J. Plant Pbysiol. Vol. 144. pp.

45 -48 (1994)

Endogenous Levels of ABA and Cytokinins and their

Relation to Stomatal Behavior in Dayflower (Commelina
communis L.)

YONG Y. WANG 1, RONG ZHOU2, and XIE ZHOU3

1 Forestry Department, Nanjing Forestry University, Nanjing 210037, PRC
2 Basic Course Department, Nanjing Agricultural University, Nanjing 210095, PRC
3 Agronomy Department, Nanjing Agricultural University, Nanjing 210095, PRC

Received August 27,1993 . Accepted January 24,1994

Summary

During adventitious root formation in dayflower, the ABA level diminished gradually, and cytokinin
(mainly isopentenyladenosine and zeatin riboside) levels in the abaxial epidermis and the transpiration
rate of leaves increased significantly. However, root excision resulted in the increase of ABA levels and
the reduction of cytokinin levels, and the transpiration rate also decreased dramatically. The stomatal be-
havior of dayflower cuttings induced by the existence or excision of adventitious root can be explained
by the concentration and ratio of endogenous cytokinin and ABA in abaxial epidermis.

Key words: Stomatal behavior, ABA, cytokinins, commelina.

Abbreviations: ABA '" abscisic acid; CTK - cytokinin; BHT = butylated hydroxy-toluene; ELISA =
enzyme linked immunosorbent assay; iPA = isopentenyladenosine; ZR = zeatin riboside; DHZR = di-
hydrozeatinriboside; BSA = bovine serum albumin; OVA = ovalbumin; PBS '" phosphate buffer solu-
tion; SE = standard error.

Introduction opening and the increase of transpiration rate, even though

It is generally accepted that stomatal movement of plants
influences the rate of gas exchange, which in turn affects
transpiration as well as photosynthesis and respiration. The
mechanism of stomatal movement is complicated. The con-
ventional view of the stomatal response to soil drying is that
as water supply is restricted, leaf water potential and turgor
decline, eventually promoting stomatal closure. There is
now a considerable amount of evidence that soil water defi-
cit can also induce stomatal closure even when leaf water sta-
tus does not change (Black mann and Davis, 1985; Gollan et
al., 1986). One explanation for this is that root-derived ABA
in response to soil drying is the primary mediator of sto-
matal closure (Zhang et al., 1987; Zhang and Davis, 1989).
Xu and Lou (1980) have found that the adventitious roots
produced from sweet potato cuttings promote stomatal
the root does not touch a layer of water, while root excision
induces stomatal closure and the reduction of transpiration
rate. In this circumstance, the stomatal responses to root ex-
istence or excision can not be explained only by root-derived
ABA.
Early speculation that soil drying would reduce CTK syn-
thesis in roots and its transport to leaves (ltai and Vaddia,
1965) has been supported by some experimental data show-
ing that soil drying reduces CTK synthesis in roots (Cf: Da-
vis et al., 1987), kinetin-like activity in root exudate (ltai and
Vaddia, 1965) and CTK content in leaves (Davis et al., 1986).
These responses imply that there may be a link between root
physiology and stomatal behavior. In this study, we report
on the effect of adventitious root existence or excision on
the levels of endogenous ABA- and CTK-like immunoreac-
tivity and the stomatal behavior of Commelina.

© 1994 by Gustav Fischer Verlag. Stuttgart

 46 YONG Y. WANG, RONG ZHOU, and XIE ZHOU

and their linear measuring ranges were all 0.064-200pmol. The

within-plate variation coefficient was from 2.0 % to 2.8 % and
that between plates was from 1.06 % to 3.4 %. Sample extract dilu-
tion curves were parallel with standard curves in ELISAs.
The crude extract was passed through a CIS Sep-pak cartridge to
Stem remove the pigments. The efflux was collected and then 600 J-IL of
Cork this solution was taken out and dried with a stream of
N 2; the residue was dissolved in 400 J-IL PBS (Om mollL, pH 9.2),
Plastic bag the pH adjusted to around 8.0 with 0.1 N HCL and extracted three
times with an equal volume of 85 % water-saturated n-butanol. The
Adventitious butanol extracts were pooled and dried with a stream of N 2 and re-
root dissolved in 600 J-IL PBS (0.01 mollL, pH 7.4) for determining the
Glass rack levels of iPAs, ZRs and DHZRs, respectively.
The flowchart described by Ross et al. (1987) was used in the
CTK assay with minor modifications. All samples were assayed in
Distilled water triplicate .
.. - .

Fig. 1: The device used for the culture of stem segments of Com-
melina. Measurement ofstomatal aperture
The stomatal aperture was expressed as transpiration rate accord-
ing to the method reported by Munns and King (1988).
Materials and Methods

Plant materials
Results
The dayflower stem segments of 8 cm long bearing one leaf were
detached in water and their lower ends immersed in distilled water Effect of the existence or excision of adventitious roots on
in a glass tube (2 cm inner diameter and 10 cm long). The experi-
foliar transpiration rate.
mental device is shown in Fig. 1. The plastic bag must be used in or-
der to keep the adventitious roots away from a layer of water. Ten It was found that the transpiration rate of detached leaves
segments per treatment were used and root excision was conducted of the cuttings decreased day by day until the appearance of
7 days after incubation. The abaxial epidermal strips (ca. 200 mg) adventitious roots, then increased significantly with elonga-
were harvested and then ground under liquid nitrogen and ex- tion of roots even when these roots had not been touching
tracted with 3 mL 80 % methanol containing 10 mg/L BHT at 4 °C the layer of water. On the contrary, it diminished dramat-
for 48 h. The supernatant was collected after centrifugation at ically after root excision (Fig. 2). These results indicated that
10,000 x g for 30 min at 4°C; the residue was retained and extracted the formation of adventitious roots promoted stomatal
again with 1 mL of the same solvent at 4 °C for 24 h and centrifuged opening, while root excision induced its closure.
again at the same conditions. The second supernatant was combined
with the first one.
Effect of the existence and excision of adventitious roots
on endogenous ABA level in abaxial epidermal strips.
The results in Fig. 3 show that ABA level in the abaxial
Determination ofABA content epidermal strips from leaves of dayfIower decreased gradu-
An indirect ELISA (Zhang et al., 1991) was used. The procedure
used to purify and fractionate ABA from the crude samples was 0.6
based on that described by Weiler (1986). C 0
'" Control ,,
'"E 0.5
Root
y ,
~.cision \
,

'.
Determination of CTK contents. ~
*
\

Three ELISAs for iPAs, ZRs and DHZRs have been developed in -..
C\
O.L A
E
the plant hormone lab of Nanjing Agricultural University (Chen '" 0.3
and Zhou, unpublished). According to the method of NaI0 4 oxi- c
dation described by Weiler (1980), iPA, ZR and DHZR were conju- -0
gated, respectively, to BSA as immunogen or OVA as immobilized '"
ii
0.2

antigen. Rabbits (about 2 kg) were immunized with an emulsion of 'c" 0 .1

the conjugates (iPA-N 9-BSA, ZR-N9-BSA and DHZR-N9-BSA, re- ~'"
spectively) in PBS and Freund's complete adjuvant, as described
previously (Hurn and Chantler, 1980). Sera were mixed with an 0
0 2 3 4 5 6 7 8
equal volume of glycerol and stored at - 20°C. Anti-iPA, -ZR and Incubation days
-DHZR antisera basically recognized iPA-group CTKs (iPAs), ZR-
group CTKs (ZRs) and DHZR-group CTKs (DHZRs). Fig.2: Effect of adventitious root formation and root excision on
The basic parameters of the 3 indirect ELISAs developed for the foliar transpiration rate of Commelina. A, B, C, D and E stand
iPAs, ZRs and DHZRs were as follows: the working concentrations for the different sampling times of determining ABA and CTK lev-
of anti-iPA, -ZR and -DHZR antisera were 1: 10,000, 1: 6000 and els. The no-tail, one-tail and two-tail arrow marks in Figs. 2, 3 and 4
1 :2000, respectively. The coating amount of iPA-N9"OVA, ZR-N9" represent the time of adventitious root appearance, formation of
OVA and DHZR-N9"OVA was 0.08, 0.11 and 0.13 J-Ig/well, respec- many roots and root excision, respectively. Averages ± SE of 3 rep-
tively. The detection limits of the 3 ELISAs were 4, 0.4 and 1 fmol, licates.
 ABA and CTK regulating stomatal behavior 47

12 Discussion
Control IT
The nature of any chemical signal moving from roots to
Root excision
o leaves in response to soil drying has been subject of much in-
E terest during the last 25 years (Zhang and Davis, 1989). Some
c:
evidence denotes that ABA plays a central role in root-to-
.'"
c:
III
shoot communication, and may convey effects of soil drying
and then control stomatal behavior (Zhang et al., 1987;
>

..
~
c:r
«
m the sap of wheat plants was not sufficient to account for the
«
Incubation days
Fig. 3: Effect of adventitious root formation and root excision on
the level of ABA in abaxial epidermis of Commelina. Averages ± SE
of 3 replicates.
ally during the formation and growth of adventitious roots
and then showed only a small increase at day 8. However,
root excision caused a marked rise in ABA level, which was
203 % of that in control plants (Fig. 3). It implied that the
significant increase of ABA level in the abaxial epidermal
strips after root excision was closely correlated to stomatal
closure and the reduction in foliar transpiration rate.
Effect of the existence and excision of adventitious roots
on endogenous CTK (iPAs, ZRs and DHZRs) levels in ab-
axial epidermal strips. . presented preliminary data that soil drying reduces the cyto-
Figure 4 illustrates that the total amount of CTKs lll-
creased considerably after adventitious root appearance and
were maintained at a higher level for several consecutive
days. However, root excision caused a marked. d~crease in
CTK levels. Of three cytokinins tested, the vanation of en-
dogenous iPA and ZR contents were very evident, but
DHZR levels showed no significant fluctuation (Fig. 4).
These results indicate that root-derived cytokinins, mainly
iPAs and ZRs, participated in the regulation of stomatal be-
havior of dayflower.
Co ntr o l
u. 28 Roo t
Zhang and Davis, 1989). However, Munns and King (1988)
and Munns (1990) showed that the concentration of ABA in
inhibitory effect of the xylem sap on stomatal conductance,
with an explanation that ABA was not the only antitranspi-
rant. The results in this paper (Fig. 3) indicate that the sto-
mata of dayflower respond to increased epidermal ABA con-
tent, which is consistent with previous reports (Zhang et aI.,
1987; Davis et al., 1987), but ABA accumulated in abaxial
epidermal strips after root excision may come from chloro-
plasts (Cornish and Zeevaart, 1984), mesophyll cytoplasm
(Hartung et al., 1981) or cytoplasm of guard cells (W~il.er et
aI., 1982; Cornish and Zeevaart, 1986). Thus, the onglll of
ABA in Commelina abaxial epidermis controlling stomatal
closure is complex in stress conditions, for it may come from
the leaves (Fig. 3; Kriedemann et aI., 1972; Munns and King,
1988) as well as the roots (Zhang et aI., 1987; Davis et aI.,
1987).
Little progress has been made in the study of the effect of
endogenous cytokinins on stomatal behavior perhaps be-
cause of the assay's technical difficulty. Davis et ai. (1986)
kinin contents in leaves. This paper is the first report that
the presence of roots, which is a prerequisite for normal
function of stomata (Xu and Lou, 1980), can raise cytokinin
levels in the abaxial epidermal strips of Commelina and fur-
thermore that root excision can reduce cytokinin levels (Fig.
4). This implied that cytokinins also controlled stomatal be-
havior as supported by the report of stomatal responses to
applied cytokinins (Blackmann and Davis, 1983) in addition
to ABA (Zhang et aI., 1987; Davis et aI., 1987).
Of three cytokinins tested, the variation of iPA and ZR
levels induced by root existence or excision is very pro-
nounced; however, there is little fluctuation in DHZR con-
tent. This may mean that DHZR has little effect on stomatal
behavior. More work in this area is clearly required.

o
eXCISion
r Plant growth and development are controlled by several
plant hormones in a complicated way (Leopold, 1982; Wang
E 21
c: and Zhou, 1991). Blackmann and Davis (1984) have found
that the variation of stomatal aperture depends on the con-
.. '4
c
to
centration of applied ABA and Zeatin. The interaction be-
> tween the increased ABA (Fig. 3) and decreased cytokinin
..
~
c:r
(Fig. 4) in abaxial epidermal strips of Commelina after root
excision appears to be the main factor resulting in stomatal
closure and the reduction of transpiration rate (Fig. 2). On
o o~Z~R~==~======~~======~~~~I~~ the other hand, from the experimental data (Figs. 3 and 4) it
2 5 6 8 was calculated that the ratio of cytokinin and ABA in ab-
IncubatIon days axial epidermis strips was about 2.2 before adventitious root
Fig.4: Effect of adventitious root formation and ~oot excision on appearance, was elevated to about 7.5 after root formation
the level of CTKs in abaxial epidermis of Commehna. T stands for and remained at a higher level up to root excision; then it
the total amounts of iPAs, ZRs and DHZRs. Averages ± SE of 3 was lowered apparently down to about one tenth of that in
replicates. the control at the same period. This means that the ratio be-
48 YONG Y. WANG, RONG ZHOU, and XIE ZHOU
tween these two hormones is also involved in the regulation
of stomatal behavior in addition to their concentrations,
which is supported by other evidence. For instance applied
cytokinins can override the influence of ABA or soil drying
on maize stomatal closure (Blackmann and Davis, 1983;
1985).
Acknowledgements
We are grateful to Prof. E. W. Weiler for participation during the
initiation of this experiment, preparation of glossy prints and help-
ful suggestions. We also thank Prof. X. D. Xu, for constructive
comments and suggestions.
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