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Seasonal Changes in the Levels of Endogenous Cytokinins in the Willow Salix babylonica L.
Seasonal Changes in the Levels of Endogenous Cytokinins in the Willow Salix babylonica L.
J. VAN STADEN and J. E. DAVEY Department of Botany, University of Natal, Pietermaritzburg 3200, Republic of South Africa Received April 6, 1981 . Accepted May 5,1981
Summary A seasonal study of the cytokinin levels in different parts of young plants of Salix babylonica revealed that for a better understanding of the role of these hormones in plant growth it is essential that a more comprehensive approach must be adopted. As would be expected a close relationship was found between the bark, leaf, xylem and root tissue analysed. It would appear as if the bark (which included the cambium, phloem, stem cortex and epidermis) in particular could play an important function in the regulation of cytokinin levels in different tissues at different times of the year.
Key words: Salix babylonica, cytokinins, seasonal changes, leaves, stems, roots.
Introduction
In a recent review it was stressed that data on seasonal levels of cytokinins in whole plants could in all probability make a considerable contribution to a better understanding of the role of these hormones in plant growth (Van Staden and Davey, 1979). Today it is generally accepted that cytokinins produced in the roots are exported via the transpiration stream to the shoots, where, depending on the organ to which they are transported, they are either utilised and/or metabolised (Letham, 1978; Van Staden and Davey, 1979). While the evidence is not conclusive there are indications that meristematic, actively dividing shoot tissues such as the cambium (Skene, 1972), and organs such as shoot apices (Kannangara and Booth, 1974) and seeds (Letham, 1963), as well as different shoot explants (Chen and Petschow, 1978; Van Staden and Choveaux, 1980) could synthesise cytokinins. Whether the presence of cytokinins in aerial organs during different stages of development reflects an ability to synthesise these compounds, or merely indicates preferential transport at a particular time, is difficult to assess. In the case of leaves it is accepted that the gradual accumulation of cytokinins in them, particularly the cytokinin glucosides or storage forms (Van Staden, 1976 b, 1977), is due to continuous transport of cytokinins from the roots and their subsequent metabolism to these storage forms (Henson, 1978; Davey and Van Staden, 1981). As the transpiration rates of young fruits and buds, which do contain very high levels of cytokinin (Burrows and Carr, 1970; Davey and
z. Pjlanzenphysiol. Bd.
104. S. 53-59. 1981.
J. VAN STADEN and J. E. DAVEY
54
Van Staden, 1978; Van Staden and Dimalla, 1981), is relatively low, it must be assumed that unless transported via the phloem (Vonk, 1979) these organs were able to synthesize a large proportion of the cytokinins that were detected in them. The physiological significance of the cytokinins detected in a particular organ or tissue at any particular time would be more readily assessable if it was known how these substances are distributed within the whole plant. By conducting a study on the seasonal levels of endogenous cytokinins in young vegetative plants of the willow Salix babyla· nica it was hoped to obtain a better understanding of their role in the control of plant growth.
Materials and Methods Plant material Three hundred plants of Salix babylonica L. were established from cuttings and grown individually in large containers for one year. Plants were maintained under natural conditions in the Southern Hemisphere and the experiment started with one-year-old plants in January 1979 and ended in December 1979. At regular intervals 25 plants were harvested and various parts such as the roots, stems (10 cm above soil level), and mature leaves collected. The stem material was subsequently divided into xylem and bark (epidermis, phloem and cambium). Each sample was massed, freeze-dried, and then ground to a homogeneous powder which was stored at - 20°C until analysed.
Cytokinin extraction and bioassay In all cases, except the buds, 3 g of dry material was homogenised with 100 ml 80% ethanol and extracted for 24 h at 5 0c. In the case of the buds 0.3 g dry material was extracted with 25 ml 80% ethanol. The extracts were filtered through Whatman No.1 paper and the residue washed with 50 m! 80% ethanol. The ethano!ic extracts were taken to dryness in vacuo at 35°C and the residues resuspended in 50 ml 80% ethanol. The pH of these extracts was adjusted to 2.5 and the cytokinins were extracted with Dowex SOW - X8 as described previously (Van Staden, 1976 a). The constituents in the concentrated extracts were subsequently separated on paper with iso·propanol : 25% ammonium hydroxide: water (10 : 1 : 1 v/v). After drying, the chromatograms were divided into 10 equal Rf strips; each strip was incorporated into a flask with 30 ml culture medium (in the case of the buds 20 ml), and the cytokinin activity determined by the soybean callus bioassay (Miller, 1965). All extractions and bioassays were repeated. Peaks of cytokinin activity significantly different from the controls were expressed as zeatin equivalents
(ZE).
Results and Discussion Material of the weeping willow Salix babylanica L. has been used on a number of occasions in attempts to gain a better understanding of the role of cytokinins in plant growth (Van Staden, 1976 a, 1977, 1979; Van Staden and Brown, 1977, 1978). In all these studies two major peaks of cytokinin-like activity were detected. A polar peak which co-chromatographed with glucosylzeatin and a non-polar peak which had the same chromatographic properties as zeatin and ribosylzeatin. During the course of this seasonal investigation the polar peak, which is thought to represent storage forms
Z. Pjlanzenphysiol. Bd. 104. S. 53-59. 1981.
Cytokinins in willow plants
55
of the endogenous cytokinins (Parker and Letham, 1973), was detected most frequently in the bark material (Fig. 1). In the mature leaves this peak was detected only in the two months immediately preceding winter (Fig. 2), and in the xylem tissue only in summer (December) (Fig. 3). No activity co-chromatographing with the 1979 -02-07
1979-01-03
2.0 1.5 1.0
U5
15
ZG~
1979-03 -14
ZG~
-
l
-~
1979-08-02
1979-06-28
L-----.JUL5---'1.~ !c-6----:::cls:------:l~
1979-{)4-25
1979-05-23
~~
~~
~
!l~~ 1979-09-05
I
1979-11-09
~J
--Q5:!-:--~~ o~1--:os~---:lb ~6----:::Q5:------;!tb
o!c-
5
Z,.,gl-'
Rf
Fig. 1: Cytokinin activity detected in extracts from 3 g dry bark material (including the cambium, phloem, stem cortex and epidermis) collected from willow plants at different times of the year. Dowex 50 purified extracts were separated on paper using iso-propanol: 25% ammonium hydroxide: water (10: 1 : 1 v/v). Z=zeatin; ZR=ribosylzeatin; ZG=glucosylzeatin. Shaded areas represent activity significantly different from the controls at the 1% level. 1979-02-07
1979-01-03
2.0
~ ZR
1979-03-'\4
Z
1979-04-25
1979-05- 23
ZG ZR
zG ~
~
~
~~ ~
~ ~ ~
15 10 ~
05
~>Vi
j U ""
20 15 10 05
!;------,;r!.s'<------7.b --coo!;;~:--~~ 1
01;--1
--,ir!.s.----t~
0\-1
-'Q5&"----.!~
oi;--l
--.!oS'<------7.,b
0\-1
Rf
Fig. 2: Cytokinin activity detected in extracts from 3 g dry mature leaf material collected from willow plants at different times of the year. Techniques and abbreviations as for Fig. 1.
Z. Pjlanzenphysiol. Ed. 104. S. 53-59. 1981.
56
].
VAN STADEN
and]. E.
DAVEY
polar peak was recorded in the roots (Fig. 4). Throughout the investigation most of the cytokinin-like activity detected in the different tissues analysed, co-chromatographed with zeatin and ribosylzeatin. To obtain a better understanding of the possible relationship between the cytokinins in the different tissues analysed, the activity recorded in Figures 1- 4 (i.e. that which was significantly different from the controls) was expressed as ng zeatin equivalents g-l dry mass. The information obtained was expressed graphically (Fig. 5). During most of the year the cytokinin activity detected in the xylem tissue was very low. However, immediately after winter and with the advent of bud-swell (Table 1) there was an increase in the activity detected in this tissue. This finding correlated well witH other studies where it was reported that the cytokinin activity in the xylem sap of trees increased prior to bud-swell (Hewett and Wareing, 1973). The
.
1979-02-07
1979-03-14
LI_--'---_-'I
LI_--'---_-'I
1979-05-23
1979-04-25
as
"
9w
>= (f)
0.5
:3
"'" Z ",91
L-----'_---'I
wo.
05
wo.
as
as
LI_--'---_-'I
LI_--'---_-'
as
as
Wo.
Wo.
to.
Rf
Fig. 3: Cytokinin activity detected in extracts from 3 g of dry xylem material collected from willow plants at different times of the year. Techniques and abbreviations as for Fig. 1. 1979-02-07
1979-05-23
zCZR
zG ZR
1979-06i3
..
" ~
>=
.
(f)
~
"
-~
to. 05
0 1.0
05 0.
1 ~~
15 -~
~L;]
L-...,.J
1979-08-02
~ 1979-11-09
~ 00
c:::JJ
c:::ru--U
L--~L-----,I
1
0.5
r"'I
100
I 0.5
J 5
1979-12-02
~
Zpgl
LI_--'---_~
tOo
0.5
10
Rf
Fig. 4: Cytokinin activity detected in extracts from 3 g of dry root material collected from willow plants at different times of the year. Techniques and abbreviations as for Fig. 1
Z. Pjlanzenphysiol. Rd. 104. S. 53-59. 1981.
Cytokinins in willow plants
57
+
Leaves yellow
Buds swelling
150
• leaves
... Roots
• Wood
o
100
Bark
~ \
;II
\
:;
50
i? z
;:
::::'"
COLLECTION
TIME
month
Fig. 5: Total cytokinin activity in different tissues of willow plants collected at different times of the year. The activity significantly different from the controls, and which is depicted in Figures 1- 4, was expressed as ng zeatin equivalents g -1 dry mass. Bark = open circles; mature leaves = closed circles; xylem = closed squares; roots = closed triangles.
level of activity remained relatively high, and in December a peak which co-chromatographed with glucosylzeatin was also present (Fig. 3). The significance of this peak is not known as it seems unlikely that it could have been produced by the predominantly non-living xylem material, or transported from the roots where it was not recorded. There is the possibility that cytokinins could have been synthesised in the xylem parynchyma or laterally transported from the bark material. The latter possibility seems more likely as this peak was present in the bark in the preceding months. It is highly unlikely that its presence could have been due to an incomplete separation of bark and xylem material as the cambium was very active at this time and allowed Table 1: Physical condition and general appearance of different organs and tissues of plants of Salix babylonica at different times of the year. Collection time 1979-01-03 1979-02-07 1979-03-14 1979-04-25 1979-05-23 1979-06-28 1979-08-02 1979-09-05 1979-11-09 1979-12-02
Physical condition of plant parts Stem
Mature leaves
bark easily removed bark easily removed bark easily removed bark drying not easily removed bark drying not easily removed bark very dry, difficult to remove bark very dry, difficult to remove bark more easily removed bark easily removed bark easily removed
green small and flat green small and flat yellowing small and flat yellow small and flat yellow, hard and beginning to abscise small and flat yellow, hard and abscising small and flat small and flat swelling light green bursting green sprouting
Buds
z. Pjlanzenphysiol. Ed. 104. S. 53-59. 1981.
58
J. VAN STADEN and]. E. DAVEY
the bark to be stripped from the xylem with ease (Table 1). That the cytokinins present in the xylem could have been partly of root origin and partly of bark origin is suggested by the fact that high levels of cytokinin were detected in the roots and bark prior to the level increasing in the xylem. The decrease in activity recorded for the roots and bark is, however, not reflected by a large increase in the xylem which seem to suggest that these cytokinins were utilised within the plants. In a recent investigation it was reported that buds, in order to be potentiated to resume growth, could utilise large amounts of cytokinin (Van Staden and Dimalla, 1981). Within the swelling buds a relatively high level of cytokinin activity was detected (Fig. 5). The highest level of cytokinin acitivty was found in yellow senescing leaves just as they were beginning to abscise. As reported earlier (Van Staden, 1977) a large proportion of this activity was due to the presence of compounds which co-chromatographed with glucosylzeatin (Fig. 2). Prior to abscision the level of activity in the mature leaves decreased while that in the bark and roots increased. Whether these increases reflect transport from the leaves, an accumulation due to a slowdown of export to the senescing leaves, or both, is difficult to decide. In the monocarpic white lupin plant there are indications that labelled zeatin applied to the mature leaves is metabolised to glucosylzeatin and then exported from them (Van Staden and Davey, 1981). Whether this happens in deciduous and evergreen trees remains to be assessed. In the present study relatively high levels of cytokinin activity were recorded in the bark. This suggest that the bark, which in this case included the cambium, the phloem and the stem cortex, could play a major role in controlling the cytokinin balance in the willow plant. These tissues could contribute to the production of cytokinins, be involved in their transport and act as potential storage tissue. Acknowledgements The financial support of the C. S. I. R., Pretoria and the University of Natal Development Fund is gratefully acknowledged.
References BURROWS, W.]. and D. J. CARR: Cytokinin content of pea seeds during their growth and development. Physiol. Plant. 23, 1064-1070 (1970). CHEN, C.-M. and B. PETSCHOW: Cytokinin biosynthesis in cultured rootless tobacco plants. Plant Physiol. 62, 861-865 (1978). DAVEY, J. E. and J. VAN STADEN: Cytokinin acitivity in Lupinus albus. II. Distribution in fruiting plants. Physiol. Plant. 43,82-86 (1978). - - Cytokinin activity in Lupinus albus. V. Translocation and metabolism of (8-!4C)zeatin applied to the xylem of fruiting plants. Phys. Plant. 51, 45-48 (1981). HENSON, I. E.: Cytokinins and their metabolism in leaves of Alnus glutinosa L. Gaertn. Z. pflanzenphysiol. 86, 363-369 (1978). HEWETT, E. W. and P. F. WAREING: Cytokinins in Populusxrobusta Schneid.: Changes during chilling and bud burst. Physiol. Plant. 28, 393-399 (1973). KANNANGARA, T. and A. BOOTH: Diffusible cytokinins in shoot apices of Dahlia variabilis. J. Exp. Bot. 25, 459-467 (1975).
Z. Pjlanzenphysiol. Bd. 104. S. 53-59. 1981.
Cytokinins in willow plants
59
LETHAM, D. S.: Zeatin a factor inducing cell division from Zea mays. Life Sci. 2, 569-573 (1963). - Cytokinins. In: D. S. LETHAM, P. B. GOODWIN and T. J. V. HIGGINS (Eds.): Phytohormones and related compounds: A comprehensive treatise, pp. 205 - 264. Elsevier/North-Holland Biomedical Press, Amsterdam, 1978. LORENZI, R., R. HORGAN, and P. F. WAREING: Cytokinins in Picea sitchensis Carriere: Identification and relation to growth. Biochem. Physiol. Pflanzen 168, 333-339 (1975). MILLER, C. 0.: Evidence for the natural occurrence of zeatin and derivatives: Compounds from maize which promote cell division. Proc. Nat. Acad. Sci. USA 54,1052-1058 (1965). PARKER, C. W. and D. S. LETHAM: Regulators of cell division in plant tissues. XVI. Metabolism of zeatin by radish cotyledons and hypocotyls. Planta 114,199-218 (1973). SKENE, K. G. M.: Cytokinins in the xylem sap of grape vine canes: Changes in activity during cold-storage. Planta 104, 89-92 (1972). VAN STADEN, J.: Occurrence of a cytokinin glucoside in the leaves and in honeydew of Salix babylonica. Physiol. Plant. 36, 225-228 (1976 a). - Seasonal changes in the cytokinin content of Ginkgo hiloba leaves. Physiol. Plant. 38, 1- 5 (1976 b). - Seasonal changes in the cytokinin content of the leaves of Salix babylonica. Physiol. Plant.
40,296-299 (1977).
- Changes in the endogenous cytokinin levels of excised buds of Salix babylonica L., cultured aseptically. Bot. Gaz. 140, 138-141 (1979). VAN STADEN, J. and N. A. C. BROWN: The effect of ringing on cytokinin distribution in Salix babylonica. Physiol. Plant. 39, 266-270 (1977). - - Changes in the endogenous cytokinins of bark and buds of Salix babylonica as a result of stem girdling. Physiol. Plant. 43, 148 -153 (1978). VAN STADEN, J. and N. A. CHOVEAUX: Cytokinins in internodal stem segments of Salix babylo· nica. Z. Pflanzenphysiol. 96, 153 -161 (1980). VAN STADEN, J. and J. E. DAVEY: The synthesis, transport and metabolism of endogenous cytokinins. Plant, Cell & Environ. 2, 93 -106 (1979). - - Cytokinin activity in Lupinus albus. VI. Translocation and metabolism of [8)4C]zeatin applied to the leaves and fruits of fruiting plants. Physiol. Plant. 51, 49-52 (1981). VAN STADEN, J. and G. G. DIMALLA: The production and utilisation of cytokinins in rootless, dormant almond shoots maintained at low temperature. Z. Pflanzenphysiol. 103, 121-129 (1981). VONK, C. R.: Origin of cytokinins transported in the phloem. Physiol. Plant. 46, 235-240 (1979).
Z. Pjlanzenphysiol. Bd. 104. S. 53-59. 1981.
J. VAN STADEN and J. E. DAVEY Department of Botany, University of Natal, Pietermaritzburg 3200, Republic of South Africa Received April 6, 1981 . Accepted May 5,1981
Summary A seasonal study of the cytokinin levels in different parts of young plants of Salix babylonica revealed that for a better understanding of the role of these hormones in plant growth it is essential that a more comprehensive approach must be adopted. As would be expected a close relationship was found between the bark, leaf, xylem and root tissue analysed. It would appear as if the bark (which included the cambium, phloem, stem cortex and epidermis) in particular could play an important function in the regulation of cytokinin levels in different tissues at different times of the year.
Key words: Salix babylonica, cytokinins, seasonal changes, leaves, stems, roots.
Introduction
In a recent review it was stressed that data on seasonal levels of cytokinins in whole plants could in all probability make a considerable contribution to a better understanding of the role of these hormones in plant growth (Van Staden and Davey, 1979). Today it is generally accepted that cytokinins produced in the roots are exported via the transpiration stream to the shoots, where, depending on the organ to which they are transported, they are either utilised and/or metabolised (Letham, 1978; Van Staden and Davey, 1979). While the evidence is not conclusive there are indications that meristematic, actively dividing shoot tissues such as the cambium (Skene, 1972), and organs such as shoot apices (Kannangara and Booth, 1974) and seeds (Letham, 1963), as well as different shoot explants (Chen and Petschow, 1978; Van Staden and Choveaux, 1980) could synthesise cytokinins. Whether the presence of cytokinins in aerial organs during different stages of development reflects an ability to synthesise these compounds, or merely indicates preferential transport at a particular time, is difficult to assess. In the case of leaves it is accepted that the gradual accumulation of cytokinins in them, particularly the cytokinin glucosides or storage forms (Van Staden, 1976 b, 1977), is due to continuous transport of cytokinins from the roots and their subsequent metabolism to these storage forms (Henson, 1978; Davey and Van Staden, 1981). As the transpiration rates of young fruits and buds, which do contain very high levels of cytokinin (Burrows and Carr, 1970; Davey and
z. Pjlanzenphysiol. Bd.
104. S. 53-59. 1981.
J. VAN STADEN and J. E. DAVEY
54
Van Staden, 1978; Van Staden and Dimalla, 1981), is relatively low, it must be assumed that unless transported via the phloem (Vonk, 1979) these organs were able to synthesize a large proportion of the cytokinins that were detected in them. The physiological significance of the cytokinins detected in a particular organ or tissue at any particular time would be more readily assessable if it was known how these substances are distributed within the whole plant. By conducting a study on the seasonal levels of endogenous cytokinins in young vegetative plants of the willow Salix babyla· nica it was hoped to obtain a better understanding of their role in the control of plant growth.
Materials and Methods Plant material Three hundred plants of Salix babylonica L. were established from cuttings and grown individually in large containers for one year. Plants were maintained under natural conditions in the Southern Hemisphere and the experiment started with one-year-old plants in January 1979 and ended in December 1979. At regular intervals 25 plants were harvested and various parts such as the roots, stems (10 cm above soil level), and mature leaves collected. The stem material was subsequently divided into xylem and bark (epidermis, phloem and cambium). Each sample was massed, freeze-dried, and then ground to a homogeneous powder which was stored at - 20°C until analysed.
Cytokinin extraction and bioassay In all cases, except the buds, 3 g of dry material was homogenised with 100 ml 80% ethanol and extracted for 24 h at 5 0c. In the case of the buds 0.3 g dry material was extracted with 25 ml 80% ethanol. The extracts were filtered through Whatman No.1 paper and the residue washed with 50 m! 80% ethanol. The ethano!ic extracts were taken to dryness in vacuo at 35°C and the residues resuspended in 50 ml 80% ethanol. The pH of these extracts was adjusted to 2.5 and the cytokinins were extracted with Dowex SOW - X8 as described previously (Van Staden, 1976 a). The constituents in the concentrated extracts were subsequently separated on paper with iso·propanol : 25% ammonium hydroxide: water (10 : 1 : 1 v/v). After drying, the chromatograms were divided into 10 equal Rf strips; each strip was incorporated into a flask with 30 ml culture medium (in the case of the buds 20 ml), and the cytokinin activity determined by the soybean callus bioassay (Miller, 1965). All extractions and bioassays were repeated. Peaks of cytokinin activity significantly different from the controls were expressed as zeatin equivalents
(ZE).
Results and Discussion Material of the weeping willow Salix babylanica L. has been used on a number of occasions in attempts to gain a better understanding of the role of cytokinins in plant growth (Van Staden, 1976 a, 1977, 1979; Van Staden and Brown, 1977, 1978). In all these studies two major peaks of cytokinin-like activity were detected. A polar peak which co-chromatographed with glucosylzeatin and a non-polar peak which had the same chromatographic properties as zeatin and ribosylzeatin. During the course of this seasonal investigation the polar peak, which is thought to represent storage forms
Z. Pjlanzenphysiol. Bd. 104. S. 53-59. 1981.
Cytokinins in willow plants
55
of the endogenous cytokinins (Parker and Letham, 1973), was detected most frequently in the bark material (Fig. 1). In the mature leaves this peak was detected only in the two months immediately preceding winter (Fig. 2), and in the xylem tissue only in summer (December) (Fig. 3). No activity co-chromatographing with the 1979 -02-07
1979-01-03
2.0 1.5 1.0
U5
15
ZG~
1979-03 -14
ZG~
-
l
-~
1979-08-02
1979-06-28
L-----.JUL5---'1.~ !c-6----:::cls:------:l~
1979-{)4-25
1979-05-23
~~
~~
~
!l~~ 1979-09-05
I
1979-11-09
~J
--Q5:!-:--~~ o~1--:os~---:lb ~6----:::Q5:------;!tb
o!c-
5
Z,.,gl-'
Rf
Fig. 1: Cytokinin activity detected in extracts from 3 g dry bark material (including the cambium, phloem, stem cortex and epidermis) collected from willow plants at different times of the year. Dowex 50 purified extracts were separated on paper using iso-propanol: 25% ammonium hydroxide: water (10: 1 : 1 v/v). Z=zeatin; ZR=ribosylzeatin; ZG=glucosylzeatin. Shaded areas represent activity significantly different from the controls at the 1% level. 1979-02-07
1979-01-03
2.0
~ ZR
1979-03-'\4
Z
1979-04-25
1979-05- 23
ZG ZR
zG ~
~
~
~~ ~
~ ~ ~
15 10 ~
05
~>Vi
j U ""
20 15 10 05
!;------,;r!.s'<------7.b --coo!;;~:--~~ 1
01;--1
--,ir!.s.----t~
0\-1
-'Q5&"----.!~
oi;--l
--.!oS'<------7.,b
0\-1
Rf
Fig. 2: Cytokinin activity detected in extracts from 3 g dry mature leaf material collected from willow plants at different times of the year. Techniques and abbreviations as for Fig. 1.
Z. Pjlanzenphysiol. Ed. 104. S. 53-59. 1981.
56
].
VAN STADEN
and]. E.
DAVEY
polar peak was recorded in the roots (Fig. 4). Throughout the investigation most of the cytokinin-like activity detected in the different tissues analysed, co-chromatographed with zeatin and ribosylzeatin. To obtain a better understanding of the possible relationship between the cytokinins in the different tissues analysed, the activity recorded in Figures 1- 4 (i.e. that which was significantly different from the controls) was expressed as ng zeatin equivalents g-l dry mass. The information obtained was expressed graphically (Fig. 5). During most of the year the cytokinin activity detected in the xylem tissue was very low. However, immediately after winter and with the advent of bud-swell (Table 1) there was an increase in the activity detected in this tissue. This finding correlated well witH other studies where it was reported that the cytokinin activity in the xylem sap of trees increased prior to bud-swell (Hewett and Wareing, 1973). The
.
1979-02-07
1979-03-14
LI_--'---_-'I
LI_--'---_-'I
1979-05-23
1979-04-25
as
"
9w
>= (f)
0.5
:3
"'" Z ",91
L-----'_---'I
wo.
05
wo.
as
as
LI_--'---_-'I
LI_--'---_-'
as
as
Wo.
Wo.
to.
Rf
Fig. 3: Cytokinin activity detected in extracts from 3 g of dry xylem material collected from willow plants at different times of the year. Techniques and abbreviations as for Fig. 1. 1979-02-07
1979-05-23
zCZR
zG ZR
1979-06i3
..
" ~
>=
.
(f)
~
"
-~
to. 05
0 1.0
05 0.
1 ~~
15 -~
~L;]
L-...,.J
1979-08-02
~ 1979-11-09
~ 00
c:::JJ
c:::ru--U
L--~L-----,I
1
0.5
r"'I
100
I 0.5
J 5
1979-12-02
~
Zpgl
LI_--'---_~
tOo
0.5
10
Rf
Fig. 4: Cytokinin activity detected in extracts from 3 g of dry root material collected from willow plants at different times of the year. Techniques and abbreviations as for Fig. 1
Z. Pjlanzenphysiol. Rd. 104. S. 53-59. 1981.
Cytokinins in willow plants
57
+
Leaves yellow
Buds swelling
150
• leaves
... Roots
• Wood
o
100
Bark
~ \
;II
\
:;
50
i? z
;:
::::'"
COLLECTION
TIME
month
Fig. 5: Total cytokinin activity in different tissues of willow plants collected at different times of the year. The activity significantly different from the controls, and which is depicted in Figures 1- 4, was expressed as ng zeatin equivalents g -1 dry mass. Bark = open circles; mature leaves = closed circles; xylem = closed squares; roots = closed triangles.
level of activity remained relatively high, and in December a peak which co-chromatographed with glucosylzeatin was also present (Fig. 3). The significance of this peak is not known as it seems unlikely that it could have been produced by the predominantly non-living xylem material, or transported from the roots where it was not recorded. There is the possibility that cytokinins could have been synthesised in the xylem parynchyma or laterally transported from the bark material. The latter possibility seems more likely as this peak was present in the bark in the preceding months. It is highly unlikely that its presence could have been due to an incomplete separation of bark and xylem material as the cambium was very active at this time and allowed Table 1: Physical condition and general appearance of different organs and tissues of plants of Salix babylonica at different times of the year. Collection time 1979-01-03 1979-02-07 1979-03-14 1979-04-25 1979-05-23 1979-06-28 1979-08-02 1979-09-05 1979-11-09 1979-12-02
Physical condition of plant parts Stem
Mature leaves
bark easily removed bark easily removed bark easily removed bark drying not easily removed bark drying not easily removed bark very dry, difficult to remove bark very dry, difficult to remove bark more easily removed bark easily removed bark easily removed
green small and flat green small and flat yellowing small and flat yellow small and flat yellow, hard and beginning to abscise small and flat yellow, hard and abscising small and flat small and flat swelling light green bursting green sprouting
Buds
z. Pjlanzenphysiol. Ed. 104. S. 53-59. 1981.
58
J. VAN STADEN and]. E. DAVEY
the bark to be stripped from the xylem with ease (Table 1). That the cytokinins present in the xylem could have been partly of root origin and partly of bark origin is suggested by the fact that high levels of cytokinin were detected in the roots and bark prior to the level increasing in the xylem. The decrease in activity recorded for the roots and bark is, however, not reflected by a large increase in the xylem which seem to suggest that these cytokinins were utilised within the plants. In a recent investigation it was reported that buds, in order to be potentiated to resume growth, could utilise large amounts of cytokinin (Van Staden and Dimalla, 1981). Within the swelling buds a relatively high level of cytokinin activity was detected (Fig. 5). The highest level of cytokinin acitivty was found in yellow senescing leaves just as they were beginning to abscise. As reported earlier (Van Staden, 1977) a large proportion of this activity was due to the presence of compounds which co-chromatographed with glucosylzeatin (Fig. 2). Prior to abscision the level of activity in the mature leaves decreased while that in the bark and roots increased. Whether these increases reflect transport from the leaves, an accumulation due to a slowdown of export to the senescing leaves, or both, is difficult to decide. In the monocarpic white lupin plant there are indications that labelled zeatin applied to the mature leaves is metabolised to glucosylzeatin and then exported from them (Van Staden and Davey, 1981). Whether this happens in deciduous and evergreen trees remains to be assessed. In the present study relatively high levels of cytokinin activity were recorded in the bark. This suggest that the bark, which in this case included the cambium, the phloem and the stem cortex, could play a major role in controlling the cytokinin balance in the willow plant. These tissues could contribute to the production of cytokinins, be involved in their transport and act as potential storage tissue. Acknowledgements The financial support of the C. S. I. R., Pretoria and the University of Natal Development Fund is gratefully acknowledged.
References BURROWS, W.]. and D. J. CARR: Cytokinin content of pea seeds during their growth and development. Physiol. Plant. 23, 1064-1070 (1970). CHEN, C.-M. and B. PETSCHOW: Cytokinin biosynthesis in cultured rootless tobacco plants. Plant Physiol. 62, 861-865 (1978). DAVEY, J. E. and J. VAN STADEN: Cytokinin acitivity in Lupinus albus. II. Distribution in fruiting plants. Physiol. Plant. 43,82-86 (1978). - - Cytokinin activity in Lupinus albus. V. Translocation and metabolism of (8-!4C)zeatin applied to the xylem of fruiting plants. Phys. Plant. 51, 45-48 (1981). HENSON, I. E.: Cytokinins and their metabolism in leaves of Alnus glutinosa L. Gaertn. Z. pflanzenphysiol. 86, 363-369 (1978). HEWETT, E. W. and P. F. WAREING: Cytokinins in Populusxrobusta Schneid.: Changes during chilling and bud burst. Physiol. Plant. 28, 393-399 (1973). KANNANGARA, T. and A. BOOTH: Diffusible cytokinins in shoot apices of Dahlia variabilis. J. Exp. Bot. 25, 459-467 (1975).
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LETHAM, D. S.: Zeatin a factor inducing cell division from Zea mays. Life Sci. 2, 569-573 (1963). - Cytokinins. In: D. S. LETHAM, P. B. GOODWIN and T. J. V. HIGGINS (Eds.): Phytohormones and related compounds: A comprehensive treatise, pp. 205 - 264. Elsevier/North-Holland Biomedical Press, Amsterdam, 1978. LORENZI, R., R. HORGAN, and P. F. WAREING: Cytokinins in Picea sitchensis Carriere: Identification and relation to growth. Biochem. Physiol. Pflanzen 168, 333-339 (1975). MILLER, C. 0.: Evidence for the natural occurrence of zeatin and derivatives: Compounds from maize which promote cell division. Proc. Nat. Acad. Sci. USA 54,1052-1058 (1965). PARKER, C. W. and D. S. LETHAM: Regulators of cell division in plant tissues. XVI. Metabolism of zeatin by radish cotyledons and hypocotyls. Planta 114,199-218 (1973). SKENE, K. G. M.: Cytokinins in the xylem sap of grape vine canes: Changes in activity during cold-storage. Planta 104, 89-92 (1972). VAN STADEN, J.: Occurrence of a cytokinin glucoside in the leaves and in honeydew of Salix babylonica. Physiol. Plant. 36, 225-228 (1976 a). - Seasonal changes in the cytokinin content of Ginkgo hiloba leaves. Physiol. Plant. 38, 1- 5 (1976 b). - Seasonal changes in the cytokinin content of the leaves of Salix babylonica. Physiol. Plant.
40,296-299 (1977).
- Changes in the endogenous cytokinin levels of excised buds of Salix babylonica L., cultured aseptically. Bot. Gaz. 140, 138-141 (1979). VAN STADEN, J. and N. A. C. BROWN: The effect of ringing on cytokinin distribution in Salix babylonica. Physiol. Plant. 39, 266-270 (1977). - - Changes in the endogenous cytokinins of bark and buds of Salix babylonica as a result of stem girdling. Physiol. Plant. 43, 148 -153 (1978). VAN STADEN, J. and N. A. CHOVEAUX: Cytokinins in internodal stem segments of Salix babylo· nica. Z. Pflanzenphysiol. 96, 153 -161 (1980). VAN STADEN, J. and J. E. DAVEY: The synthesis, transport and metabolism of endogenous cytokinins. Plant, Cell & Environ. 2, 93 -106 (1979). - - Cytokinin activity in Lupinus albus. VI. Translocation and metabolism of [8)4C]zeatin applied to the leaves and fruits of fruiting plants. Physiol. Plant. 51, 49-52 (1981). VAN STADEN, J. and G. G. DIMALLA: The production and utilisation of cytokinins in rootless, dormant almond shoots maintained at low temperature. Z. Pflanzenphysiol. 103, 121-129 (1981). VONK, C. R.: Origin of cytokinins transported in the phloem. Physiol. Plant. 46, 235-240 (1979).
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