Journal of Experimental Botany, Vol. 56, No. 413, pp. 1017–1028, March 2005
doi:10.1093/jxb/eri095 Advance Access publication 14 February, 2005
RESEARCH PAPER
Opposite patterns in the annual distribution and
time-course of endogenous abscisic acid and
indole-3-acetic acid in relation to the periodicity of cambial
activity in Eucommia ulmoides Oliv.
Kalima-N’Koma Mwange1,2,*, Hong-Wei Hou1,*, You-Qun Wang3, Xin-Qiang He1 and Ke-Ming Cui1,†
1
Peking University, College of Life Sciences Beijing 100871, PR China
2
Commissariat Général à l’Energie Atomique, Centre Régional d’Etudes Nucléaires de Kinshasa
(CGEA/CREN-K), B.P. 868, Kinshasa XI, Rép. Dém. du Congo
3
China Agricultural University, College of Biology, Beijing 100094, PR China
Received 23 November 2004; Accepted 3 December 2004
Abstract
The seasonal change of free abscisic acid (ABA) and
indole-3-acetic acid (IAA) and their relationship with
the cambial activity in Eucommia ulmoides trees were
investigated by ABA and IAA immunolocalization using
primary polyclonal and rhodamine-red fluorescing secondary antibodies, ABA and IAA quantification using
high performance liquid chromatography (HPLC), and
systematic monitoring of vascular cell layers production. ABA and IAA clearly displayed opposite annual
distribution patterns. In the active period (AP), both
immunolocalization and HPLC detected an abrupt
decrease of ABA, reaching its lowest level in the
summer. During dormancy, ABA started increasing
in the first quiescence (Q1) (autumn), peaked in the
rest (winter), and gradually decreased from the onset
of the second quiescence (Q2) (the end of winter). IAA
showed a reverse pattern to that of ABA: it sharply
increased in AP, but noticeably decreased from the
commencement of Q1. Longitudinally, the ABA distribution increased apico-basally, contrasting with IAA.
Laterally, most of the ABA was located in mature
vascular tissues, whereas the IAA essentially occurred
in the cambial region. The concomitant IAA–ABA
distribution and seasonal changes in vascular tissues
greatly correlated with xylem and phloem cell production, and late wood differentiation and maturation.
Interestingly, the application of exogenous ABA to
quiescent E. ulmoides branches, in a water-culture
system, inhibited external IAA action on cambial activity reactivation. These results suggest that, in
E. ulmoides, ABA and IAA might probably interact in
the cambial region. The annual cambial activity could
be influenced by an IAA:ABA ratio; and ABA might
play a key role in vascular cambium dormancy in
higher plants. The relationship between hormonal
changes and the (particular) annual life cycle of
E. ulmoides is also discussed.
Key words: ABA, annual periodicity, cambial region, dormancy,
Eucommia ulmoides, IAA, vascular tissues.
Introduction
The vascular tissues are among the most intensely
studied plant tissues (Catesson et al., 1995; Lachaud et al.,
1999; Ye, 2002). However, because of the high complexity
and diversity in the plant kingdom (Fahn and Werker, 1990),
many phenomena that occur during secondary growth in
higher plant species remain poorly understood. The annual
fluctuations in cambial activity are generally related to the
alternation of cold and warm seasons or dry and rainy
seasons (Lachaud et al., 1999). During these seasonal
fluctuations, several changes occur in the cambial cells, for
example, in their structure and biochemistry (Riding and
Little, 1984; Mellerowicz et al., 1992), cytoplasm (Farrar
* These authors contributed equally to this study.
y
To whom correspondence should be addressed. Fax: +86 10 62751526. E-mail: [email protected]
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1018 Mwange et al.
and Evert, 1997; Yin et al., 2002), nuclei (Mellerowicz et al.,
1993; Llyod et al., 1996; Yin et al., 2002), and cytoskeletoncell wall continuum (Chaffey et al., 1997; Bourquin et al.,
2002). Most of these changes are internally regulated by
phytohormones (Sundberg and Uggla, 1998; Wang and Cui,
1999), including both growth instigators (i.e. indole-3-acetic
acid [IAA] and gibberellic acid [GA]) and inhibitors (i.e.
abscisic acid [ABA] and ethylene).
Seasonal changes in the cambial activity in perennial
plants have often been attributed to modifications in the
concentrations of phytohormones in association with environmental conditions. As a matter of fact, the IAA and GA
concentrations are found to be highest in tissues in which
cell division is heavily active (MacMillan, 2002). With
improved biochemical and immunological techniques, the
proposed direct link between the auxin concentration and
cambial activity (Wareing et al., 1964) is currently being
verified. The findings by Uggla et al. (1996, 1998), and
Tuominen et al. (1997), which clearly established a correlation between IAA gradient widths and cambial activity,
have been particularly informative.
In the same manner, the role of ABA in periodic cambial
activity is gradually being elucidated. Two to three decades
ago, reports by Jenkins and Shepherd (1974) on Pinus
radiata, Dumbroff et al. (1979) on Acer saccharum, and
Little and Wareing (1981) on Picea sitchensis have
stated that the ABA level in tree stems was at its minimum
just before cambial reactivation, low during the cambial
active period, and increased during the formation of late
wood at the beginning of dormancy. However, these studies
have only covered the active period and did not examine
in detail the ABA quantification and distribution pattern in
the vascular tissues of these plants.
On the other hand, there is growing evidence that
phytohormones, rather than acting separately, interact with
each other co-ordinately to regulate cellular processes in
plants. The cross-talk of IAA with other phytohormones
has been convincingly demonstrated in the last decade
(see review by Swarup et al., 2002). However, information
on the relationship between IAA and ABA has been
mainly limited to the regulation of stomatal aperture
(Armstrong et al., 1995), bud dormancy (Moore et al.,
1995), root growth (Wilson et al., 1990), programmed
cell death in maize endosperm (Young and Gallie,
2000), and seed dormancy and germination (Corbineau
and Côme, 2002; Fellner et al., 2001; Zeng et al., 2003). The
role of IAA and ABA in the vascular cambium of temperate
woody species, particularly in the dormancy phases (quiescence and rest), has not been widely investigated.
This study, conducted during the active and dormant
(quiescence–rest) periods, aimed at evaluating, quantitatively and qualitatively, the annual variations of IAA and
ABA levels and determining whether a relationship exists
between their distribution and anatomical changes in the
vascular tissues of the Eucommia ulmoides trees.
Materials and methods
Plant material, sampling, and sample treatment
The study was conducted for one year between January and
December. Healthy 20-year-old trees of E. ulmoides growing on
the campus of Peking University (Beijing, China) were used. At each
time point, samples from four different trees were harvested at three
tree levels (Fig. 1A): the top, consisting of 2–3-year-old small twigs
on the highest branches; the middle, located on the main stem just
above the lowest branches; and the base, at 50 cm above the ground.
For IAA and ABA quantification, three fractions of cell layers
were carefully excised with a clean sharp knife, and consisted of
the cambial region, mature xylem, and mature phloem fractions
(Fig. 1B). As revealed by light microscopy observations, the
cambial region comprised the cambium, immature phloem, and
immature xylem cells. Here, the term ‘immature cell layers’ includes
all cells located between the maturing xylem and maturing phloem as
illustrated by Lachaud et al. (1999). Mature xylem and phloem
contained lignified tracheary and sieve elements, respectively. The
harvested samples (2–2.5 g for each fraction) were collected
separately in 2.5 ml plastic vials and immediately immersed in
liquid nitrogen for at least 1 h before storage at ÿ70 8C until further
analysis.
For IAA and ABA immunolocalization, tissue blocks of about
0.5 cm3 were excised from the same tree levels with a sharp knife and
included tissues located between the mature phloem and mature
xylem (Fig. 1B). Upon their removal from the tree, samples were
prefixed, post-fixed, embedded, and sectioned as previously described by Mwange et al. (2003a).
IAA and ABA immunolocalization
The immunolocalization procedure and sections observation, including negative and positive controls, were performed following
Mwange et al. (2003a). The primary antibodies used were polyclonal
anti-IAA and anti-ABA antibodies raised in rabbits against carboxyllinked IAA and ABA, respectively (China Agricultural University,
Beijing, China). The cross-reactivity of these antibodies was
Fig. 1. Sampling locations of E. ulmoides tissues in the tree (A) and in
the vascular tissues (B). (A) Top, 2–3-year-old small twigs on the highest
branches; middle, just above the lowest branches; base, 50 cm above the
soil. (B) The vascular tissue was divided into three fractions: mature
phloem (MP), the cambial region (CR), and mature xylem (MX). Crosssection (B), bar=50 lm.
ABA and IAA distribution in Eucommia vascular tissues 1019
measured by an enzyme-linked immunosorbent assay (ELISA). The
compounds tested for competition with immobilized IAA–BSA
included a series of indole derivatives, tryptophol, and DL-tryptophan,
and for ABA–BSA comprised isomers of farnesol, xanthoxin, and
(–)enantiomers of ABA. These substances all showed <2% and <0.1%
cross-reactivity, respectively, with IAA and ABA. The secondary
antibody used was a rhodamine-red-conjugated goat-anti-rabbit
antibody (Zhongshan Corp. Ltd, Beijing, China).
IAA and ABA analysis and quantification
Free IAA and ABA extraction, purification, and measurement using
HPLC was conducted as described earlier (Mwange et al., 2003a).
Each purified aliquot of IAA and ABA was analysed three times.
Counting of cambial cell layers
Tissue blocks for anatomical observation were fixed in formalin–
alcohol–acetic acid (FAA). The blocks were then dehydrated in an
alcohol series and embedded in paraffin wax. Sections (9–10 lm
thick) were cut with a rotary microtome and stained with Delafield’s
haematoxylin–safranin as described by Cui and Wang (1997).
Cambial growth was measured by counting the radial cell layers of
the cambium as well as the immature and mature phloem and xylem
according to Sundberg et al. (1991). Cell layers were counted in six
tissue sections obtained from each tree level.
ABA and IAA application in a water-culture system
E. ulmoides 1-year-old quiescent (Q2) branches were harvested in
early February. Following complete manual removal of the buds, the
branches were divided into eight groups of five branches and treated as
follows: lanolin alone, IAA alone, IAA+10 nM ABA, IAA+1000 nM
ABA, IAA+10 000 nM ABA, 10 nM ABA alone, 1000 nM ABA
alone, and 10 000 nM ABA alone. The water culture system was set up
in a growth chamber with a 16 h photoperiod, light of 12 000 lx,
temperatures of 24/17 8C (day/night), and a relative humidity of
7567%. ABA (from Jiangsu Shuanggou Co, China) was added to the
water in 150 ml glass bottles and IAA (from Gibco) was applied (1 mg
IAA gÿ1 lanolin) to the excised tops of branches according to Luo
et al. (1995) and Cui and Luo (1996). The ABA and IAA application
was refreshed weekly. After 2 weeks, small blocks of vascular tissues
were excised from the branches, fixed in a solution of 4% paraformaldehyde and 1% glutaraldehyde, dehydrated in an alcohol series,
embedded in LR white resin (sigma), cut into 4 lm sections, stained
with toluidine blue O (TBO), and observed under a Zeiss Axioskop 2
Plus microscope (Germany) equipped with a computer-assisted digital
camera. Sections from branches that were freshly harvested on the
sampling day were fixed and embedded as above for use as controls.
Statistics
The measured and calculated parameters (IAA and ABA content,
number of cell layers) were statistically analysed with one-way
ANOVA. Four to six replicates were performed, and the means were
tested by LSD at P=0.05 using the SAS software package. Data from
each sampling date were analysed separately.
Results
Seasonal distribution of IAA and ABA in E. ulmoides
vascular tissues
The immunolocalization technique used revealed a clear
seasonal pattern in the distribution of IAA and ABA in
E. ulmoides vascular tissues. Traces of free IAA were
almost not detected in samples harvested in December at all
the tree levels (Fig. 2A1–3). From mid-February, a weak but
steady IAA signal was clearly observed in the cambial zone
at the tip of the tree (Fig. 2B1), but was very slightly
perceptible in sections of samples from the middle (Fig.
2B2) and extremely low in the base (Fig. 2B3) of the tree.
By late February, sections from the middle and base levels
exhibited stronger IAA labelling (Fig. 2C1–2) than observed
previously. Nevertheless, the cambial zone at the base level
remained faintly stained (Fig. 2C3). In early March (Fig.
2D), the free IAA signal in all three tree levels had
increased, but decreased in intensity from the top toward
the bottom of the tree (top > middle > base) (Fig. 2D1–3).
The IAA staining became more consistent in mid-March
(Fig. 2E1–3) and was very high by the last 10 d of March (Fig.
2F1–3). Around early April (Fig. 2F1–3), the intensity of IAA
staining was similar in all three levels, with staining in the
cambial zone slightly greater than in the mature xylem and
phloem layers. This IAA distribution pattern was maintained until late July (results not shown). By late October
(Fig. 2G), the IAA traces had decreased selectively, not
only in the different tree levels (top < middle < base) but
also in the different tissues (cambial region > mature xylem
and phloem).
In contrast to IAA, the ABA signal (Fig. 3) was more
intensely visualized in tissue sections of samples harvested
in winter (December) (Fig. 3A). By late February, the ABA
staining had visibly decreased (Fig. 3B). Samples taken in
spring (Fig. 3C) and summer (Fig. 3D) had the lowest ABA
signal, as shown by weaker rhodamine red fluorescence in
the vascular tissues investigated. Later, in September (Fig.
3E), the ABA signal slowly increased with the approach of
winter.
Interestingly, ABA also showed non-uniform staining in
the different tree levels and vascular tissues. The levels of
ABA increased from the top of the tree to the base level
(base > middle > top) at all sampling dates (Fig. 3A1–3–E1–3).
In the vascular tissues, most of the ABA visualized in each
sample section was in the xylem (MX), followed by the
phloem (MP) tissues. The cambial region (CR) was faintly
stained in sections collected in mid- and late winter (Fig.
3A1–B3) as well as in late autumn (Fig. 3E1–3). Furthermore,
the CR remained almost unlabelled during the spring (Fig.
3C1–3) and summer (Fig. 3D1–3).
Seasonal variations in the ABA and IAA content in
E. ulmoides vascular tissues
There were noticeable variations in the ABA and IAA
content in the mature phloem and xylem as well as in the
cambial region at the top, middle, and base levels of the
E. ulmoides tree (Fig. 4). Free ABA was detected in samples collected on all dates, from January to December, in
all of the tree levels and vascular tissue layers (Fig. 4).
The ABA content decreased from January to April and
May. This decrease was initially slow (January–February),
1020 Mwange et al.
Fig. 2. Seasonal IAA distribution in E. ulmoides vascular tissues as revealed by fluoro-immunolocalization. Tissues harvested during the rest period in
winter (A, 26 December) did not display a high rhodamine-related IAA signal. In the quiescence-2 phase (B, 18 February), an IAA signal first appeared
in the cambial region (CR) at the top of the tree (B1), but not at the middle (B2) or base (B3) levels. From the onset of the active period (C, 24 February),
IAA was detected not only at the top (C1), but also less intensely at the two lower levels (C2, C3). During the active period in spring (D, 10 March; E, 21
March; F, 1 April), all three locations on the tree (1, 2, and 3) exhibited IAA signals of different longitudinal (top > middle > base) and radial (MP < CR >
MX) intensities. During the quiescence-2 developmental phase in autumn (G, 20 September), a significant reduction in the intensity of the IAA signal
was detected at all three levels (G1, G2, G3). Cross-sections, bar=80 lm.
became more rapid from around early March to May, and
reached the lowest level between June and July. A gradual
increase in ABA concentration was then observed beginning late August, indicating that the hormone was probably
being actively synthesized from that period.
The ABA contents in the different levels of E. ulmoides
trees indicate that the most ABA accumulated at the base
level, followed by the middle level (Fig. 4). The tip of the
tree had the lowest ABA content. Among the different
vascular tissues, the mature xylem contained the highest
amounts of ABA over the year, except in December and
January, when the ABA content of mature xylem and
mature phloem was not significantly different. In addition,
the ABA content of mature phloem was higher than that of
the cambial region at every sampling time.
Free IAA production was first detected in the top level of
the tree at the end of February. By contrast with ABA, the
IAA content strongly increased beginning in March, and
was detected in the cambial region and mature xylem in
both the middle and base tree levels. However, in mature
phloem, this phytohormone was detected for the first time
in early and late March in the middle and base levels,
respectively. The IAA content of the whole tree peaked
between May and July. Thereafter, it drastically declined
during August and, from the second half of November to
early February, the extraction procedure used did not allow
the detection of the weak free IAA content, that is required
for maintaining the meristem identity during the resting
time.
On average, at each sampling date between late February
and June in the three levels of the tree, significantly more
IAA accumulated in the cambial region than in the mature
phloem and mature xylem. Interestingly, between late July
and late August, the IAA contents of the cambial region and
mature phloem did not differ significantly. From September
to November, more IAA accumulated in the mature phloem
than in the cambial region and the mature xylem.
Anatomical changes in E. ulmoides vascular tissues
To determine whether a relationship exists between the
ABA–IAA variations and the seasonal cambial activity, the
lateral growth of E. ulmoides was periodically measured by
counting vascular cell layers. The counts showed that the
cambium was highly productive between late February and
early July (Fig. 5).
The cambial reactivation resulted in a sharp production
of xylem (inward) and phloem (outward), and began in all
three tree levels with the generation of immature xylem cell
layers. This production of cell layers was greatest between
mid-June and early July, after which it decreased sharply,
reaching the lowest point before mid-October.
ABA and IAA distribution in Eucommia vascular tissues 1021
of quiescent and freshly harvested branches contained an
average of 3–4 cell layers (Fig. 6J). The cambium of IAAtreated branches (Fig. 6F) was more strongly reactivated
than the ABA-treated (Fig. 6C–E) and control (Fig. 6A)
branches. In branches treated exogenously with both ABA
and IAA (Fig. 6G–I), no cambial activity was observed, as
determined by counting the cell layers in the cambial region
(Fig. 6J). This suggests that ABA negatively influenced the
reactivation of the cambium by IAA. In the future,
exogenous treatment of branches with lower concentrations
of ABA (<10 nM) may aid in assessing the relationship
between the two phytohormones.
Discussion
The present study was conducted over one calendar year in
Beijing. Previously, Luo et al. (1995), Cui and Luo (1996),
and Mwange et al. (2003b) identified, in addition to the
active period, three consecutive stages in the E. ulmoides
dormancy period, named the quiescence-1 (Q1), rest (R),
and quiescence-2 (Q2) stages. The terms quiescence and
rest here refer to the definitions proposed by Little and
Bonga (1974).
Results in this paper show obvious connections between
(i) annual E. ulmoides development (throughout all of the
above developmental periods), (ii) endogenous variations
in ABA and IAA distribution and quantity, and (iii) cell
division and differentiation in vascular tissues.
Fig. 3. Seasonal ABA distribution in E. ulmoides vascular tissues as
revealed by fluoro-immunolocalization. Rhodamine-stained ABA was
more prominent in both phloem (MP) and xylem (MX) than in the
cambial region (CR). The ABA signal was stronger in winter (A, 26
December) than in autumn (E, 20 September) or the quiescence-2 (B, 24
February) and active (C, 21 March; D, 01 June) developmental stages.
The longitudinal distribution of ABA in E. ulmoides vascular tissues was
observed to increase with the following trend: top (1) < middle (2) < base
(3); bar=80 lm.
Cell division in the cambial zone ceased in late July in
the top tree level; then in the middle level and, by late
August, in the bottom level (see arrows in Fig. 5). The
decreases in the numbers of immature xylem and phloem
cell layers were associated with increases in the mature
xylem and phloem cell layers, respectively (Fig. 5).
Effect of exogenous IAA-ABA application on cambial
activity in a water-culture system
To test for a mutual influence of ABA and IAA on the
cambial activity of E. ulmoides, quiescent (1–2-year-old)
branches were treated with ABA, IAA, or both ABA and
IAA, and the branches were then grown in a water-culture
system. On the sampling date (Fig. 6A), the cambial region
Annual variations in ABA, IAA, and cambial periodicity
in E. ulmoides
The ABA–IAA quantification results correlated strongly
with the ABA–IAA localization (Figs 2, 3) and clearly
showed the annually opposite patterns of these growth
regulators in E. ulmoides vascular tissues, suggesting
a possible conflict in the concurrent expression of the two
hormones.
In January, E. ulmoides was in the rest stage (Fig. 7), and
the cambial cells in the G1 phase (Zhong et al., 1995).
Earlier research (Mwange et al., 2003b) has shown that,
during the quiescence-2 stage, this species performs physiochemical processes, such as protein synthesis and gene
duplication (S phase) that are required for the activation of
internal activity when the proper temperature, light, and soil
moisture conditions appear in springtime.
The influence of the rest time was still considerable at the
beginning of the quiescence-2 phase, which was still
marked by a high ABA level, almost undetectable free
IAA, and no change in cell layers number (Figs 2, 3, 4, 5).
The cambial activity began at the same time as an abrupt
augmentation in the IAA content, a strong decline in the
ABA content, and a remarkable increase in cell division in
the cambial zone at the top as well as at the middle and
bottom of the tree. The concomitant occurrence of these
1022 Mwange et al.
Fig. 4. Annual changes in free ABA and IAA patterns in E. ulmoides vascular tissues at top (A), middle (B), and base (C) of the tree. The fluctuations in
ABA and IAA occurred as opposite trends throughout the seasons in different tree levels and vascular tissues. With the extraction technique used, free
IAA (histograms) was detected only between late February and mid-November, with a considerable increase in springtime, a plateau in summer, and
a relatively strong decrease in autumn. However, the levels of ABA (lines) progressively decreased from February to July and gradually increased from
the beginning of the dormancy period in August. The highest ABA content was observed in winter (from late November to early February). The values
shown are the means of four replicates. Vertical bars indicate standard error.
processes is an indication that polar transport of IAA, which
is synthesized in buds, induces both cambial cell division
and derivative differentiation (Bennett et al., 1998; AvsianKretchmer et al., 2002). Cell division stopped with the
cessation of IAA transport, but mature xylem cell and
phloem cell differentiation continued for some time (Fig.
5), as this process may require a relatively low amount of
IAA (Jacobs, 1984; Raven et al., 1992; Mwange et al.,
2003a).
Cambial activity in plants is strongly related to changes
in endogenous IAA levels (Siddiqi, 1991; Lachaud et al.,
1999): high IAA quantities are related to cell division,
supporting the primary role of IAA in maintaining the
meristematic character of cambial cells. With the increase
ABA and IAA distribution in Eucommia vascular tissues 1023
Fig. 5. Annual changes in the cell layers in E. ulmoides vascular tissues
at the top, middle, and base levels. New cell layers (immature phloem and
xylem) were intensely produced during the active period from early
March to late July, whereas late wood and mature phloem were more
actively formed between late July and early October, when the IAA levels
were sharply decreasing. Cambial cell division at the top level started at
the end of February (arrowhead), earlier than at the other levels (early and
late March at the middle and bottom levels, respectively). The vascular
cambium activity (arrow) ceased around the end of July at the top and
middle levels and late August at the base level. The values shown are the
means of six replicates. Vertical bars indicate standard error.
in IAA content during the active period (Fig. 4), the cambial
zone gained more cell layers, and the subsequent cell
division resulted in significant production of immature
xylem and phloem cells (Fig. 5). The number of cells across
the cambial zone during the active period usually reflects
the balance between the rates of production and differentiation of cambial derivatives (Lachaud et al., 1999). The
cessation of cell division in the cambial zone, due to the
decrease of IAA concentration in quiescence-1 phase (Fig.
5, arrows) confirms the role of IAA in inducing cambial cell
division and the differentiation of cambial derivatives in
either xylem or phloem cells (Catesson et al., 1995; AvsianKretchmer et al., 2002). It is important to mention here that
Fig. 6. Effects on cambial activity from ABA and IAA treatments of
water-cultured twigs of 1–2-year-old E. ulmoides trees. ABA was applied
at 10 nM (ABA1), 1000 mM (ABA2), or 10 000 nM (ABA3) by addition
to the water in the glass culture bottles. IAA was carried in lanolin (1 mg
IAA gÿ1 lanolin) and applied to the excised tops of the branches. The
culture conditions are described in the Materials and methods. Compared
with control sections from branches freshly harvested on the sampling
date (A), the treatments of lanolin alone (B), ABA1 (C), ABA2 (D), ABA3
(E), IAA+ABA1 (G), IAA+ABA2 (H), or IAA+ABA3 (I) did not
significantly stimulate cambial activity, as shown by counts (J) of the cell
layers in the cambial region (CR). Treatment with IAA alone (F) resulted
in a significant augmentation of the number of cell layers in the CR. MP,
mature phloem; MX, mature xylem. Bar=50 lm.
in E. ulmoides, xylem production ceased almost two
months earlier than in Broussonetia papyrifera, as observed
by Cui et al. (1995a). The present study is the first to
describe such an early cessation of cambial activity in
a temperate woody species. The above phenomenon has
previously been observed only in tree species grown under
1024 Mwange et al.
photoperiod stress (Mellerowicz et al., 1992). In Beijing,
late July to August is still a long-day period, with temperatures among the highest of the year (>30 8C). The present
finding supports the assertion by Guo (1992) that
E. ulmoides could originate from tropical biotopes. The
high temperatures of this period would trigger the vascular
cambium to enter the quiescence stage gradually.
E. ulmoides could have inherited from its tropical ancestors
this dormant characteristic that acts during the dry and hot
season (Cui and Luo, 1996).
The quiescence-1 phase in E. ulmoides begins when
ABA levels are rapidly increasing and IAA levels abruptly
decreasing, and ends when no more free IAA is detected in
the cambial region of the top level (Fig. 7). This phase
corresponds to the time between the initiation of DNA
fragmentation and the nuclear decomposition previously
observed in E. ulmoides leaves (Cao et al., 2003). This
timing suggests that DNA fragmentation in leaves leads to
the cessation of IAA biosynthetic gene activity and implies
an important role for intact DNA in controlling IAA
Fig. 7. Relationship between the seasonal ABA–IAA changes and the
annual cambial periodicity in E. ulmoides. Free ABA was highly detected
during the rest (R) time (winter), but began to decrease at the end of the
quiescence-2 (Q2) stage, and attained the lowest level during the active
(A) period (spring and summer), and then gradually increases in the
quiescence-1 (Q2) stage. The pattern of the variation in free ABA was the
opposite of that of IAA at the same developmental phases of the tree. Free
IAA was detected in the R phase.
synthesis in buds and leaves (Bartel, 1997). Lower IAA
production, subsequent to the programmed cell death
process in leaves in June and July (Cao et al., 2003) or to
its change into conjugated forms, could lead to a gradual
IAA deficiency in E. ulmoides conductive tissues. The
resulting decrease in IAA probably favours the differentiation of mature phloem and xylem cells (Fig. 5).
Since, at this time of the year, all leaves have been shed
from the E. ulmoides branches, ABA biosynthesis may not
be a leaf-dependent process. ABA could be produced in
plant roots as asserted by Cutler and Krochko (1999) and
Sauter et al. (2001), supporting the down-up decreasing
gradient of ABA discussed in this paper (Fig. 8B).
Do ABA and IAA interact in E. ulmoides vascular
tissues?
Results in this paper broadly show that, during E. ulmoides
annual development, the vascular tissues displayed increasing ABA latero-centrifugal and apico-basal gradients,
as well as increasing IAA latero-centripetal and baso-apical
gradients in vascular tissues (Fig. 8). ABA was more
abundant in the basal parts of the tree, characterized by
a relatively weak rhythm of cell division. Thus, the vascular
tissues near the apical and active meristem has received less
ABA.
Plant growth regulation is a global interactive phenomenon in which the perception of one hormone may affect the
concentration, perception, and responses of others (Grossmann and Hanses, 2001). This study revealed contrasting
patterns of ABA and IAA distribution, with greater production in the dormant and active periods, respectively
(Figs 2, 3, 4). Although some authors have attributed the
increase of ABA levels in trees to water stress in late
Fig. 8. IAA–ABA radial (A) and longitudinal (B) gradients in
E. ulmoides. (A) The ABA content is higher in the fully differentiated
phloem (MP) and xylem (MX) than in the cambial region (CR), and the
IAA content shows the opposite pattern in these vascular tissues. Crosssection, bar=50 lm. (B) The ABA content increases going upwards in the
tree, whereas the decrease in IAA content is oriented downwards.
ABA and IAA distribution in Eucommia vascular tissues 1025
summer (Little and Wareing, 1981), this annual IAA–ABA
contrasting pattern, that has also been reported in previous
studies (Emery et al., 1998; Kojima et al., 1999; Wijayanti
et al., 1995; Peres et al., 1997) should attract serious
scientific attention. For example, the divergent tendencies of IAA and ABA observed during the recovery of
E. ulmoides bark after manual bark ringing (Mwange et al.,
2003a) are unlikely to have been related to water deficiency,
as the plants were well protected from desiccation. This
could suggest that the probable IAA–ABA crosstalk is
instigated by a mutual inhibition process between the two
phytohormones, either directly or indirectly.
Although a definitive conclusion on the possible reciprocal inhibition between ABA and IAA in vascular
tissues seems premature at this stage, an environmentallyregulated relationship between IAA and ABA action may
exist in higher plants. It can also be hypothesized that the
substance that inhibits ABA or IAA synthesis may not be
IAA or ABA itself, but other biocomponents (enzymes, . . .)
directly or indirectly involved in the biosynthesis and/or
expression of these hormones.
As a matter of fact, preliminary studies conducted in this
laboratory have shown that ABA inhibits the expression of
the ABP1 gene, which encodes the putative IAA receptor
IAA binding protein (ABP1) (HW Hou, YT Zhou, KN
Mwange, WF Li, XQ He, and KM Cui, unpublished
results). This may lead to an inhibition of IAA effects on
cambial activity and derivative differentiation.
It is also possible that regulation of the annual cambium
activity might be linked to an IAA:ABA ratio. A higher
ratio (implicating a high IAA concentration) could induce
cambial activity in plants during the active period. However, when the ratio gradually approaches its lowest level
(indicating a significant increase in the ABA content)
during dormancy, the cambial activity slows down, promoting the active maturation of late wood. The decrease in
the IAA:ABA ratio triggers the cambium to enter the
quiescence-1 phase in autumn as the tree begins to
experience unfavourable environmental conditions. In winter, with the complete loss of free IAA in the vascular tissue
and ABA content reaching its highest levels, the cambium
undergoes its rest period.
Moreover, roles may be played by peroxidases, production of which is often significantly induced in plants by
increased ABA (Cottle and Kolattukudy, 1982; Roberts and
Kolattukudy, 1989; Hou et al., 2004). It is likely that
a gradual production of ABA-stimulated peroxidase at
quiescence-1 leads to the oxidation and, consequently, to
a progressive decrease of free IAA that takes place from
quiescence-1 through winter in E. ulmoides vascular tissues
(Fig. 7).
The water-culture system is a simple and reliable way to
investigate certain biological phenomena in plants, in
particular, the effects of plant growth regulators. Application of exogenous IAA in this system usually instigates
cambial activity in quiescent branches (Cui et al., 1995b;
Wang and Cui, 1999; Mwange et al., 2003b). This increase
might be due to the increase in the IAA:ABA ratio owing to
both the IAA application and the artificial improvement in
the growth conditions, including temperature, light, and
water availability. Therefore, a lack of root-ABA supply
in water-cultured quiescent branches could favour an
increase in the IAA:ABA ratio, resulting in cambial cell
reactivation in the branches. This may explain why watercultured branches of quiescent E. ulmoides, supplemented
with different ABA concentrations in this experiment
did not show increased cambial activity, whereas the
cambium in branches treated with IAA was reactivated
(Fig. 6F, J). ABA treatment would contribute to the
reduction in the IAA:ABA ratio. However, it is unknown
why identical IAA treatments to fully resting branches in
the water-culture system did not instigate cambial reactivation (Catesson, 1994; Wang and Cui, 1999).
ABA and cambium dormancy
Dormancy has been extensively studied in seeds (Corbineau
and Côme, 2002; Fellner et al., 2001; Zeng et al., 2003) and
buds (see reviews by Anderson et al., 2001), where the role
of ABA in the process has been systematically shown.
Consistent research work related to its implication in
cambial meristem dormancy have, to date, remained relatively scant.
In a previous work, Heide (1986) noticed that the
application of ABA in lanolin to apical buds of Picea
abies seedlings suspended shoot elongation for about 3
weeks in the majority of plants, but failed to induce normal
winter buds. He concluded that the role of ABA in the
induction of dormancy was uncertain in conifers as well as
in deciduous woody plants.
Later, however, numerous bud-related studies have established a direct control of bud dormancy by ABA. However, with the development of investigation techniques,
suggestions from recent findings have attributed the bud
dormancy mechanism to cytokinins- and IAA-induced
synthesis of ethylene (Trewavas, 1991; Salisbury and Ross,
1992; Moore et al., 1995). ABA could be an ethylenetriggered second hormonal messenger that acts during plant
growth regulation (Grossmann and Hansen, 2001). Rohde
et al. (2002) have recently demonstrated the expression of
the poplar ABSCISIC ACID-INTENSIVE3 (ABI3) protein
at the initiation of bud set and dormancy. As the growth of all
of the meristems is nearly suppressed during winter, it is
expected that, in both buds and the cambium, dormancy is
regulated by similar molecular mechanisms.
The present study supports the possible cold-stressstimulated biosynthesis of ABA in plants (Figs 3, 4).
During quiescence-1, E. ulmoides begins to face serious
environmental stresses such as low temperatures, short
photoperiod, and water deficiency, which become more and
more drastic with the approaching winter. The high ABA
1026 Mwange et al.
expression in E. ulmoides lateral meristems discovered in
this trial could be more closely related to the dormancy
phase, as suggested by Leung and Giraudat (1998), Okubo
(2000), and Rock (2000), than to leaf senescence and
shedding (Rohde et al., 2000).
In a recent study, by describing a genomics approach to
investigate cambial dormancy using a combination of largescale expressed sequence tag (EST) sequencing and global
transcript profiling, Schrader et al. (2004) have provided
a better and more promising tool for understanding the
effect of hormonal and environmental signals on the induction and maintenance of dormancy via activation and
repression of diverse gene expression programmes in
cambial meristems.
Conclusion
Associating both quantitative and immunolocalization of
IAA and ABA in the vascular tissues of E. ulmoides, the
present study, conducted over a whole year in apical to
basal parts of the tree, has revealed opposite concentration
gradients of the two hormones. A possible opposite
function of these hormones in regulating the annual cambial
activity is suggested. IAA and ABA concentrations are
high, respectively, in the active and dormant periods. If
numerous studies have already demonstrated the high
implication of IAA in cell proliferation and differentiation
(occurring in the active period), little, however, is known on
the role of ABA on the arrest of cambial meristem activity
(during dormancy). As for IAA, related-ABA signal transduction studies are needed in dormant vascular cambium,
and the confirmation of IAA–ABA crosstalk in higher
plants will require additional molecular assays involving
ABA-sensitive mutants.
Acknowledgements
This study was funded by the State Key Basic Research and
Development Plan of China (G1999016002) and the National
Natural Science Foundation of China (30170056, 30471367,
30070614). Authors greatly appreciate the scholarship award to Dr
KN Mwange by the Chinese and Congolese (RD Congo) Governments. Special thanks are addressed to Professor Liu Hu-Wei
(College of Chemistry and Molecular Engeneering, Peking University) and his laboratory staff members for their priceless scientific
support and help in performing the HPLC technique. Authors greatly
thank Ms Pang Yu for her technical support and the anonymous
reviewers for their constructive criticism.
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