全 文 ：Vol. 29 No.4
Dec. 2011
第 29 卷 第 4 期
2011 年 12 月
经 济 林 研 究
Nonwood Forest Research
Dynamic activity of endogenous plant hormones in Zelkova
schneideriana during the growth of seedlings
ZHANG Ri-qing1a, YANG Jie2, JIN Xiao-ling1b, TANG Chun-fang1 a, LIU Hai-long1 a,3
(1a. College of Forestry; b. College of Landscape Architecture, Central South University of Forestry & Technology,
Changsha, Hunan 410004, China; 2. Zhuzhou Foreign Language School, Zhuzhou 412000, Hunan, China;
3. Biotechnology Research Institute, Guangxi Academy of Forestry, Nanning 530001, Guangxi,China)
Abstract: Zelkova schneideriana is a valued hardwood used for timber and gardening. In order to fi nd the relationship
between the content of endogenous plant hormones and the seedling growth condition of Z. schneiderian, the levels of
endogenous IAA, GA3, ZT, and ABA in seedlings were detected by RP-HPLC. The results showed that the contents of
four endogenous hormones fi rst decreased, and then increased, fi nally dropped. Specifi cally, there were high levels of
IAA and ZT but low levels of GA3 and ABA when the terminal bud was bourgeon at March. After the period of budding,
the contents of the four hormones decreased slowly. Each hormone′s content increased steadily in early May. The levels
of IAA and ABA reached the maximum in mid-July and then declined. The levels of ZT and GA3 reached their highest
level at the end of August and reduced in the subsequent phase of growth. There was a decrease in the ABA content in late
July and then it stabilized thereafter. This steady level of ABA showed a correlation with inhibition of bud growth in cool
autumn. The seedlings came into a physiological dormancy at October. The ratios of ABA to GA3 and IAA to ZT showed
a trend like letter “W” during the growth of seedlings. It suggests that May and August are two fl ourishing growth periods
for Z. schneideriana seedlings.
Key words: endogenous plant hormones; HPLC; Zelkova schneideriana; seedling
榉树实生苗苗期内源激素含量的动态变化
张日清 1 a，杨 婕 2，金晓玲 1b，汤春芳 1 a，刘海龙 1 a，3
(1. 中南林业科技大学 a. 林学院；b. 风景园林学院，湖南 长沙 410004；
2. 株洲外国语学校，湖南 株洲 412000；
3. 广西林业科学研究院 生物技术研究所，广西 南宁 530001)
摘 要： 榉树是一种用材和园艺观赏硬质阔叶树种。为了了解内源植物激素与榉树实生苗生长状况的关系，采用反
相高效液相色谱法测定了榉树实生苗内源 IAA、GA3、ZT 和 ABA 的水平。结果表明，这 4 种内源激素含量呈现先降
低、再升高、然后下降的趋势。当 3 月顶芽萌发的时候，IAA 和 ZT 水平较高，而 GA3 和 ABA 水平较低。过了萌芽
期后，4 种激素含量缓慢下降。在 5 月初，4 种激素的含量稳步升高。在 7 月中旬，IAA 和 ABA 含量达到最高值，
随后开始下降。在 8 月末，ZT 和 GA3 含量达到最高值，随后开始下降。7 月下旬 ABA 含量有所下降，随后保持稳定。
ABA 含量的稳定说明其在秋季对芽生长有一定的抑制作用。10 月，苗木开始了生理休眠。在苗木的整个生长周期中，
ABA 和 GA3 ，IAA 和 ZT 含量的比值均呈现“W”形的变化趋势，说明 5 月和 8 月是榉树苗木的 2 个快速生长期。
关键词： 内源植物激素；HPLC；榉树；实生苗
中图分类号： S687 文献标志码： A 文章编号： 1003-8981(2011)04-0001-05
收稿日期：2011-10-11
基金项目：国家林业公益性行业科研专项“榉树优良种源选择及快速繁育技术研究 ”(200904011) 。
作者简介：张日清 (1959—)，男，湖南醴陵人。教授，博士，博士研究生导师，主要从事经济林栽培育种和林业生物技术应用方面的教学与
科研工作。E-mail：hanszhangriqing@yahoo.com.cn。
DOI:10.14067/j.cnki.1003-8981.2011.04.018
2 第 4 期张日清，等：榉树实生苗苗期内源激素含量的动态变化
specific tissues or cells needs some techniques which
allow us to accurately determine the distribution of
these compounds. Recently, because of the introduction
of advanced analytical methods, high performance
liquid chromatography (HPLC), a wide spectrum of
endogenous plant hormones has been identified and
quantifi ed in plants [8]. The objectives of this study were:
(1) to elucidate the variation in four endogenous plant
hormones (IAA, GA3, ZT, and ABA) levels in Zelkova
seedlings during all-year progress of seedlings growth;
(2) and to know the relationships between the changes
of endogenous plant hormones and the seedling growth.
1 Materials and Methods
1.1 Cultivation and sampling of plant materials
Seeds of Zelkova (Z. schneideriana) were
scattered over the ground for growing in a nursery in
Central South University of Forestry & Technology in
spring 2009. The plants grew under natural conditions.
The leaves of seedlings were sampled from the first
pair of leaves to the defoliation. To determine the
endogenous plant hormones, the fresh leaves were
washed and dried. Finally, the samples were weighed
and stored under -80 ℃ for hormone extraction.
1.2 Determination of endogenous plant hormones
Plant hormones were extracted and purified
following the method by Ruozhong Wang, et al[9] with
some modification. 2.8 g fresh samples were ground
in 11 mL (5, 2, 2, 2 mL) cold 80% methanol with a
pestle and mortar under dim light, and the mixture was
permeated for 15 h under 4℃ . Then, the mixture was
centrifuged (Thermo, LEGEND MACH 1.6R Centrifuge,
USA) for 20 min at 5 000 g under 4 ℃ and repeated three
times. The collected supernatant was frozen to dryness
with freezer (Thermo Fisher, MODVLYOD-230, USA).
The dry powder was redissolved with 8 mL NH4Ac
(0.1 mol·L-1, pH 9.0), the solution was centrifuged
for 20 min at 15 000 g under 4 ℃ . The supernatant was then
filtered through Polyvinylpolypyrrolidone (PVPP,
Sigma, USA) column, DEAE sephadex A-25 (Sigma,
USA) and Sep-Pak C18 Cartridges (Waters, Ireland).
The endogenous plant hormones of the Sep-Pak
C18 Cartridges were extracted with 50%(volume ratio)
methanol. The filtrate of each sample was frozen to
Zelkova schneideriana Hand.-Mazz., ranking
the second class in the state-protected plant species,
is a deciduous hardwood tree belonging to the genus
Ulmus[1]. It is outstanding for ornamental use with paler
green in spring and orange or rusty red leaves in fall.
Zelkova produces excellent timber with massive, hard,
sheeny, and durable features. In addition, it has good
cold hardiness and strong resistance to air pollutants and
drought. In many countries, zelkova can be planted in
parking lots, and used as median strip plants, shade tree,
sidewalk cutout (tree pit), and residential street tree. It
can successfully grow in urban areas where there is air
pollution, poor drainage, compacted soil, and/or drought.
Z. schneideriana is one of the most important
landscape species because of its large crown and high
disease resistance [2-3]. It is highly valued in China,
Korea, and other East Asian countries [4]. Now there is an
increasing demand for this tree in China. However, it has
become a rare and endangered species in China due to
uncontrolled commercial logging and lacking of effective
propagation methods [5]. Resource shortage in Zelkova is
a problem that allows no negligence in China.
Endogenous plant hormones, a variety of low
relative molecular mass compounds, are capable of
affecting almost every aspect of plant′s life. Though there
has been a century research about the diverse range of
their biological effects, there is still rare understanding
of their action mode at the cellular and molecular level.
The physiological mechanisms of seedling growth
are still unknown, even though the information about
plant hormone regulation of the seedling growth is
indispensable to better planting. Fortunately, plenty of
analytical chemistry means have elucidated the chemical
nature of these bioactive molecules [6]. Besides auxins,
cytokinins, ethylene, gibberellins and abscisic acid, the
five classical families of plant growth substances are
being supplemented with brassinosteroids and jasmonic
acid. Some new derivatives and actives structures are still
being discovered [7].
Quantitative analysis of endogenous plant
hormones has already shown several correlations
between the developmental physiological processes and
the changes in the levels of plant growth substances.
The study of the role of endogenous plant hormones in
3第 29 卷 经 济 林 研 究
dryness then redissolved in the fi rst isocratic elution.
Four endogenous plant hormones were pooled,
evaporated, and subjected to ODS reverse-phase (C18)
column (4.6×250 mm, 5 μm particle, Waters, USA)
fitted with a C18 guard column, UV detection (254
nm, Shimadzu, SPD-20A, Japan) and gradient elution
is the mixture of methanol and 0.8% acetic acid. The
mobile phase was programmed to hold, 0-5 min, the
fi rst isocratic elution with a mixture of 8 ︰ 92 (volume
ratio) methanol: 0.8% acetic acid, then followed by
a linear gradient elution from 5 min to a composition
of 50 ︰ 50 (volume ratio) at 18 min, another linear
gradient elution from 18 min to a composition of
75 ︰ 25 (volume ratio) at 21 min, holding this mixture
for 9 min, and fi nally followed by linear gradient elution
from 30.01 min to a composition of 8 ︰ 92 (volume
ratio) at 40 min. Run with a fl ow rate of 0.8 mL·min -1
at 25 ℃ . The peak areas were recorded and calculated
by a computer with LC Solution data acquisition and
integration soft ware (Shimadzu, Japan). Recoveries
were calculated for all hormones and the concentrations
of of IAA, GA3, ZT and ABA were corrected on
international standard.
1.3 Data analysis
Data were initially analyzed to determine whether
the variances of homogenous in the experiments. In all
cases, the variances of homogenous and the data from
three repeat experiments were pooled. The samples
were measured and the means standard deviations of
three replicates were calculated. Statistical analyses of
variance of pooled data for all attributes were carried
out using Microsoft Excel software.
2 Results
2.1 Concentration changes of four endogenous plant
hormones during seedling growth of Zelkova
In the period of seedling growth, the contents of
IAA, GA3, ZT and ABA showed a fluctuant dynamic
activity. In general, the contents of four endogenous
plant hormones first decreased, and then increased,
fi nally dropped.
The dynamic change of content of GA3, ZT, IAA
and ABA during the seedling growth of Zelkova are
Results are expressed as the mean of three independent experiments. Vertical bars
represent SD are within the symbols.
Fig. 1 Content of GA3 during seedling growth of Zelkova
Results are expressed as the mean of three independent experiments. Vertical bars
represent SD are within the symbols.
Fig. 2 Content of ZT during seedling growth of Zelkova
Results are expressed as the mean of three independent experiments. Vertical bars
represent SD are within the symbols.
Fig. 3 Content of IAA during seedling growth of Zelkova
Results are expressed as the mean of three independent experiments. Vertical bars
represent SD are within the symbols.
Fig. 4 Content of ABA during seedling growth of Zelkova
4 第 4 期张日清，等：榉树实生苗苗期内源激素含量的动态变化
shown in Fig.1-4, respectively. There were high levels
of IAA and ZT but low levels of GA3 and ABA (the
levels of GA3 and ABA were litter higher compared with
the levels from late March to mid-June, but were far
lower than that from mid-June to late September) in the
growth phase of the fi rst pair of compound leaves when
terminal bud will bourgeon (the time course of shoot
growth is from March to early May). The contents
of four endogenous plant hormones kept low levels
(GA3 5.98 ng·g-1, ZT 6.15 ng·g-1, IAA 0.02 ng·g-1,
ABA 0.67 ng·g-1) form April to early May. Then, the
contents of four hormones began to increase steadily. The
contents of ZT and GA3 rose rapidly at July and reached
highest levels (ZT 74.91 ng·g-1, GA3 707.92 ng·g-1) at the
end of August, and then declined in the subsequent phase.
Moreover, the contents of IAA and ABA increased
and reached their maximum (IAA 13.38 ng·g-1, ABA
41.26 ng·g-1) in mid-July. However, ABA content
always kept a stable level.
2.2 The ratios of ABA to GA3 and IAA to ZT during
seedling growth of Zelkova
According to the data of the contents of four
endogenous hormones, the concentration ratios of ABA to
GA3 and IAA to ZT were fi gured out, which reveal plant
growth conditions, such as development or dormancy.
The ratio of ABA to GA3 showed a trend like letter
“W” (Fig.5). It declined slowly in early March (the fi rst
pair of leaves growth phase) and reached its first low
point in mid-June. After that, it rose impermanent and
then declined reaching its second low point in late of
August. The ratio rose in the subsequent phase.
The ratio of IAA to ZT also showed a trend
like letter “W” (Fig.5). The high ratio of IAA to ZT
appeared in early March and declined to its first low
point in late April (a low ratio kept from late April to
early May). It then rose at later growth stages and a
remarkable rate appeared from May to mid-June. The
ratio declined gradually and reached its second low
point in late August. Finally, it increased slowly in the
subsequent phase.
3 Discussion
In this study, changes of four endogenous plant
hormones and ratios of ABA to GA3 and IAA to ZT in
seedling of Zelkova were investigated. These results
were observed during the seedling growth of Zelkova
and were associated with developmental changes in the
seedlings.
3.1 The change of IAA, GA3, ZT and ABA content
during the growth of seedlings
IAA, GA3, ZT and ABA are intensively involved
in plant growth process[10]. During the stage of having
terminal buds, there were high levels of IAA and ZT
but low levels of GA3 and ABA in the seedlings, which
is similar to the changes in endogenous phytohormone
content of Larch seedlings during terminal bud growth [11].
Xuesha Ke and Wei Li [12] reported that the values of
IAA and ZT were positively significantly correlated
with growth. Their results are in agreement with our
results for Zelkova, which shows that high levels of IAA
and ZT are the basic of the terminal bud germination.
However, the low level of ABA suggests that ABA is
not required in this period of development. Compared
with the level of ABA in the prophase development,
the higher level of ABA showed a correlation with
inhibition of bud growth in cool autumn [13-14]. During
the growth of seedlings, the level of GA3 increased at
late April and reach a obvious peak (707.92 ng·g-1) at
the end of August. GA3 is best known for promoting cell
elongation. GA3 often stimulates bud growth
[15], and
its endogenous level increases concomitantly with bud
elongation [13], our study also showed a similar result.
The result also showed that an increasing level of GA3
stimulated the growth of the seedlings.
3.2 The ratio of ABA to GA3
The role that ABA and GA3 regulating plant
development was different, but it was sure that the cooperation
Vertical bars represent SD are within the symbols.
Fig. 5 Ratios of ABA to GA3 and IAA to ZT
5第 29 卷 经 济 林 研 究
of ABA and GA3 could regulate plant development. It has
been known that the positive correlations existed between the
ratio of ABA to GA3 and the freezing tolerance of this plant.
The increase in freezing tolerance of this plant was caused
by the high ABA content, low GA3 content, and high ratio
of ABA to GA3. Constance N White et al
[16] reported that
the ratio of ABA to GA3 revealed the plant development or
dormancy. The high ratio that means the high level of ABA
showed that seedlings came into physiological dormancy,
which has been proved in the opposite way. The low level of
GA3 and high ratio of ABA to GA3 have shown the inhibition
of seedlings growth and promotion of maturation [16-17]. The
two low points appeared in mid June and late August during
the seedlings growth periods (Fig.5). It showed that the
endogenous quantity of GA3 in this plant was more than that
of ABA, and the seedlings were in bloom. In addition, the
peak contents of ABA to GA3 appeared when seedlings grew
slowly in October. The result suggests that seedlings come
into physiological dormancy.
3.3 The ratio of IAA to ZT
It is well established that a low ratio of IAA to
ZT promotes the growth of seedling, and a high ratio
promotes the development of root [18]. The two low
points appeared from late April to early May and late
August during the seedlings′ growth periods (Fig.5)
showing the seedlings are in bloom and May and
August are the two flourishing growth periods of
Zelkova seedlings. The seedlings specially showed the
best fl ourishing growth in May. It was noticeable that
the lowest ratio of ABA to GA3 was appeared earlier
than that of IAA to ZT, which is probably correlated
with IAA promoting GA3
[19]. This supposal needs to be
proved with further studies in future.
In this study, findings of correlation analyses
suggest that the levels of endogenous plant hormones
play an important role in the seedling growth of Z.
schneiderian. Dynamic activity of endogenous plant
hormones is an indeed difficult problem. The authors
just made some pilot study at the present time, so more
studies were needed in future.
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