they exist, the explanation given by me would fail.
Besides the reversible system of the amylase action other rever-
sible processes concerned in the sugar-starch metabolism may
also exist; the result of their joint activity is reflected in the
movements.

The results of the measurements of the osmotic values of the
upper and the lower half of the pulvinar tissue have shown that
in an uplifted position of the leaf the osmotic value of the cells
in the lower half was higher than that of the cells in the upper
half and reversely. This fact matches with the statements by
Zimmermann (1929) and others; all data indicate that changes
in the osmotic value of the cells run parallel to changes in
volume. The greater the volume, the higher the osmotic value
of the cell sap. The objection of BiInning (1936), that this paral-
lelism precludes the possibility that the volume changes are

caused by changes in the osmotic value, does not hold. If changes
m volume may be induced by alterations of the sugar content
of the cells, the effect (the volume decrease or -increase) will
rapidly follow alterations of the osmotic values, since the time
of latency only depends on the water permeability of the mem-
branes. This latention lasts for so short a time (perhaps several
minutes, perhaps half an hour), that it is practically impossible
to state the sequence of processes suggested by BiiNNiNG.

When plants of Phaseolus are inverted, the movements con-
tinue, but their period is also rapidly inverted (within 2 or 3
hours). This phenomenon can be explained as follows: in the
normal position the weight of the lamina tends to enlarge the
volimie of the cells in the upper half and to decrease that of
the cells in the lower half. When the plant is inverted, the weight
acts in an opposite way, so that the cell volume in the anatomical
upper half is mechanically decreased and in the anatomical lower
half is enlarged, compared with the former, normal position. Now
the cell volumes that were decreased will increase (and vice
versa), according to the same mechanism of starch-sugar conver-
sion, which accounts for the movements in the normal position
of the plant. It may be remembered that it is a general feature
of the system, that the volumes which have been decreased tend
to increase, while those which have been enlarged tend of de-
crease. I draw the attention to the fact, that the weight of the leaves
(acting in downward direction) is no conditio sine qua non for
the occurrence of movements, since with considerably reduced
laminae the pulvini continue their movements. The fact, that
the plants stop moving when fixed on the horizontal axis of a
rotating clinostat indicates, that the continuously varying way
in which the weight of the laminae influences the processes in
the tissue, in that case, balances the changes in amylase activity
in the cells. The movements on the clinostat have not been studied
by registering them (technical difficulties prevented this); the
occurrence of movements has been valued. Therefore it still
remains possible, that slight movements (in daily periodic rhythm)
have escaped the observation of the investigators. Perhaps a
detailed study of the influence of gravity on the daily periodic
movements might reveal new and important data for the know-
ledge of the motile mechanism.

SUESSENGUTH (1922) has discussed, among other subjects, the
influence of wilting on the action of amylases in plants. His con-
clusion from a survey of literature is that wilting causes the

hydrolysing enzymes to grow active. This remarkable effect —
for, while wilting withdraws water from the cell, by hydrolysis
water is bound — has been explained in two ways. Von Molisch
(1921) thinks of an increase of the enzyme concentration (besides
that of all other solved substances), as a result of the loss of
water. Suessenguth has mentioned the possibility that adsorbtion
interfaces are set free by the withdrawal of water, and so the
substrate becomes accessible for the enzyme (or the enzyme for
the substrate).

The explanation of von Molisch might as well be applicable
to the changes in water content during the movements of the
motile pulvini. If the total amount of amylase were constant in
both halves of a pulvinus, the concentration of the am.ylase would
be altered with the cell volume. This change of the amylase
concentration, by a decreased (or increased) production of osmo-
tically active substances, would counteract the direction of alter-
ation of the volume, just as supposed in the hydrogen-ion-
amylase system described in the last §. However, the differences
in amylase concentration should be measurable and this has
not appeared to be possible (chapter VI).

Lepeschkin (1934) deduced from his formulae that the turgor
pressure of a tissue depends on the concentration of the fluids
outside the tissue, with which it is in contact. To illustrate this
thesis (which generally of course, holds true), he points to the
fact that, when the lamina of a leaf is totally cut off, the move-
ments stop in a few days. According to Lepeschkin the lack of
a transpiration stream through the vascular bundle causes, in
this case, the concentration of the osmotically active substances
outside the motile tissue gradually increase, until the turgor of
the motile tissue has so much decreased that no movement can
occur. — This conception does not match the facts, since the
pulvini (with removed lamina) keep their rigidity and therefore
their turgor has not decreased.

The phenomenon of the stopping of the movements after cutting
off the lamina, can be much easier explained in the following
way: the starch content of the starch layer in the pulvinus seems
not only to be supplied by the photosynthesis of the pulvinar
tissue itself but, for an important part, by that of the leaf. If the
lamina has been cut off, the starch of the pulvinar starch layer will
gradually be consumed in the motile mechanism and then the
movements will stop. — In my experiments I have often used
plants, of which the lamina had been cut off for the greatest part.
These plants kept on moving in the normal day- and night

succession but, once placed in constant darkness, they stopped
moving within a few days, since the reduced starch content of
the starch layer cannot maintain the motile mechanism for a
longer time.

I add the results of some experiments which support this view
(table 31 and fig. 40).

Fig. 40. A sketch illustrating the
extent to which the lamina had
been cut off in the experiments
of table 31.

AD 8a ) ^ still moving fairly well on Nov. 8th/9th, movements
AD 9a \ I slowly decreasing from Nov. 8th to 14th.

Resuming all experimental and theoretical data given in the
preceding chapters, we may conclude that the mechanism of the
pulvinar movements in constant darkness can be explained by
enzymatic processes in the tissue. Changes in the osmotic values

of the cell contents, and thus changes of the volumes, interact
with these enzymatic actions.

No differences of amylase quantities at different stages of the
movement could incontestably be observed. Some results pointed
to differences, but these are so inconsistent that, in a theory on
the mechanism, the possibility of differences in amylase quantities
cannot yet be accounted for.

The alterations of the potential in the pulvinus found a satis-
factory explanation in the shifting of ionic concentrations during
the movement, and at the same time they strongly endorse the
possible occurrence of changes in the hydrogen-ion concentrations
in the motile cells.

The theory still is rather vague as far as the details of the
process are concerned, but this was unavoidable since, hitherto,
reliable data on the details of the motile mechanism are too
scanty. No independent study in different fields of plant physio-
logy ever will be able to reveal complicated processes such as
underlie the movements of variation of the pulvini of Phaseolus
multiflorus and allied species. Only the combination of as many
aspects of the problem as possible may finally result in a detailed
knowledge of all processes involved in the motile mechanism.

1.nbsp;The mechanism of the pulvinar movements of Phaseolus
multiflorus L. was studied in several ways.

2.nbsp;A comparison of the osmotic values of the cell content in
the upper and in the lower half of a pulvinus at various positions
of the leaves, showed that in an uplifted position of the leaf the
lower half has a higher suction power than the upper half, while
in a drooped position of the leaf the upper half has a higher
suction power. These results confirm those of Zimmermann (1929)
and Weidlich (1930).

3.nbsp;The relation between the presence of starch in the starch
layer of the pulvini and the occurrence of movements was inves-
tigated. The results of the experiments show that, as long as
starch is present, movements may take place; if the starch has
disappeared from the starch layer the movements stop. The
starch probably acts as a source of energy for the processes
resulting in movement.

4.nbsp;The methods of extracting amylase from the tissue and
of testing its activity were studied. — In some cases a difference
in amylase activity of the upper and the lower half of the pulvinar

motile tissue could be detected. In other cases no differences at
all were found. No conclusions as to a periodic change in the
amylase activity or -quantities could be drawn from these
experiments.

5.nbsp;The amylase extracted from the pulvinar tissue showed a
remarkable difference in activity at different hydrogen-ion con-
centrations. The pH of the pulvinar tissue appeared to lie between
5,5 and 6,5, and it is just in this region that a decrease in the
amylase activity was demonstrated.

In 3 series of experiments, in collaboration with J. B. Thomas,
the potential differences on petiole and pulvinus were measured.
A parallelism between movement and P.D. variations was demon-
strated, which made it probable that mainly shifting of H-ion
concentrations was measured.

6.nbsp;A theory was given that enabled to account for all pheno-
mena described by me and by earlier investigators. In short the
hypothesis is this: the volume of the cells depends (in upper and
lower antagonistic halves) on the osmotic value of the cell
content (sugar); sugar is removed constantly by respiration and
by diffusion, it is supplied from starch by the action of amylase;
the amylase thus may influence the total amount of sugars in
the cells, and at the same time its activity is regulated by the
pH; it was made probable that fluctuations of the pH parallel
to changes in volume occur.

7.nbsp;In this way the principles of an endonomous automatism
were suggested and a mechanism was described liable to all
kinds of conditional (internal and external) factors.

I owe much to the kind interest and the encouragement of
Prof. dr. V. J. Koningsberger.

Bois, E. et A. Nadeau, 1936, Contribution à l'étude d'Acer saccharum. Les
amylases de la sève d'érable et le pouvoir-tampon. Canad. J. Res. 14,
B, 373.

Primärblattgelenkes von Phaseolus multiflorus. Planta 18, 288.
Brouwer, G., 1926, De periodieke bewegingen van de primaire bladeren bij

Canavalia ensiformis. Thesis, Utrecht.
Bünning, E., 1931, tJber die autonomen tagesperiodischen Bewegungen der
Primärblätter von Phaseolus multiflorus. Jb. Bot. 75, 439.

Ceemer, H., 1923, Untersuchungen über die periodischen Bewegungen der
Laubblätter. Z. f. Bot. 15, 593.

Gmi, K. Venkata and A. Sreenivasan, 1937, Das Amylasesystem des Reis-
kornes während des Reifens und Keimens. Bloch. Zs. 289, 155

Gkooi, G. J. de, and J. B. Thomas, 1938, On bioelectric potentials and the
movements of the motile leaf pulvini in Phaseolus multiflorus L
Proc. Acad., Amsterdam, 41, 403.

Hilbueg, C., 1881, Über Turgoränderungen in den Zellen der Bewegungs-
gelenke. Unters, bot. Inst. Tübingen 1, 23.

Hofmeister, W., 1862, Über die Mechanik der Reizbewegungen von Pf lan-
zentheilen. Flora 45 (N.R. 20), 497.

Jost, L., 1898, Beiträge zur Kenntnisnbsp;der nyktitropischen Bewegungen. Jb.
Bot. 31, 345.

Kerstan, K., 1909, Über den Einflusznbsp;des geotropischen und heUotropischen
Reizes auf den Turgordruck in den Geweben. Beitr. Biol. Pflanz. 9

Kleinhoonte, A., 1929, Über die durch das Licht regulierten Bewegungen
der Canavalia-BVatter. Arch. Neerl. Sei. ex. et nat. Illb, 5, 1.

-— 1932, Untersuchungen über die autonomen Bewegungen der Pri-
märblätter von Canavalia ensiformis DC. Jb. Bot. 75, 679.

Klinkenberg, G. A. van, 1931, Over de scheiding en de werking van de
beide moutamylasen. Thesis, Utrecht.

Lepeschkin, W. W., 1909, Zur Kenntnis des Mechanismus der Photonasti-
schen Variationsbewegungen. Beih. Bot. Zbl. 24, 308.

- 1934, Zur Analyse des Turgordrucks der Gewebe,' seiner Varia-
tionen und des Mechanismus der Variationsbewegungen. Ber. D.B.G.
52, 475.

LiNDERsra0M-LANG, K., amp; H. Holter, 1933, Studies on enzymatic Histo-
chemistry. V. A micro-method for the estimation of sugars. C rend
Lab. Carlsberg 19, No. 14.

Molisch, H., 1921, Über den Einflusz der Transpiration auf das Verschwin-
den von Starke in den Blättern. Ber. D.B.G., 39, 339.

Oparin, A., 1934, Die Wirkung der Fermente in der lebenden Zelle
Ergebn. Enz. forsch. 3, 57.

Oparin, A. amp; S. Riskina, 1932, Über die Aktivität der Amylase in den
Blattern der Zuckerrübe. Bioch. Z. 252, 8.

Orru, A 1930, Azione del latice del Ficus carica suU' amido. Boll Soc
ital. Biol. sper. 5, 176.

Panta^li, E., 1901, Studi d'anatomia e fisiologia sui pulvini motori di
Robinia Pseudacaeia e Porliera hygrom.etrica. Modena.

Przylecki, St. J. von, 1935, Über die intrazelluläre Reguherung der Enzym-
reaktionen mit besonderer Berücksichtigung der Amylasewirkung,
Erg. Enz. forsch. 4, III.

Sachs, J., 1857, Über das Bewegungsorgan und die periodischen Bewe-
gungen der Blätter von Phaseolus un Oxalis. Bot. Ztg. 15, 793.

schwendener, S., 1887, Die Gelenkpolster von Phaseolus und Oxalis. Sitz,
ber. Akad. Wiss. Berlin 12, 176.

Stoppel, R., 1932, Welcher Faktor ist für die Tagesrhythmik der Pflan-
zen verantwortlich zu machen? Ber. D.B.G. 50, 486.

Suessenguth, K., 1932, Über das Wirksamwerden pflanzlicher Enzyme.
Ergebn. Biol. 1, 364.

Ursprung, A., 1930, Zur Terminologie und Analyse der osmotischen Zu-
standsgröszen. Z. Bot. 23, 183.

Weidlich, H., 1930, Die Bewegungsmechanik der Variationsgelenke. Bot.
Arch. 28, 219.nbsp;. ,

wiederscheim, W., 1904, Studien über photonastische und thermonastische
Bewegungen. Jb. Bot. 40, 230.

wijsman, H. P., 1889, De diastase, beschouwd als mengsei van maltase
en dextrinase. Thesis, Amsterdam.

Zimmerman, W., 1929, Die Schlafbewegungen der Laubblätter. Tubmg.
naturwiss. Abh. 1929, H 12, 16.

A.nbsp;Micro-photographs of cross sections of the pulvini of the experiment
of fig. 9 (See text p. 784). The Roman numerals refer to the time of
fixation of the pulvinus, as indicated in fig. 9. The photographs f, g and h
give a detailed picture of the starch layer at the resp. moments I, VI and
XI of fig. 9.

B.nbsp;One example of a print and one of an enlargement made from an
■'enzymographic platequot; (see p. 789).

Volumeveranderingen van cellen in een weefsel kunnen niet
verklaard worden uitsluitend met behulp van veranderingen van
de plasmamembranen wat betreft hun permeabiliteit voor water
of voor opgeloste stoffen.

Om kwantiteit of activiteit van enzympreparaten onderling
te vergelijken, is het noodzakelijk om, door bepaling van ten-
minste twee punten, het initiaal verloop van de hydrolyse-curve
vast te leggen.

De volgens Miss Green door Furtado gemaakte „extraordinary
blundersquot; zijn geen blunders; het artikel van Furtado behelst
een ernstige kritiek op de opvattingen van Miss Green.

De Interationale Nomenclatuurregels behooren een artikel te
bevatten, dat inhoudt, dat men alleen een systematische naam
aan een hybride mag geven, indien aannemelijk is gemaft, dat
de hybride constant of homozygoot is.

Het is wenschelijk het begrip phagocytose uitsluitend te ge-
bruiken voor het opnemen door cellen van niet, of gedeeltelijk,
verteerde vaste stoffen.

De onderzoekingen van Harnisch rechtvaardigen niet zyn con-
clusie, dat primaire en secundaire oxybiose berusten op chemisch
verschillende processen.

De voorstellingen van Renner en Noack, over de erfelijkheid
van bont in bet geslacht Hypericum, kunnen zonder veel moeite
in één zienswijze vereenigd worden.

Het is niet noodig om ter verklaring van het gedrag van be-
paalde Notch-deficiencies in Drosophila melanogaster, een „Gen-
wirkungsausfallquot; als werkhypothese aan te nemen.

De onderzoekingen van BSrner bewijzen niet, dat de „intra-
cellulaire staafjesquot; in viruszieke planten niet als vroegtijdig
symptoom van de virusziekte mogen worden opgevat.

Het is wenschelijk door grondig physiologisch en chemisch
onderzoek na te gaan in hoeverre een tekort aan zeldzame
elementen in den bodem oorzaak kan zijn van achteruitgang in
stand en productie van cultuurgewassen na de eerste cultuur-
jaren.

Zuiver wetenschappelijk biologisch onderzoek is, ook in tijden
van economische depressie, noodzakelijk, aangezien het ten
allen tijde de gegevens, die voor het toegepast landbouwkundig
onderzoek den grondslag vormen, moet kunnen verschaffen.

	shortening in % of-the length in glucose
objects	epidermis	parenchjTna	parench. near vase, cylinder	vascular cylinder
El. upper half lower half E2. upper half lower half E3. upper half upper half lower half lower half	11,3% 2,3% 19,5% 12,5% 11,8% 9,5% 12,7% 5,8%	10,8% 6,5% 19,7% 15,7% 12,7% 24,3% 27,8% 30,4%	6,7% 1,5% 11,9% 14,7% 7,5% 7,7% 13,2% 17,0% 1	0% 0% 0% 0% 0% 0%

		nimiber of
series	object	measure- ments	shortening	difference	3 X mv
G	upper half lower half	5 5	18,8% 28,9%	10,1	10,26
O	upper half lower half	8 6	26,6% 37,5%	11,5	6,24
H	upper half lower half	6 7	17,8% 23,3%	5,5	6,99

ratio:		al	samples: a2	a3
actual ratio		16	: 4 :	1
1 read from	hydrolysing 1 hour	16	: 4 :	1
the graph after:	hydrolysing 2 hours	12,5	: 4 :	1
the graph after:	hydrolysing 4 hours	7	: 3,7 :	1

0 ! 2 J	* £ 6 TABLE 8.	9 8 9 Influence of	ether.
time of hydrolysis	a		b
2h30 8h30 21hOO	3,29 9,64 12,98	4,12 12,20	3,72 10,76 13,42	4,83 13,94

time of hydrolysis	a		b
OhOO IhSO 7h00 20h00	1,53 2,78 4,15 7,08	1,52 2,75 4,10 7,02	0,43 3,03 10,02 13,72	0,41 2,89 9,63

kind of movement	the movement induced by:	H ^ s ^ n	direction of the movement determined by:	il	growth or variation	examples:
endowmous	internal agent	CQ § i ri	plant		growth	nutation, rotati- on, growth itself
	internal agent	}H 1 ffl	plant		variation	part of nyctinas- tic movements
nastic	external agent		plant	q	growth	photonasty thermonasty
	external agent	1			variation	seismonasty (Dionaea)
tropic	external agent	1 a	agent	o amp;		phototropism, geotropism, etc.

time of hydrolysis	k		1
1 h	3,30	2,92	5,17	7,25
2 h	—	—	5,54	7,78
3 h	5,18	4,59	—	—

time of	time of extraction:
hydrolysis	6	8	11V2 hours
lh30	1,60	2,12	3,00

	time of extraction:
	IV2	3	4 hoirrs
time of hydrolysis	(lh30) 11,05 (2h45) 14,13 (4hl5) 14,55	(lh30) 12,25 (3h00) 14,40	(IhOO) 12,73 (2h00) 14,58

time of hydrolysis	a			b
IV2 h 3 h	4,01 5,17	4,33 5,58	3,98 5,05	3,36 4,26;

temp.	time of hydrolysis:
temp.	Ya		3%	6VJ	11 hours
35° C. 0° C.	5,07 2,27	5,26 3,29	5,69 4,54	5,69 5,05	5,30

time of				b
hydrolysis		3
1 h	9,79	9,79	6,45	6,30
31/2 h	14,70	14,70	12,85	12,80

time of hydrolysis	a		b
V2 h 1 h IV2 h	4,47 6,13 7,60	4,47 6,13 7,60	4,05 5,85 7,45	4A3 5,96 7,59

a	-17,1	mg upper	half
b	— 16,3	mg lower	half
c	— 13,0	mg upper	half
d	— 15,9	mg lower	half
e-	— 20,4	mg upper	half
f	— 24,2	mg lower	half
g	— 25,0	mg upper	half
h	— 21,3	mg lower	half

	__		--0/

' f		e - 20.2 mg
III	1 1 1 1 1 .	f - 24,2 mg 1 1 1 1 1 1	1 ; 1

time of hydrolysis	a	b	c	d	e	f	1 ^	h
1 h 3 h	1,80 4,90	2,18 5,77	2,17 5,31	1 2,74 1 6,69	2,36 5,52	2,70 6,36	2,59 5,73	2,73 6,14

	6''00	ngt;'m	1S''00		^quot;00
-		jd		/ƒ
	b			/ƒ	,h
-	/ ^	Y /'		' ,s
-	Igb- 1.78	// i •	// t9f-	t.80	/ tg h . 1.68
	tga • 1.5*	'/ tg c- 158	tg e -	158	Ig g ■ 1.55
1	O.U 1	0,37 lt; 1	1 1	o.n	0.13 .1 1

	0^00	6'^00		n^oo	1â''00 .h
	la				/
			,d		À
	/				Y /
				f	/ /
				f/e	/1
-			.c	//	!
- 1)	tga = IM	// tgc	■ /.«J	// ig e ' 10	tgg= 2,n
	tgb -- 1.98	tga	-- 2, Ob	tgf ' 1,?4	igh-- 2,8S
	0M6 1 1	1 1	-0,63	-0,05 -1-1_	-0,71 1 1

time of hydrolysis	pH: 2,0	2.8	4.0	5.0	6.0	7.0
1 h	0	1,45	2,35	2,55	2,45	2,65
2 h	0	2,70	3,90	4,20	3,75	4,45
7V2 h	0	4,15	5,15	5,05 ,	4,15	5,85

time of hydrolysis	pH: 3.0	5.2	5.6	5.8	6.0	7.0	7.6
5 min.	0.30	0.60	0.50	0.60	0.60	0.60	0.40
40 min.	2.60	—	3.90	3.70	4.00	3.80	3.60
100 min.	3.00	4.40	4.20	4.05	4.20	4.50	4.50

time of hydrolysis	pH: 4.6	7.0	105
0 min. 60 min. 180 min.	6,95 6,93 6,90	7,40 7,40 7,70	6,40 6,68 7,15	substrate: Maltose
0 min. 90 min		0,00 7,67		substrate: Starch

amylase activity		more active i less active
quantity of sugars		t more sugar I less sugar
volume of ttie cells		1 enlargement 1 diminishing
hydroqen-ion cont	mtration___	[optimal (J amylase