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Evidence of the presence in xylem sap of substances with kinetin-like activity
Life Sciences Yol . 5 pp . 2061-207, 1966. Printed in Great Brita~n.
Pergamon Press Ltd.
EVIDENCE OF TF~ PRESENCE IN XYLEM SAP OF SUBSTANCF3 WITH KII~TIN-LDCE ACTIVITY D . J . Carr and W . J. Burrows Botany Department, Queen's University, Belfast, Northern Ireland (Received 13 July 1966 ; in final form 6 September 1966) Substances with biological activity similar to that of kinetin (6-ilirfutyl aminopurine) have been shown to exist in extracts from various species of Pruita (2,12,14,21,25,36), seeds (3,4,12,13,17,18,19), tumour cells of Vinoa roses (33), pea seedling~ (34) and coconut milk (16,24,29,35) .
An active compound
isolated by Letham (12) from maize has been named zeatin (13) . This substance has been chemically characterised as 6-(4=hydroxy3-methylbut-2-e~1) aminopurine (15) .
An active compound isolated
Prom plum fruitlets (14) bears a close resemblance to zeatin and a factor found by Miller (19) in maize has also been identified as zeatin (37) .
In repent years evidence has accumulated suggesti.r~
the possibility of the export Prom the root to the shoot of substances with biological activity similar to that oP kinetin . Tomato plants deprived of roots suffer ahlorosis and a reduction oP stem growth which can be partially suppresaed~by coconut milk(32) . This observation led Went (31) to postulate the production of e hormone ("oaulocaline") by roots and its transport to the shoot
*Mr. Burrawa is a Post-graduate Scholar of the Agricultural Research Council . 2061
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gINETIN-LISE ACTIVITY
system, regulati~ig its growth and maintenance .
In studies on
flooding injury Jackson (8) concluded that "some factor is supplied by the unflooded root system which is essential for the maintenance of green colour in the leaves _ . ." .
Chibnall (5)
also postulated a factor transported from roots to leaves, maintaining their metabolism and preventing the fall in protein levels Fihich accompanies detachment of the leaf from the plant . The formation of roots on the petioles of detached leaves results in increase in the protein-synthesizing capacity of the leaves, due,
according to Parthier (23) and Mothes et al . (20)
to a root-synthesized hormone regulating senescence in the leaves .
Richmond and Lang (26) discovered that kinetin applied
to detached leaves of Xanthium pennsylvanicum delays the loss of protein and the breakdown of chlorophyll,
thus indicating the
possible nature of the postulated root-hormone as a kinetin-like substance .
Kulaeva (11) obtained evidence that the sap bleeding
from the decapitated root system does indeed contain substances with activity in chlorophyll-retention resembling that of kinetin. Further evidence of the export of such substances from roots to shoots was obtained by Kende (9,10) Loeffler and van Overbeek (16) and Nitsch and Nitsch (21) .
Weirs and Vaadia (30)
suggest that
the region of the root with the highest kinin-like activity is its apex .
Itai and Vaadia (7) have further proposed that the root
system of a water-stressed sunflower plant exports less of the kinin-like substance to the shoot than does that of a control plant .
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SINETIN-LIRE ACTIVITY
The experiments reported below were initiated after a qualitative demonstration in this laboratory of the presence of factors in the bleeding sap of Xanthium pennaylvanicum able to delay the lose of chlorophyll in detached leaves of the same species, and therefore with properties resembling those of kinetin (Mukherjee, unpublished) .
~e report the exploration of the spe-
cificity of a chlorophyll-retention bioassay similar to that of Kende (9) and its use in the detection of active subatanéea in the bleeding sap of a number of herbaceous plants . Materials and Methods Standardised bioassay .
In the form of bioassay finally developed
barley seeds (var . Dea)
are germinated, without previous soaking
(c .f . Kende, 9), in sand in the dark at 25° for 2 days .
The
seedlings are raised under vFIO fluorescent lamps with a 20-hr . daily photoperiod at a temperature between 20 and 25° .
After 7
days, when the first leaf has expanded and the second is about to appear, leaves of a uniform height and width are selected .
Frown
each, the segment between 3 and 4 cm . from the tip is excised and floated on distilled water at 25° in the dark for 24 hours . To allow for discards, due to unevenness in chlorophyll content then apparent, an excess of 25916 over the number of segments required for a given experiment is cut .
Four uniform segmenta are
floated, abaxial surface uppermost, on 1 ml . of teat solution containing 250 units of penicillin in a sealed 3-dram vial for 48 hours in darkness at 25° . up for each test solution .
At least three replicates are set The chlorophyll is extracted by two
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BINETIN-LIRE AC'PIYITY
Yol .
5, No . 22
successive 5 ml . lots of 80% acetone each acting for 12 hours in the dark at room temperature .
The extracts are combined, made up
as required to 10 ml . and the OD measured using the total volume in a 4 cm .glass cell in a Unicam SP 600 spectrophotometer at 645, 652 and 663 m~ .
The chlorophyll content is calculated by
Arnon's method (1) . Colleçtion of
xylem sap .
Plants were raised in sand cultures .
They were supplied with ICI "Solufeed" at the rate of 1 g . per litre supplemented with 5 ml . ti . Ca(N03 ) 2 per litre . the plants were watered with deionised water . species were used :
Otherwise,
The following
Pisum arvense L ., Lupinus angustifolius L .,
Xanthium pennsylvanicum Wallr ., and Impatiens glandulifera Royle .
On the day of harvest, the plants were watered at 7 am .
At 11 am they were de-topped, and the stumps were fitted with silicone rubber sleeves .
The liquid exuding from the cut stumps
was collected every hour or every two hours, depending on the rate of exudation, for 24 hours .
Immediately after collection,
each sample was deep frozen and stored at -22° prior to use .
In
all experiments except those on the exudates from these plants, the OD of an aliquot of each chlorophyll extract was measured in a 1 cm . glass cell .
Otherwise, the procedures were as described
above . Stat istical treatment of data .
In all experiments prior to those
on plant exudates the mean chlorophyll content was calculated per set of 4 leaf segments, together with its 95% confidence limits . Any value in excess of the control whose range does not overlap
Yol . 5, No . 22
SINETIN-LISE ACTIVITY
that of the control is held to be significant .
2065
In bioassays of
chromatograms, the results have been subjected to analysis of variance and the error mean square term used to calculate a least significant difference (LSD) for 95% probability .
Values exceed-
ing the control value by more than the LSD are held to be signicant . Experimental Results The initial experiments were directed towards optimisation and determination of the specificity of the bioassay . (a)
Differential sensitivity of barley varieties .
Seedlings of
eight varieties were raised as described above and tested, using kinetin .
The results of these tests are shown in Fig . 1 .
The
most sensitive varieties, Dea and Rika, gave responses to 0 .003 4~g" ml'1 kinetin significantly different fra~m that of controls without kinetin .
The other varieties were either insensitive or
too variable to be of use .
Since similar tests on subsequent
occasions showed Dea to be more consistent than Rika, Dea has been chosen for the bioassay . (b)
Temperature for optimum response .
Suitable temperatures for
the bioassay were found to be 15° and 25° (Fig . 2) . is more convenient to maintain and has been selected .
The latter Senescence
occurred too rapidly at 30° for kinetin at law concentration to exert consistently an effective delaying action . (c)
Orientation of segments to teat solution .
Since the leaves
are some~nlhat difficult to wet, it was suspected that the orientation of the segments towards the solution might affect the res-
Yol. 5, No . 22
HIIQETIN-LIHE ACTMTY
30
V
ts
0
(
L
3a10~
3x10 3
KIeeNe
FIG . 1 .
1 1
3x103
3alÔ~
Kinetin
3x10 1
p~ " rel -1
Kinetin-induced chlorophyll retention in eight barley varieties . Ordinates : log .mean chlorophyll content per 4 leaf segments . I~g . Half standard errors are indicated . sponse .
0
e
3 a10 1
~u0 .ml-1
FIG . 2 . Effect of temperature on kinetin-induced chlorophyll retention in Dea barley . Ordinates: as in Fig . 1 .
Submerged segments became limp and remained green, even
in the absence of kinetiny chlorophyll could be extracted frown them by boiling acetone,. but not by acetone at roam temperature . No significant differences could be detected due to floating the segments with the abaxial surface either upwards or downwards on the solutions .
However, in order to obviate variation which might
arise from a random orientation, segments are floated on their allaxial surlaces in the standard bioassay . (d)
Specificity :
the response to amino-acids.
The amino com-
pounds listed in Table 1 were tested in the standard bioassay at
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KINETIN-LIKE ACTIVITY
concentrations in the ranges stated .
These ranges were chosen so
as to extend far above those of amino-acids likely to be present in the extracts of the bleeding sap of the plants to be used in experiments . instance .
No significant positive response was obtained in any
Alanine, which Sechs (28) found in a qualitative test
on chlorophyll retention in oat leaves to be active at O .1M, is not a normal constituent of bleeding sap of herbaceous plants . (e)
Specificity :
the re sponse to gibberellic acid and auxin . A
very wide range of concentrations of both GA9 and IAA was tried . As Fig . 3 shows, no significant effect was observed in any of these treatments .
The lack of response to GA S is in marked con-
tract to the findings of Fletcher and Osborne (6) who used leaf disks of Taraxacum officinale , but is in agreement with those of Kendo (10) and Sechs (28) . (f)
Specifitv :
kinetin analogues _and synergists .
In the callus
proliferation bioassay (24) synergism has been shown between myoinositol and kinins, and myo-inositol has been identified by Letham (14) in extracts of plum fruitlets, in which it is synergistic with the naturally-occurring kinin .
~yo-inositol is neith-
er active by itself nor synergistic with kinetin in the barley leaf bioassay .
Table 2 demonstrates the similarly negative re-
sulta of testa of sucrose and mannitol .
Kende (9) has shown that
his barley leaf (var . Delta) bioassay is insensitive to adanine~ glucose and inorganic nitrogen compounds up to O .1M.
Tests of
three analogues of kinetin gave results in agreement with those of Ruraishi (12) using the expansion of radish leaf disks (Fig . 4) .
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KINETIN-LIKE ACTIVITY
Vol . S, .No. 22
TABLE 1 Compound
Concentration range tested mg ./ml .
Aspartic acid Asparagine Homoserine Glutamine Glutamic acid Proline Lysine . Threonine Arginine Histidine Glutathione (reduced form) Alanine Cysteine
0 .01 - 1 .0
Valine Leucine Iso-leucine Phenylalanine Tyrosine Serine Glycine Methionine
0 .001 - 0 .1
'15
~13
~003 0" 03
0~3
3
30
PPT .
300
FIG . 3 . Absence of chlorophyll-retention activity of GAa and~ïAA. Ordinates as in Fig . 1 .
O . ~ 00 03
0~03
0~3
3
FIG . 4 . Activity of analogues of kinetin. Ordinates as in Fig . 1 .
DPm~
30
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gINETIN-LIRE ACTIVITY
Only marginal loss of activity follows replacement of the furan ring by a 6-C aromatic ring, but considerable reduction follows replacement by a methyl group. TABLE 2 Treatment
Chlorophyll, Ng per 4 leaf segments 18 .83
t
0 .623
29 .98
±
0 .750
19 .67
t
0 .624
10 " -1 M . Mannitol 10 -a M .
19 .67
t
0 .695
19 .55
t
0 .645
" 10 -1 M.
19 .56
f
0 .678
Control Kinetin, Sucrose,
0 .03 4~g ml-1 10-a M .
Having defined the optimal conditions of the bioassay and to some extent explored its specificity to some of the compounds likely to be present in detectable amounts in xylem sap, attention was turned to bioassay of the samples of xylem sap collected as previously described.
An aliquot of~such a sample was frozen-
dried, the residue taken up in the minimum of water and subjected to TLC on silica gel G (Merck) spread on 20 x 20 cm glass plates at 0 .5 mm thickness .
The sample was spotted on the start line 3cm
frown the edge of the plate ; after drying and equilibration with the solvent for 45 minutes more solvent was added to the tank and the front allowed to ascend 10 cm from the start line .
The plate
was then removed, dried in a stream of cold air and observed under W.
Each 1 cm strip corresponding to 0 .1 Rf value was eluted with
ethyl acetate acidified with acetic acid .
Chromatograms of aque-
ous fractions of partitioned extracts were eluted with ethyl acetats alone .
Each elutes was blown down to dryness and the residue
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BINETIN-LIRE ACTIVITY
taken up in 4 ml distilled water .
Vol .
5, No . 22
The control treatment in each
bioassay involved the use of the elutee similarly treated from a 1 cm strip prior to the start line .
The solvent systems used
were : Solvent A.
Tertiary amyl alcohol :formic acid :H 2 0, 3 :2 :1 by vol .
Solvent B .
Ha0-saturated secondary butanol .
Solvent C .
Boric acid, 0 .03M adjusted to pH 8 .4 with NaOH .
Solvents B and C are adopted from Miller (19) . Bioassay of whole~xylean sap .
A 700-m1 sample of sap collected
from balsam plants ( Impatiens balsaminea ) was frozen dried, the residue taken up in a minimum of distilled water and subjected to TLC using solvent A.
Examination of the developed and dried
plate under W revealed a dark zone, Rf 0 .1-0 :3, and dark zones in the regions of Rf 0 .6 and 0 .7 .
Bioassay of the eluates showed
two regions of kinin activity (Fig . 5a) . That at the higher Rf value could, according to the W observation, be due to two separate compounds .
The streaking of the activity at low Rf values
could result from breakdown of a compound during handling and chromatography .
To test this, a further 350 ml of balsam sap was
subjected to TLC using the same procedure as before .
A strip cor-
responding to Rf 0 .1-0 .3 was eluted in bulk and re-chromatography$ using solvent C .
The result appears to confirm the hypothesis of
breakdown (Fig . 5b) resulting in the production of another active compound which with this solvent migrates to Rf 0 .9 . Since these results suggest the presence in balsam sap of çompounds with properties remarkably similar to those of the
Vol . 5, No . 22
KINETIN-LIâE ACTIVITY
2071
so
10
I~pafi~n~
olo11i111iF~~a
FIG. 5 (a)
Chromatography of whole sap of Itgpatiens balsaminea . Ordinates, 4sg chlorophyll per set of ~ leaf segments . The lower of the two horizontal lines zspresents the chlorophyll con tent of control segments, the upper, the 95% confidence limit. Shaded areas represent statistically significant activity .
(b)
Re-chromatography of Zone 1, using solvent C .
(c)
Re-chromatography of active fraction eluted frasa Rf p .1-0 .3 of the TLC of the ethanol-soluble fraction of balsam sap (see Fiq . 6), using solvent B .
kinins of immature maize grain reported by-Miller (19) we have adapted his fractionation procedure to xylem eap as follows :
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KINETIN-LIKE l1CTIVITY
Vol . F, No . 22
XX 1_y em sap f~ree~ze dry Solids extract 4 times with one tenth the original volume 70% ethanol,filter, combine filtrates . Residue
Ethanol lution
inactive solids
evaporate off ethanol, extract remaining solution 4 times with equal vols . nbutanol . Combine butanol extracts . Butanolphase chromatography bioassay
Chromatography of an aqueous solution of the ethanol-insoluble residue from 500 ml of sap using solvent A, followed by bioassay of the eluates of the chromatogram yielded no activity .
We may
assume therefore that the active substances are all extracted into the ethanol . balsam sap,
The n-butanol phase from the same sample of
fractionated according to the scheme outlined above,
was subjected to TLC using solvent C .
Bioassay of the chromato-
gram ahowed two zones of activity one at Rf 0 .5 the other at Rf 0 .9-1 .0 (Fig . 6) .
Chromatography, using solvent B, and bioassay
of the aqueous fraction revealed only one peak of activity on the chromatogram (Fig . 6) . Two peaks of activity similar to those found in the butanol phase of balsam sap extracts were obtained by similar treatment of 1 litve of eap fron Xanthium and also from 330 ml of field pea sap (Fig . 6) .
The butanol phase of extracts from 350 ml of lupin sap
Vol . ~, No . 22 AQUEOUS
203
BINETIN-LIGE ACTIVITY FACTION
~UTANOI
n
Iwp~ti~n~
IEACTION
~I~ndvlif~r~
~~ n oO e r r jf re n )IantAiuw
L
p~nn~ylv~nie~w
~
r r
n I .l.
st . ..
r O
LuPlnu .
a n~u~liF~llu~
FIG . 6 . Chlorophyll-retention activity in xylem sap of four species of flowering plants . The quantities of sap were : 500 ml, balsam : 1 litre, Xanthium ; 330 ml, field pea ; 350 ml . lupin . The sap was fractions end as~cescribed in the text . Chromatograms were developed with solvents B (aqueous fraction), C (butanol Praotion) .
showed only a single peak, corresponding to the faster-moving of the two found in the other species .
Activity at Rf 0.5 could, how-
20`]4
gINETIN-LIgE ACTIVITY
Vol . ~, No . 22
ever, be masked by substances at the same Rf inhibitory to the bioassay .
The aqueous fraction of the sap extracts from all these
plants contained an active substance moving slowly in chromatography, similar to that found in balsam sap (Fig . 6) .
In the aqueous
fraction from the extracts from Xanthium and field pea sap there was a fast-moving secondary peak at Rf O .g .
As previously indica-
ted for entire balsam sap, this might arise from breakdown of the slow-moving factor during prolonged handling .
It was possible to
demonstrate that the labile fraction is water-soluble rather than butanol-soluble .
Following chromatography of the ethanol-soluble
fraction of 350 ml of balsam sap using solvent A, the compounds at Rf 0 .1-0 .3 were eluted .
The elutee was re-ehromatographed using
solvent B and the chromatogram bioassayed (Fig . 5C) .
The faster
peak in the aqueous fraction appears to be derived from the slower . Discussion The results of these bioassays clearly demonstrate the presenee in xylem sap of substances resembling kinetin in their ability to delay the breakdown of chlorophyll in barley leaf segments . The chromatographic mobility of these substances on silica gel G is strikingly like that of the active compounds in maize extracts in Miller's experiments (1g) in which the support was Whatman No .l paper .
Thus activity at Rf 0 .5 could well be due to a compound
like the zeatin of maize and that at Rf 0 .9 to a substance such as its nucleoside .
Miller (19)~has suggested that his slow-moving
factor is the monophosphate nucleotide of zeatin . phosphate would yield the activity at Rf O .g .
Removal of the
A similar breakdown
Vol . 5, No . 22
gINETIN-ZIgE ACTIVITY
2075
of a chromatographically slow-moving compound from the xylem sap of balsam, Xanthium and field pea to yield a fast-moving, active substance has been demonstrated .
In all these respects the sap-
borne compounds with chlorophyll-retention activity strikingly re semble those of extracts of maize kernels .
We have not yet been
able to compare the unknown substances from xylem sap directly with authentic zeatin or its derivatives.
Further work is required
to substantiate the suggestions made above concerning the nature of the active compounds in xylem sap .
In preliminary experiments,
we have been able to demonstrate their activity in the soybean callus bioassay of Miller (see also Kende, 10) ; it therefore seems probable that the active substances can be classified as phytokinins .
It is of importance that these substances are present in a
biological fluid likely to be representative of that moving under normal physiological conditions from the root to the shoot .
At
present it cannot, of course, be excluded that these substances are present as a result of the removal of the shoot system ; conversely, there is as yet no rigorous evidence of their synthesis in the root system itself .
Experiments are planned to elucidate
further the nature and origin of these substances and their significance to the physiology of the plant . Summary The first part of the paper describes optimisation and tests of the specificity of a bioassay for substances active in chlorophyll retention, using segments of barley leaves .
This bioassay
is used to deaoonstrate the presence in whole xylem sàp, and in
Vol . 5, No . 22
KINETIN-LIKE ACTIVITY
206
ethanolic extracts of sap,
of balsam, Xanthium , field pea and blue
Lupin of compounds with kinetin-like activity .
Further fractiona-
tion of ethanolic extracts yields three active components .
The
first is a water-soluble compound which breaks down to yield a component which resembles in its activity and chromatographic mobility one of the two more butanol-soluble components .
Similar
compounds are present in the first three species examined, and in part these similarities extend also to blue lupin.
In the discus-
sion coanparisons are made between the kinetin-like compounds of xylem sap and the published data on those of extracts from maize kernels . References 1.
D . I . ARNON, Plant Physiol .,
24, 1 (1949) .
2.
W. BOTTOMLEY,N . P . KEFFORD, J. A. ZWAR, and P . L . GOLDACRE, Au stral . J . Biol . Sci ., 16, 395 (1962) .
3.
G. BEAUCHESNE, in Plant Growth Regulation , p. 667, Univ . Pres$, Amea (1961) .
4.
G . BEAUCHESNE, M . LEBOEUF and R . GOUTAREL, in Recrulateurs naturels de la Croissance végétale , p . 119, C.N .R .S ., Paris (1963) .
5.
A . C . CHIBNALL, Protein Metabolism in the Plant , Yale Univ . Press, New Haven (1939) .
6.
R . A. FLETC.HRR and D . J . OSBORNE, Nature (Lund.) 2 (1965) .
7.
C . ITAI and Y. VAADIA, Physiol . Plant ., = 941 (1965) .
8.
W. T . JACKSON, Am . J . Bot ., 43, 496 (1956) .
9.
H . KENDE, Science , 145, 1066 (1964) .
10 .
H. KENDE, Proc . Nat. Acad . Sci .(Wash .,) ~,
1302
11 .
O . N. KUhAEVA, Fiziol . Rasten ., 9, 229 (1962) .
Iawa State
, 1176
(1965) .
12 .
S . KURAISHI, Sci . Pap. Coll . Gen. Educ . Univ . Tokyo , 9, 67 (1959) .
13 .
D. S. Letham, in Re ug late urs naturels de la croissance végé tale , p. 569, C .N .R .S . Paris (1963) .
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20~~
KINETIN-LISE ACTIVITY
14 .
D . S . LETHAM, Life Sciences , 3, 152 (1963) .
15 .
D . S, LETHAM . J . S . SHANNON and I . R, McDONALD . Proc . Chem . Soc ., p . 230, July, 1964 .
16 .
J. E, LOEFFLER and J. VAN OVERBEEK, in Regulateurs naturels de la croissance végétale , p . 77, C .N .R .S ., Paris (1963) .
17 .
C. 0 . MILLER, Proc . Nat . Acad . Sci . (Wash .), 47, 170 (1961) .
18 .
C, O, MILLER and F . H, WITHAM, in Regulateura naturels de la croissance végétale , p . 1 (erratum), C.N,R .S ., Paris (1963) .
19 .
C. O . MILLER, Proc . Nat . Acad . Sci . (Wash.), 54, 1052
20 .
K, MOTHES, L, ENGELSRECHT and L. KULAEVA, Flora, L47, 445 (1959) .
21 .
J. P . NITSCH, Bull, soc, bot . Fr . 107,
22 .
J. P .~NITSCH and C. NITSCH, Bull . soc, bot . Fr ., (1965) .
23 .
B . PARTHIER, Flora, 154,
24 .
J. K . POLLARD, E . M . SHANTZ and F . C, STEWARD, 36, 492 (1961) .
25 .
L. E . POWEtL and C. PRATT, Nature (tond .), 204, 602 (1964) .
26 .
A. E, RICHMOND and A. LONG, Science, 125, 650 (1957) .
27 .
J. H, ROGOZINSKA, J . P . HELGESON and F . SKOOG, Plant Physiol 40, 469 (1965) .
28 .
T . SACHE , On the mechanism of apical dominance, Ph .D .Thesis, Haxvard, 1965 .
29 .
M. SHAW and B, I, S . SRIVASTAVA, Plant Physiol., (1964) .
30 .
C, WEISS and Y. VAADIA, Life Sciences , 4, 1323 (1965) .
31 .
F . W, WENT, Plant Physiol ., 18, 51 (1943) .
32 .
F . W. WENT and D . M. BONNER, Arch . Biochem., 1, 439 (1943) .
33 .
H . N, WOOD, in Regulateurs naturels de la croissance végé tale , p . 97 . C,N.R,S Paris (1963) .
34 .
J . A. ZW~R and F, SKOOG, Austral . J. Biol . Sci ., 16, 129 (1962) .
35 .
J . A. ZWAR, W, BOTTOMLEY and N . P . KEFFORD, Austral . J . Biol . Sçi ., 16, 407 (1962) .
36 .
J, A, ZWAR, M, I . BRUCE, W, BOTTOMLEY and N. P . KEFFORD, Regulateurs naturels de la croissance végétale , p. 123, C,N,R.S ., Paris (1963) .
37 "
D . S . LETHAM and C . 0. MILLER, Plant and Cell PY~yaiol ., 6 . 355 (1965)
(1965) .
263 (1960) . 112, 11
230 (1964) . Plant Physiol
39, 528
Pergamon Press Ltd.
EVIDENCE OF TF~ PRESENCE IN XYLEM SAP OF SUBSTANCF3 WITH KII~TIN-LDCE ACTIVITY D . J . Carr and W . J. Burrows Botany Department, Queen's University, Belfast, Northern Ireland (Received 13 July 1966 ; in final form 6 September 1966) Substances with biological activity similar to that of kinetin (6-ilirfutyl aminopurine) have been shown to exist in extracts from various species of Pruita (2,12,14,21,25,36), seeds (3,4,12,13,17,18,19), tumour cells of Vinoa roses (33), pea seedling~ (34) and coconut milk (16,24,29,35) .
An active compound
isolated by Letham (12) from maize has been named zeatin (13) . This substance has been chemically characterised as 6-(4=hydroxy3-methylbut-2-e~1) aminopurine (15) .
An active compound isolated
Prom plum fruitlets (14) bears a close resemblance to zeatin and a factor found by Miller (19) in maize has also been identified as zeatin (37) .
In repent years evidence has accumulated suggesti.r~
the possibility of the export Prom the root to the shoot of substances with biological activity similar to that oP kinetin . Tomato plants deprived of roots suffer ahlorosis and a reduction oP stem growth which can be partially suppresaed~by coconut milk(32) . This observation led Went (31) to postulate the production of e hormone ("oaulocaline") by roots and its transport to the shoot
*Mr. Burrawa is a Post-graduate Scholar of the Agricultural Research Council . 2061
2062
Vol . 5, No . 22
gINETIN-LISE ACTIVITY
system, regulati~ig its growth and maintenance .
In studies on
flooding injury Jackson (8) concluded that "some factor is supplied by the unflooded root system which is essential for the maintenance of green colour in the leaves _ . ." .
Chibnall (5)
also postulated a factor transported from roots to leaves, maintaining their metabolism and preventing the fall in protein levels Fihich accompanies detachment of the leaf from the plant . The formation of roots on the petioles of detached leaves results in increase in the protein-synthesizing capacity of the leaves, due,
according to Parthier (23) and Mothes et al . (20)
to a root-synthesized hormone regulating senescence in the leaves .
Richmond and Lang (26) discovered that kinetin applied
to detached leaves of Xanthium pennsylvanicum delays the loss of protein and the breakdown of chlorophyll,
thus indicating the
possible nature of the postulated root-hormone as a kinetin-like substance .
Kulaeva (11) obtained evidence that the sap bleeding
from the decapitated root system does indeed contain substances with activity in chlorophyll-retention resembling that of kinetin. Further evidence of the export of such substances from roots to shoots was obtained by Kende (9,10) Loeffler and van Overbeek (16) and Nitsch and Nitsch (21) .
Weirs and Vaadia (30)
suggest that
the region of the root with the highest kinin-like activity is its apex .
Itai and Vaadia (7) have further proposed that the root
system of a water-stressed sunflower plant exports less of the kinin-like substance to the shoot than does that of a control plant .
Yol . 5, No . 22
2063
SINETIN-LIRE ACTIVITY
The experiments reported below were initiated after a qualitative demonstration in this laboratory of the presence of factors in the bleeding sap of Xanthium pennaylvanicum able to delay the lose of chlorophyll in detached leaves of the same species, and therefore with properties resembling those of kinetin (Mukherjee, unpublished) .
~e report the exploration of the spe-
cificity of a chlorophyll-retention bioassay similar to that of Kende (9) and its use in the detection of active subatanéea in the bleeding sap of a number of herbaceous plants . Materials and Methods Standardised bioassay .
In the form of bioassay finally developed
barley seeds (var . Dea)
are germinated, without previous soaking
(c .f . Kende, 9), in sand in the dark at 25° for 2 days .
The
seedlings are raised under vFIO fluorescent lamps with a 20-hr . daily photoperiod at a temperature between 20 and 25° .
After 7
days, when the first leaf has expanded and the second is about to appear, leaves of a uniform height and width are selected .
Frown
each, the segment between 3 and 4 cm . from the tip is excised and floated on distilled water at 25° in the dark for 24 hours . To allow for discards, due to unevenness in chlorophyll content then apparent, an excess of 25916 over the number of segments required for a given experiment is cut .
Four uniform segmenta are
floated, abaxial surface uppermost, on 1 ml . of teat solution containing 250 units of penicillin in a sealed 3-dram vial for 48 hours in darkness at 25° . up for each test solution .
At least three replicates are set The chlorophyll is extracted by two
2064
BINETIN-LIRE AC'PIYITY
Yol .
5, No . 22
successive 5 ml . lots of 80% acetone each acting for 12 hours in the dark at room temperature .
The extracts are combined, made up
as required to 10 ml . and the OD measured using the total volume in a 4 cm .glass cell in a Unicam SP 600 spectrophotometer at 645, 652 and 663 m~ .
The chlorophyll content is calculated by
Arnon's method (1) . Colleçtion of
xylem sap .
Plants were raised in sand cultures .
They were supplied with ICI "Solufeed" at the rate of 1 g . per litre supplemented with 5 ml . ti . Ca(N03 ) 2 per litre . the plants were watered with deionised water . species were used :
Otherwise,
The following
Pisum arvense L ., Lupinus angustifolius L .,
Xanthium pennsylvanicum Wallr ., and Impatiens glandulifera Royle .
On the day of harvest, the plants were watered at 7 am .
At 11 am they were de-topped, and the stumps were fitted with silicone rubber sleeves .
The liquid exuding from the cut stumps
was collected every hour or every two hours, depending on the rate of exudation, for 24 hours .
Immediately after collection,
each sample was deep frozen and stored at -22° prior to use .
In
all experiments except those on the exudates from these plants, the OD of an aliquot of each chlorophyll extract was measured in a 1 cm . glass cell .
Otherwise, the procedures were as described
above . Stat istical treatment of data .
In all experiments prior to those
on plant exudates the mean chlorophyll content was calculated per set of 4 leaf segments, together with its 95% confidence limits . Any value in excess of the control whose range does not overlap
Yol . 5, No . 22
SINETIN-LISE ACTIVITY
that of the control is held to be significant .
2065
In bioassays of
chromatograms, the results have been subjected to analysis of variance and the error mean square term used to calculate a least significant difference (LSD) for 95% probability .
Values exceed-
ing the control value by more than the LSD are held to be signicant . Experimental Results The initial experiments were directed towards optimisation and determination of the specificity of the bioassay . (a)
Differential sensitivity of barley varieties .
Seedlings of
eight varieties were raised as described above and tested, using kinetin .
The results of these tests are shown in Fig . 1 .
The
most sensitive varieties, Dea and Rika, gave responses to 0 .003 4~g" ml'1 kinetin significantly different fra~m that of controls without kinetin .
The other varieties were either insensitive or
too variable to be of use .
Since similar tests on subsequent
occasions showed Dea to be more consistent than Rika, Dea has been chosen for the bioassay . (b)
Temperature for optimum response .
Suitable temperatures for
the bioassay were found to be 15° and 25° (Fig . 2) . is more convenient to maintain and has been selected .
The latter Senescence
occurred too rapidly at 30° for kinetin at law concentration to exert consistently an effective delaying action . (c)
Orientation of segments to teat solution .
Since the leaves
are some~nlhat difficult to wet, it was suspected that the orientation of the segments towards the solution might affect the res-
Yol. 5, No . 22
HIIQETIN-LIHE ACTMTY
30
V
ts
0
(
L
3a10~
3x10 3
KIeeNe
FIG . 1 .
1 1
3x103
3alÔ~
Kinetin
3x10 1
p~ " rel -1
Kinetin-induced chlorophyll retention in eight barley varieties . Ordinates : log .mean chlorophyll content per 4 leaf segments . I~g . Half standard errors are indicated . sponse .
0
e
3 a10 1
~u0 .ml-1
FIG . 2 . Effect of temperature on kinetin-induced chlorophyll retention in Dea barley . Ordinates: as in Fig . 1 .
Submerged segments became limp and remained green, even
in the absence of kinetiny chlorophyll could be extracted frown them by boiling acetone,. but not by acetone at roam temperature . No significant differences could be detected due to floating the segments with the abaxial surface either upwards or downwards on the solutions .
However, in order to obviate variation which might
arise from a random orientation, segments are floated on their allaxial surlaces in the standard bioassay . (d)
Specificity :
the response to amino-acids.
The amino com-
pounds listed in Table 1 were tested in the standard bioassay at
Yol . 5, No . 22
2067
KINETIN-LIKE ACTIVITY
concentrations in the ranges stated .
These ranges were chosen so
as to extend far above those of amino-acids likely to be present in the extracts of the bleeding sap of the plants to be used in experiments . instance .
No significant positive response was obtained in any
Alanine, which Sechs (28) found in a qualitative test
on chlorophyll retention in oat leaves to be active at O .1M, is not a normal constituent of bleeding sap of herbaceous plants . (e)
Specificity :
the re sponse to gibberellic acid and auxin . A
very wide range of concentrations of both GA9 and IAA was tried . As Fig . 3 shows, no significant effect was observed in any of these treatments .
The lack of response to GA S is in marked con-
tract to the findings of Fletcher and Osborne (6) who used leaf disks of Taraxacum officinale , but is in agreement with those of Kendo (10) and Sechs (28) . (f)
Specifitv :
kinetin analogues _and synergists .
In the callus
proliferation bioassay (24) synergism has been shown between myoinositol and kinins, and myo-inositol has been identified by Letham (14) in extracts of plum fruitlets, in which it is synergistic with the naturally-occurring kinin .
~yo-inositol is neith-
er active by itself nor synergistic with kinetin in the barley leaf bioassay .
Table 2 demonstrates the similarly negative re-
sulta of testa of sucrose and mannitol .
Kende (9) has shown that
his barley leaf (var . Delta) bioassay is insensitive to adanine~ glucose and inorganic nitrogen compounds up to O .1M.
Tests of
three analogues of kinetin gave results in agreement with those of Ruraishi (12) using the expansion of radish leaf disks (Fig . 4) .
2068
KINETIN-LIKE ACTIVITY
Vol . S, .No. 22
TABLE 1 Compound
Concentration range tested mg ./ml .
Aspartic acid Asparagine Homoserine Glutamine Glutamic acid Proline Lysine . Threonine Arginine Histidine Glutathione (reduced form) Alanine Cysteine
0 .01 - 1 .0
Valine Leucine Iso-leucine Phenylalanine Tyrosine Serine Glycine Methionine
0 .001 - 0 .1
'15
~13
~003 0" 03
0~3
3
30
PPT .
300
FIG . 3 . Absence of chlorophyll-retention activity of GAa and~ïAA. Ordinates as in Fig . 1 .
O . ~ 00 03
0~03
0~3
3
FIG . 4 . Activity of analogues of kinetin. Ordinates as in Fig . 1 .
DPm~
30
Vol . 5, No . 22
2069
gINETIN-LIRE ACTIVITY
Only marginal loss of activity follows replacement of the furan ring by a 6-C aromatic ring, but considerable reduction follows replacement by a methyl group. TABLE 2 Treatment
Chlorophyll, Ng per 4 leaf segments 18 .83
t
0 .623
29 .98
±
0 .750
19 .67
t
0 .624
10 " -1 M . Mannitol 10 -a M .
19 .67
t
0 .695
19 .55
t
0 .645
" 10 -1 M.
19 .56
f
0 .678
Control Kinetin, Sucrose,
0 .03 4~g ml-1 10-a M .
Having defined the optimal conditions of the bioassay and to some extent explored its specificity to some of the compounds likely to be present in detectable amounts in xylem sap, attention was turned to bioassay of the samples of xylem sap collected as previously described.
An aliquot of~such a sample was frozen-
dried, the residue taken up in the minimum of water and subjected to TLC on silica gel G (Merck) spread on 20 x 20 cm glass plates at 0 .5 mm thickness .
The sample was spotted on the start line 3cm
frown the edge of the plate ; after drying and equilibration with the solvent for 45 minutes more solvent was added to the tank and the front allowed to ascend 10 cm from the start line .
The plate
was then removed, dried in a stream of cold air and observed under W.
Each 1 cm strip corresponding to 0 .1 Rf value was eluted with
ethyl acetate acidified with acetic acid .
Chromatograms of aque-
ous fractions of partitioned extracts were eluted with ethyl acetats alone .
Each elutes was blown down to dryness and the residue
200
BINETIN-LIRE ACTIVITY
taken up in 4 ml distilled water .
Vol .
5, No . 22
The control treatment in each
bioassay involved the use of the elutee similarly treated from a 1 cm strip prior to the start line .
The solvent systems used
were : Solvent A.
Tertiary amyl alcohol :formic acid :H 2 0, 3 :2 :1 by vol .
Solvent B .
Ha0-saturated secondary butanol .
Solvent C .
Boric acid, 0 .03M adjusted to pH 8 .4 with NaOH .
Solvents B and C are adopted from Miller (19) . Bioassay of whole~xylean sap .
A 700-m1 sample of sap collected
from balsam plants ( Impatiens balsaminea ) was frozen dried, the residue taken up in a minimum of distilled water and subjected to TLC using solvent A.
Examination of the developed and dried
plate under W revealed a dark zone, Rf 0 .1-0 :3, and dark zones in the regions of Rf 0 .6 and 0 .7 .
Bioassay of the eluates showed
two regions of kinin activity (Fig . 5a) . That at the higher Rf value could, according to the W observation, be due to two separate compounds .
The streaking of the activity at low Rf values
could result from breakdown of a compound during handling and chromatography .
To test this, a further 350 ml of balsam sap was
subjected to TLC using the same procedure as before .
A strip cor-
responding to Rf 0 .1-0 .3 was eluted in bulk and re-chromatography$ using solvent C .
The result appears to confirm the hypothesis of
breakdown (Fig . 5b) resulting in the production of another active compound which with this solvent migrates to Rf 0 .9 . Since these results suggest the presence in balsam sap of çompounds with properties remarkably similar to those of the
Vol . 5, No . 22
KINETIN-LIâE ACTIVITY
2071
so
10
I~pafi~n~
olo11i111iF~~a
FIG. 5 (a)
Chromatography of whole sap of Itgpatiens balsaminea . Ordinates, 4sg chlorophyll per set of ~ leaf segments . The lower of the two horizontal lines zspresents the chlorophyll con tent of control segments, the upper, the 95% confidence limit. Shaded areas represent statistically significant activity .
(b)
Re-chromatography of Zone 1, using solvent C .
(c)
Re-chromatography of active fraction eluted frasa Rf p .1-0 .3 of the TLC of the ethanol-soluble fraction of balsam sap (see Fiq . 6), using solvent B .
kinins of immature maize grain reported by-Miller (19) we have adapted his fractionation procedure to xylem eap as follows :
2072
KINETIN-LIKE l1CTIVITY
Vol . F, No . 22
XX 1_y em sap f~ree~ze dry Solids extract 4 times with one tenth the original volume 70% ethanol,filter, combine filtrates . Residue
Ethanol lution
inactive solids
evaporate off ethanol, extract remaining solution 4 times with equal vols . nbutanol . Combine butanol extracts . Butanolphase chromatography bioassay
Chromatography of an aqueous solution of the ethanol-insoluble residue from 500 ml of sap using solvent A, followed by bioassay of the eluates of the chromatogram yielded no activity .
We may
assume therefore that the active substances are all extracted into the ethanol . balsam sap,
The n-butanol phase from the same sample of
fractionated according to the scheme outlined above,
was subjected to TLC using solvent C .
Bioassay of the chromato-
gram ahowed two zones of activity one at Rf 0 .5 the other at Rf 0 .9-1 .0 (Fig . 6) .
Chromatography, using solvent B, and bioassay
of the aqueous fraction revealed only one peak of activity on the chromatogram (Fig . 6) . Two peaks of activity similar to those found in the butanol phase of balsam sap extracts were obtained by similar treatment of 1 litve of eap fron Xanthium and also from 330 ml of field pea sap (Fig . 6) .
The butanol phase of extracts from 350 ml of lupin sap
Vol . ~, No . 22 AQUEOUS
203
BINETIN-LIGE ACTIVITY FACTION
~UTANOI
n
Iwp~ti~n~
IEACTION
~I~ndvlif~r~
~~ n oO e r r jf re n )IantAiuw
L
p~nn~ylv~nie~w
~
r r
n I .l.
st . ..
r O
LuPlnu .
a n~u~liF~llu~
FIG . 6 . Chlorophyll-retention activity in xylem sap of four species of flowering plants . The quantities of sap were : 500 ml, balsam : 1 litre, Xanthium ; 330 ml, field pea ; 350 ml . lupin . The sap was fractions end as~cescribed in the text . Chromatograms were developed with solvents B (aqueous fraction), C (butanol Praotion) .
showed only a single peak, corresponding to the faster-moving of the two found in the other species .
Activity at Rf 0.5 could, how-
20`]4
gINETIN-LIgE ACTIVITY
Vol . ~, No . 22
ever, be masked by substances at the same Rf inhibitory to the bioassay .
The aqueous fraction of the sap extracts from all these
plants contained an active substance moving slowly in chromatography, similar to that found in balsam sap (Fig . 6) .
In the aqueous
fraction from the extracts from Xanthium and field pea sap there was a fast-moving secondary peak at Rf O .g .
As previously indica-
ted for entire balsam sap, this might arise from breakdown of the slow-moving factor during prolonged handling .
It was possible to
demonstrate that the labile fraction is water-soluble rather than butanol-soluble .
Following chromatography of the ethanol-soluble
fraction of 350 ml of balsam sap using solvent A, the compounds at Rf 0 .1-0 .3 were eluted .
The elutee was re-ehromatographed using
solvent B and the chromatogram bioassayed (Fig . 5C) .
The faster
peak in the aqueous fraction appears to be derived from the slower . Discussion The results of these bioassays clearly demonstrate the presenee in xylem sap of substances resembling kinetin in their ability to delay the breakdown of chlorophyll in barley leaf segments . The chromatographic mobility of these substances on silica gel G is strikingly like that of the active compounds in maize extracts in Miller's experiments (1g) in which the support was Whatman No .l paper .
Thus activity at Rf 0 .5 could well be due to a compound
like the zeatin of maize and that at Rf 0 .9 to a substance such as its nucleoside .
Miller (19)~has suggested that his slow-moving
factor is the monophosphate nucleotide of zeatin . phosphate would yield the activity at Rf O .g .
Removal of the
A similar breakdown
Vol . 5, No . 22
gINETIN-ZIgE ACTIVITY
2075
of a chromatographically slow-moving compound from the xylem sap of balsam, Xanthium and field pea to yield a fast-moving, active substance has been demonstrated .
In all these respects the sap-
borne compounds with chlorophyll-retention activity strikingly re semble those of extracts of maize kernels .
We have not yet been
able to compare the unknown substances from xylem sap directly with authentic zeatin or its derivatives.
Further work is required
to substantiate the suggestions made above concerning the nature of the active compounds in xylem sap .
In preliminary experiments,
we have been able to demonstrate their activity in the soybean callus bioassay of Miller (see also Kende, 10) ; it therefore seems probable that the active substances can be classified as phytokinins .
It is of importance that these substances are present in a
biological fluid likely to be representative of that moving under normal physiological conditions from the root to the shoot .
At
present it cannot, of course, be excluded that these substances are present as a result of the removal of the shoot system ; conversely, there is as yet no rigorous evidence of their synthesis in the root system itself .
Experiments are planned to elucidate
further the nature and origin of these substances and their significance to the physiology of the plant . Summary The first part of the paper describes optimisation and tests of the specificity of a bioassay for substances active in chlorophyll retention, using segments of barley leaves .
This bioassay
is used to deaoonstrate the presence in whole xylem sàp, and in
Vol . 5, No . 22
KINETIN-LIKE ACTIVITY
206
ethanolic extracts of sap,
of balsam, Xanthium , field pea and blue
Lupin of compounds with kinetin-like activity .
Further fractiona-
tion of ethanolic extracts yields three active components .
The
first is a water-soluble compound which breaks down to yield a component which resembles in its activity and chromatographic mobility one of the two more butanol-soluble components .
Similar
compounds are present in the first three species examined, and in part these similarities extend also to blue lupin.
In the discus-
sion coanparisons are made between the kinetin-like compounds of xylem sap and the published data on those of extracts from maize kernels . References 1.
D . I . ARNON, Plant Physiol .,
24, 1 (1949) .
2.
W. BOTTOMLEY,N . P . KEFFORD, J. A. ZWAR, and P . L . GOLDACRE, Au stral . J . Biol . Sci ., 16, 395 (1962) .
3.
G. BEAUCHESNE, in Plant Growth Regulation , p. 667, Univ . Pres$, Amea (1961) .
4.
G . BEAUCHESNE, M . LEBOEUF and R . GOUTAREL, in Recrulateurs naturels de la Croissance végétale , p . 119, C.N .R .S ., Paris (1963) .
5.
A . C . CHIBNALL, Protein Metabolism in the Plant , Yale Univ . Press, New Haven (1939) .
6.
R . A. FLETC.HRR and D . J . OSBORNE, Nature (Lund.) 2 (1965) .
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C . ITAI and Y. VAADIA, Physiol . Plant ., = 941 (1965) .
8.
W. T . JACKSON, Am . J . Bot ., 43, 496 (1956) .
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H . KENDE, Science , 145, 1066 (1964) .
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H. KENDE, Proc . Nat. Acad . Sci .(Wash .,) ~,
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11 .
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12 .
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13 .
D. S. Letham, in Re ug late urs naturels de la croissance végé tale , p. 569, C .N .R .S . Paris (1963) .
Vol . 5, No . 22
20~~
KINETIN-LISE ACTIVITY
14 .
D . S . LETHAM, Life Sciences , 3, 152 (1963) .
15 .
D . S, LETHAM . J . S . SHANNON and I . R, McDONALD . Proc . Chem . Soc ., p . 230, July, 1964 .
16 .
J. E, LOEFFLER and J. VAN OVERBEEK, in Regulateurs naturels de la croissance végétale , p . 77, C .N .R .S ., Paris (1963) .
17 .
C. 0 . MILLER, Proc . Nat . Acad . Sci . (Wash .), 47, 170 (1961) .
18 .
C, O, MILLER and F . H, WITHAM, in Regulateura naturels de la croissance végétale , p . 1 (erratum), C.N,R .S ., Paris (1963) .
19 .
C. O . MILLER, Proc . Nat . Acad . Sci . (Wash.), 54, 1052
20 .
K, MOTHES, L, ENGELSRECHT and L. KULAEVA, Flora, L47, 445 (1959) .
21 .
J. P . NITSCH, Bull, soc, bot . Fr . 107,
22 .
J. P .~NITSCH and C. NITSCH, Bull . soc, bot . Fr ., (1965) .
23 .
B . PARTHIER, Flora, 154,
24 .
J. K . POLLARD, E . M . SHANTZ and F . C, STEWARD, 36, 492 (1961) .
25 .
L. E . POWEtL and C. PRATT, Nature (tond .), 204, 602 (1964) .
26 .
A. E, RICHMOND and A. LONG, Science, 125, 650 (1957) .
27 .
J. H, ROGOZINSKA, J . P . HELGESON and F . SKOOG, Plant Physiol 40, 469 (1965) .
28 .
T . SACHE , On the mechanism of apical dominance, Ph .D .Thesis, Haxvard, 1965 .
29 .
M. SHAW and B, I, S . SRIVASTAVA, Plant Physiol., (1964) .
30 .
C, WEISS and Y. VAADIA, Life Sciences , 4, 1323 (1965) .
31 .
F . W, WENT, Plant Physiol ., 18, 51 (1943) .
32 .
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33 .
H . N, WOOD, in Regulateurs naturels de la croissance végé tale , p . 97 . C,N.R,S Paris (1963) .
34 .
J . A. ZW~R and F, SKOOG, Austral . J. Biol . Sci ., 16, 129 (1962) .
35 .
J . A. ZWAR, W, BOTTOMLEY and N . P . KEFFORD, Austral . J . Biol . Sçi ., 16, 407 (1962) .
36 .
J, A, ZWAR, M, I . BRUCE, W, BOTTOMLEY and N. P . KEFFORD, Regulateurs naturels de la croissance végétale , p. 123, C,N,R.S ., Paris (1963) .
37 "
D . S . LETHAM and C . 0. MILLER, Plant and Cell PY~yaiol ., 6 . 355 (1965)
(1965) .
263 (1960) . 112, 11
230 (1964) . Plant Physiol
39, 528