Cytokinins in the root pressure exudate of Citrus jambhiri Lush. colonized by vesicular-arbuscular mycorrhizae
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Tree Physiology 4,9-18 (1988).
0 1988 Heron Publishing-Victoria, Canada.
Cytokinins in the root pressure exudate of Citrus jambhiri Lush.
colonized by vesicular-arbuscular mycorrhizae
R. K. DIXON’, H. E. GARRETT2 and G. S. COX2
’ School of Forestry, Auburn University, Auburn, AL 36849-5418, USA
’ School of Forestry, Fisheries and Wildlife, University of Missouri, Columbia, MO 45211, USA
Received April 22, 1987
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Summary
The influence of vesicular-arbuscular mycorrhizal (VAM) symbiosis on the transport of cytokinins
from the root to the shoot of Citrus jumbhiri Lush. seedlings inoculated with cultures of Glomus
etunicatum (Becker and Gerd.), G. fusciculatum (Thaxt.) Gerd. and Trappe, or G. mosseae (Nichol.
and Gerd.) was investigated. Cytokinins collected from root exudates over a 90-day period were
analyzed by high-performance liquid chromatography, mass spectrometry and a bioassay. The flux of
cytokinins was independent of root exudate flux. Seedlings inoculated with G. fasciculatum or G.
mosseae yielded a greater flux of zeatin, dihydrozeatin and zeatin riboside than non-inoculated
seedlings. The flux of zeatin riboside was significantly greater than the flux of zeatin in seedlings
inoculated with VAM symbionts. Vesicular-arbuscular mycorrhizal relationships apparently contri-
buted to, or inguenced, the export of cytokinins from the root. The elevated cytokinin flux of
inoculated seedlings was associated with improved tissue phosphorus nutrition and a significant
increase in seedling biomass.
Introduction
It is generally accepted that, in vascular plants, cytokinins are produced by the
root system and transported to the shoot, where they are required for the mainten-
ance of chlorophyll, cell division, ion transport, and photosynthesis (Torrey 1976,
van Staden and Davey 1979). Transport of exogenous cytokinins from root to
shoot has been demonstrated in several woody genera including Acer, Betulu
(Wareing 1980), Citrus (Mozes and Altman 1977, Hutton and van Staden 1983),
Malus and Populus (van Staden and Davey 1979).
The levels of cytokinin-like substances are higher in vesicular-arbuscular
mycorrhizal (VAM) monocotyledons (Allen et al. 1980) and dicotyledons (Edriss
et al. 1984, Dixon et al. 1988) than in non-inoculated plants. Dixon et al. (1988)
suggested that enhancement of cytokinin production in Citrus colonized by vesicu-
lar-arbuscular mycorrhizae is associated with the VAM infection rather than
improved mineral nutrition. Miller (1967) and Crafts and Miller (1974) have
demonstrated zeatin and zeatin riboside production by several ecto- and ectendo-
mycorrhizal fungi in vitro. There is evidence that cytokinins may reduce plant
resistance to fungal invasion (Azcon et al. 1978, Haberlach et al. 1978).
The presence of specific cytokinins in rough lemon (Citrusjumbhiri Lush.) root
pressure exudate has not been demonstrated. Moreover, the influence of VAM on
cytokinin flux during seedling development has not been evaluated. The objectives10 DIXON, GARRETT AND COX
of this study were to: (1) establish the presence of cytokinins in root pressure
exudate of Citrus jumbhiri, and (2) characterize profiles of cytokinin flux in root
pressure exudate in relation to seedling ontogeny, phosphorus nutrition and VAM
development. The relationship between VAM development and cytokinin flux was
evaluated for three fungal symbionts.
Materials and methods
Plant culture
Plants were grown from seed in 750-ml containers filled with a loamy sand
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containing 7, 35, 36,40, 112, 10, 2, 1, and 1 mg g-l of available NO3 N, P, K,
Mg, Ca, Fe, Mn, Zn, and Cu, respectively. Soil pH was 6.1 and organic matter
content was 0.5%. Soil was analyzed as described by Menge et al. (1978).
Vesicular-arbuscular mycorrhizal inoculum was obtained from cultures of
Glomus fusciculatum (Thaxt.) Gerd. and Trappe, G. etunicatum (Becker and
Gerd.) and G. mosseae (Nichol. and Gerd.) maintained in pots with soybean
(Gfycine mux L.). Soil-borne inoculum of each fungus was used to inoculate the
seedlings (Bethenfalvay and Yoder 1981). Ten g of soil inoculum containing
approximately 300 spores was thoroughly mixed with the upper one-third of the
container soil. The non-inoculated control treatment received 50 ml of sieved
inoculum leachate to standardize associated microflora (Menge et al. 1978). Half-
sibling Citrus jumbhiri seeds were surface sterilized with 30% Hz02 for three
minutes, rinsed, and planted in each container. The treatments were randomized
on benches with each Citrus-fungus combination replicated. Seedlings emerged
within three days.
Plants were maintained in a greenhouse in a 16-h photoperiod with natural
illumination supplemented with high intensity sodium lamps (700 pmol m-2 s-l).
Ambient temperatures ranged from 28-30 “C during the day to 20-24 “C at night.
Soil water potential was maintained near -0.1 MPa by means of gravimetric
methods. The growth medium of each seedling was supplemented weekly with 50
ml of half-strength Hoagland’s solution minus P (Hoagland and Amon 1950).
Collection of plant material
Root pressure exudate was collected at 15-day intervals, 15 to 90 days after
seedling emergence. Exudate was always collected three hours after sunrise to
avoid endogenous plant rhythms (van Staden and Wareing 1972). On the day of
collection, seedlings were covered with clear polyethylene bags to standardize
transpiration. Shoots were excised at the root collar and exudate collected through
latex tubing into glass collection tubes held on ice (Morris et al. 1976, Heindl et
al. 1982). Cross-contamination with phloem exudate was avoided by stripping
away tissue external to the xylem to a distance of 1 cm below the point of
excision. At the end of the collection period, the exudate was weighed, centri-
fuged (500 g) for 30 min, reduced to dryness by flash evaporation at 35 “C and
stored at - 20 “C.CYTOKININS IN ROOT EXUDATE OF CITRUS 11
Seedlings were harvested at 1.5day intervals after emergence. Dry weights of
roots, shoots, and leaves (dried at 80 “C for 72 h) were measured. Colonization by
VAM fungi was ascertained after clearing and staining the root systems (Phillips
and Hayman 1970). The percentage of vesicular-arbuscular mycorrhizal root
length was computed for each seedling root system by a grid intersect method
(Giovanetti and Mosse 1980). Following the study, chlamydospores of G. fuscicu-
latum and G. mosseae were identified from growth media subsamples by wet
sieving and decanting (Gerdeman and Nicholson 1963). Leaf and root samples
collected for nutrient analysis were washed to remove any residue (Smith and
Storey 1976). After digestion with perchloric acid (Mader and Hoyle 1964), P was
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analyzed in leaves and roots by means of an inductively coupled plasma spectro-
photometer (Barnes 1978). Seedling growth data were subjected to analysis of
variance.
Extraction, purijication and bioassay of cytokinins
Exudate from single plants yielded quantities of cytokinins approaching the lower
limits of detectability (< 5 ng plant-‘). Therefore, the exudate from two or three
plants was pooled before storage. Because the length of the exudate collection
period and the number of plants sampled varied, presentation of the results was
standardized by expressing cytokinin fluxes as pmol plan-’ h-l. The reported
fluxes are means of the transport rates of cytokinins and exudate.
The frozen samples were analyzed for cytokinins as described by Horgan and
Kramers (1979). The cytokinin residue was redissolved in glass-distilled water,
adjusted to pH 4.0 with 1 N HCI, partitioned against equal volumes of glass-
distilled normal butanol and water, and passed through a Dowex 5OW-X8 cation
exchange resin (H+ form, 200-400 mesh, 0.5 X 10 cm) column at a flow rate of
15 ml h-l. Cytokinins were eluted from the column with 50 ml of 5 N NHdOH.
Subsequently, the ammonium solution was flash evaporated and the cytokinin
residue redissolved in 2 ml of glass-distilled water and the pH adjusted to 4.0.
Recovery of cytokinins was determined by means of internal standards (dihydro-
zeatin, zeatin, zeatin riboside) from Sigma Chemical Company, St. Louis, MO,
USA (Horgan and Kramers 1979).
Partially purified cytokinin sample extracts were separated and quantified by
high-performance liquid chromatography (HPLC) on a Partisil 10 ODS CI~ column
as described by Horgan and Kramers (1979). Gradient elution from the reverse
phase column was accomplished with glass-distilled acetonitrile delivered by high
pressure pumps controlled by a solvent programmer. Chromatography was carried
out with a Perkin-Elmer (Norwald, CT, USA) series 3B pump equipped with 20
pl-loop injection valves, an LC-75 variable wavelength detector fitted with an
8-ml flow-through cell set at 260 nm, an LC-75 autocontrol and a Sigma IOB data
station. A chromatogram of cytokinins from Citrus jumbhiri xylem exudate is
presented in Figure 1. Fractions of selected samples were collected and the authen-
ticity of cytokinins was determined by the soybean callus bioassay (Miller 1967).
Trimethylsilyl derivatives of standards and samples were also analyzed by massDIXON, GARRETT AND COX
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Time (min.)
Figure 1. High-performance liquid chromatography of cytokinins from the root pressure exudate of
Citrus jambhiri.
spectrometry as described by Heindl et al. (1982).
Results
Significant (P = 0.05) VAM colonization of Citrus jumbhiri by all three fungal
symbionts was evident 15 days after seedling emergence (Figure 2). Seedling
colonization with G. fasciculatum or G. mosseae was approximately 70% at the
end of the study. Colonization of seedling root systems by G. etunicatum reached
Figure 2. Percent colonization of Citrus jambhiri root systems by Glomus etunicatum (Ge), Glomus
fasciculatum (Gf) and Glomus mosseae (Gm) 90 days after seedling emergence. Bars indicate
standard error.CYTOKININS IN ROOT EXUDATE OF CITRUS 13
a plateau of approximately 50% after 45 days. Identification of chlamydospores
sieved from soil samples in each VAM treatment did not reveal any cross-contami-
nation among fungal species. Control seedlings were non-mycorrhizal.
Total seedling dry weights of the VAM inoculated and non-inoculated seedlings
are presented in Figure 3. Seedlings inoculated with G. fusciculutum or G.
mosseae grew rapidly throughout the 90-day period. After 45 days, the non-
inoculated plants and those inoculated with G. etunicatum were significantly (P =
0.05) smaller than seedlings in the other treatments. The increase in total seedling
dry weight was associated with increases in P concentrations of leaves and roots
(Table 1). Plants inoculated with G. fusciculatum or G. mosseae had significantly
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(P = 0.05) greater concentrations of tissue P than those that were either not
inoculated or inoculated with G. etunicatum.
Figure 3. Mean dry weight of Citrus jumbhiri inoculated with Glomus etunicatum (Ge), Glomus
fusciculatum (Gf) and Glomus mosseae (Gm) 90 days after seedling emergence. Bars indicate
standard error.
Table 1. Phosphorus concentration (%) in leaves and roots of Citrus jambhiri seedlings inoculated
with three vesicular-arbuscular mycorrhizal (VAM) fungi, 4, 8 and 12 weeks after seedling
emergence.
VAM fungi Week
4 8 12
Leaf Root Leaf Root Leaf Root
G. mosseae 0.14a’ 0.14a 0.19a 0.16a 0.21a 0.17a
G. etunicatum 0.12a 0.13a 0.18a 0.16a 0.20a 0.17a
G. fasciculatum 0.12a 0.14a 0.18a 0.16a 0.20a 0.16a
Non-inoculated control 0.05b 0.06b 0.08b 0.08b 0.08b 0.07b
i Values in each column not followed by a common letter are significantly different (P = 0.05).14 DIXON, GARRETT AND COX
The patterns of cytokinin flux throughout the early development of C. jumbhiri
were similar in all seedlings (Figures 4, 5 and 6). An initially low flux slowly
increased with an increase in plant size. The flux of root pressure exudate did not
always coincide with the flux of cytokinins. However, cytokinin fluxes varied
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30 60 90
DAYS AFTER EMERGENCE
Figure 4. Mean flux of zeatin riboside and of root pressure exudate of Citrus jambhiri root systems
inoculated with Glomus etunicatum (Ge), Glomus fasciculatum (Gf), Glomus mosseae (Gm) and non-
inoculated control 90 days after seedling emergence. Bars indicate standard error.
Exudate
Gm P
Gf 6 Gc f
DAYS AETER EMERGENCE
Figure 5. Mean flux of zeatin and of root pressure exudate of Citrus jambhiri root systems inoculated
with Glomus etunicatum (Ge), Glomus fasciculatum (GO, Glomus mosseae (Gm) and non-inoculated
control 90 days after seedling emergence. Bars indicate standard error.CYTOKININS IN ROOT EXUDATE OF CITRUS 15
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Figure 6. Mean flux of dihydrozeatin and of root pressure exudate of Cirrus jumbhiri root systems
inoculated with Glomus etunicatum (Ge), Glomus fasciculatum (Gf), Glomus mosseae (Gm) and non-
inoculated control 90 days after seedling emergence. Bars indicate standard error.
significantly (P = 0.05) with the VAM association of the seedling.
The zeatin, dihydrozeatin and zeatin riboside fluxes of the non-inoculated seed-
lings were significantly lower than those of seedlings inoculated with G. fusciculu-
turn or G. mosseae (Figures 4, 5 and 6). Cytokinin flux of seedlings inoculated
with G. etunicatum was generally lower than that of seedlings inoculated with G.
fusciculutum or G. mosseue. The flux of zeatin riboside was significantly (P =
0.05) greater than the flux of corresponding zeatin in seedlings inoculated with
VAM fungal symbionts (Figures 4 and 5). Dihydrozeatin flux of seedlings inocu-
lated with G. fusciculutum was nearly twice that of plants that were either not
inoculated or inoculated with G. mosseue or G. etunicutum (Figure 6).
Analysis of selected zeatin and zeatin riboside samples by mass spectrometry
(MS) verified that the HPLC procedures utilized for isolation and analysis were
reliable. Mass spectra of trimethylsilyl zeatin riboside samples contained ions
(relative intensities in parenthesis) of m/z 639 (M+, 3.5), 624 (4.2), 550 (6.0),
536 (9.7), 319 (5.0), 230 (9.4), 202 (19.3) and 73 (TMS, 100). Trimethylsilyl
zeatin samples contained ions and relative intensities of m/z 261 (M+ , 13.0), 230
(83.8), 216 (16.5), 188 (38.4), 162 (5.0), 135 (5.2), 133 (6.2) and 73 (TMS, 100).
Ions with these m/z ratios were also characteristic of trimethylsilyl zeatin and
zeatin riboside standards and previous reports of zeatin and zeatin riboside mass
spectra (Morris et al. 1976, Heindl et al. 1982). Samples of dihydrozeatin were
not analyzed by MS because of the relatively small quantities recovered at any
sample date. Analysis of selected cytokinin fractions by the soybean bioassay
further confirmed our chromatographic and mass spectral results (Figure 7).DIXON, GARRETT AND COX
2 DHZ ZR
-cb---4-
1.0
. & d
IO IO
I :“:”
TIME (MIN) TIME (MIN) TIME (MIN)
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G. mOSSeae G. fasiculatum G. etunicatum Zeatin (pg 1-j)
Figure 7. Cytokinin activity of root pressure exudate from VAM-inoculated and non-inoculated Citrus
jumbhiri as determined by soybean callus bioassay after high-performance liquid chromatography.
Cross hatched areas represent activity significantly different from the control (P = 0.05). ZR, zeatin
riboside; Z, zeatin; DHZ, dihydrozeatin.
Discussion
We have demonstrated the presence of cytokinins in the xylem exudate of Citrus
jumbhiri and shown that significant changes in cytokinin flux occur during seed-
ling development (cf. Morris et al. 1976, Horgan and Wareing 1980). The flux of
cytokinins in the xylem exudate of seedlings generally increased with increases in
plant size and VAM development. This finding is consistent with the observation
that acropetal transport of cytokinins to terminal buds and developing leaves
stimulates growth in dicotyledons (Mozes and Altman 1977, Menary and van
Staden 1978, Palmer et al. 1981, Hutton and van Staden 1983). In Citrus, changes
in cytokinin flux with seedling development were not closely related to changes in
root exudate flux. A lack of correlation between root exudate flux and cytokinin
flux has also been reported for annual dicotyledons (Davey and van Staden 1976,
Heindl et al. 1982).
The cytokinin content of xylem exudate was significantly altered by VAM
colonization of seedlings and the extent of the change was dependent on symbiont
species. The flux of dihydrozeatin differed significantly between seedlings inocu-
lated with G. fusciculutum and those inoculated with G. mosseae even though
both groups of seedlings had similar total dry weights and P nutrition. Changes in
cytokinin activity in roots and leaves of Citrus jumbhiri as a result of VAM
colonization have been reported previously (Dixon et al. 1988). The significantly
greater xylem flux of cytokinin in VAM Citrus jumbhiri seedlings compared with
non-mycorrhizal seedlings is consistent with reports of in vitro production of
zeatin and zeatin riboside by mycorrhizal fungi (Slankis 1973, Crafts and Miller
1974, Barea and Azcon-Aguilar 1982). Although it cannot be assumed that the
cytokinin flux from the whole root system is a reflection of either VAM fungi
cytokinin content or export, there is circumstantial evidence of phytohormone
transport from ectomycorrhizal fungi to host plant. For example, exudates ofCYTOKININS IN ROOT EXUDATE OF CITRUS 17
Suillus edulis, which contain IAA- and gibberellin-related compounds and a
cytokinin, induced morphological changes in the roots of Scats pine (Pinus
sylvestris L.) similar to those that occur during formation of mycorrhizal roots
(Slankis 1973).
The root-to-shoot flux of zeatin riboside in Citrus was generally greater than the
fluxes of other cytokinins in seedlings inoculated with VAM fungi. Even when
concentrations of cytokinins in the exudate were low, the ribosides were usually
present in greater quantities than the free bases. Zeatin riboside is the major
transport form of cytokinins in other plant genera including Acer (Horgan and
Wareing 1980), Glycine (Heindl et al. 1982), Lupinus (Davey and van Staden
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1978), Lycopersicon (Carnes et al. 1975, Davey and van Staden 1976), and
Populus (Hewett and Wareing 1973). The significance of the high ratio of ribo-
sides to free bases in Citrus seedling xylem exudate is unknown, although it has
been shown that, in Lycopersicon esculentum Mill., the ribose moiety, which
increases zeatin solubility, faciiitates xylem transport of cytokinins (van Staden
and Dimalla 1977).
Acknowledgments
The financial assistance of Allied Corporation is gratefully acknowledged. The authors thank Drs.
Kalus 0. Gerhardt and Roy H. Rice for providing facilities and for conducting the GS-MS analyses.
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