Plant Growth Regulation 35: 233237, 2001.

233
2001 Kluwer Academic Publishers. Printed in the Netherlands.

Effects of plant growth regulators on secondary wall thickening of cotton

fibres

Yang You-Ming 1, Xu Chu-Nian 1,*, Wang Bao-Min 1 and Jia Jun-Zhen 2

1
College of Crop Science, China Agricultural University, 100094, Beijing, China; 2College of Crop Science,
College of Basic Science and Technology, China Agricultural University, 100094, Beijing, China; *Author for
correspondence (phone: 86 010 62892556; fax: 86 010 62891298)
Received 25 August 2000; accepted in revised form 21 February 2001

Key words: Cotton (Gossypium hirsutum) fibre, Ovule culture, Plant growth regulators, Secondary cell wall

Abstract

The effect of plant growth regulators on the secondary wall thickening of cotton fibre was studied. The results
indicated that the GA S and iP + iPA levels in the fibre of field-grown cotton plants remained almost constant but
the IAA and ABA levels changed considerably during fibre development. Although the change in both IAA and
ABA levels seemed not to be closely related with the rate of cellulose accumulation, there was still a relationship
between the ratio of ABA to IAA and secondary wall thickening. In in vitro studies, ABA (50 molL 1) mark-
edly enhanced the accumulation of dry matter and cellulose in the fibre cell wall during secondary wall thick-
ening, but no similar effect was observed with NAA, GA 3 or kinetin treatments. The role of ABA in secondary
wall thickening of cotton fibre is discussed.

Abbreviations: ABA abscisic acid, DPA days post anthesis, DW dry weight, ELISA enzyme-linked im-
munosorbent assay, FW fresh weight, GA 3 gibberellic acid, GA S gibberellins, IAA indoleacetic acid,
iP(A) isopentenyladen(os)ine, NAA naphthalene acetic acid, SD standard deviation

Introduction cotton fibre properties (such as length, strength and

Cotton fibres, the most important economical parts of
cultivated cotton, are single-celled trichomes devel-
oped from individual epidermal cells on the outer in-
tegument of ovules. Fibre development can be di-
vided into four distinct but somewhat overlapping
phases. Phase 1, fibre initiation, occurs at anthesis.
Phase 2, fibre elongation, starts soon after phase 1 and
continues for 3 4 weeks. Phase 3, secondary wall
thickening, overlaps with late phase 2 and continues
until the fibre is matured. It is characterised by the
deposition of almost pure cellulose and an overall
wall thickening. Phase 4 is the stage when the fibre
reaches full maturity and become dead and desiccated
(Basra and Malik 1984; DeLanghe 1986). Fibre prop-
erties are established during fiber development. For
example, fibre strength is determined mainly by the
characteristics of the secondary wall. Improvement of
fineness) is required by modern spinning industries
(Meredith et al. 1991). Therefore, it is relevant to
study the mechanism of plant growth regulators on
the development of cotton fibres.
Considerable investigations have been focused on
the effect of plant growth regulators on the initiation
and elongation of cotton fibres (reviewed in Holt and
Stewart (1994); Kosmidou-Dimitropoulou (1986)).
Based on these studies, it was commonly agreed that
IAA and GA stimulate or activate fibre development,
whereas ABA, kinetin and ethylene inhibit fibre
growth. The action of GA occurs during initiation and
the early elongation phase, while that of IAA contin-
ues until the late elongation phase.
There are two different viewpoints about the effect
of plant growth regulators on cellulose synthesis and
secondary wall thickening. Kosmidou (1986) consid-
ered that auxin is necessary for secondary wall thick-
234
ening and ABA had an adverse effect (Kosmidou-
Dimitropoulou 1986). However, some investigators
reported that GA 3 and IAA had no effect on cellulose
synthesis, kinetin inhibited it slightly, but ABA was
stimulatory (Francey et al. 1989; Jaquet et al. 1982;
Pillonel and Meier 1985). It is important to obtain
new information on the roles of plant growth regula-
tors on cellulose synthesis and secondary wall thick-
ening. Hence the present study investigated the levels
of endogenous IAA, GA S, ABA and iP + iPA in the
developing fibres of field-grown cotton plants and
also explored the effect of NAA, GA 3, kinetin and
ABA on secondary wall thickening of the cultured fi-
bre.
Materials and methods
Gossypium hirsutum L.cv Zhongmiansuo-12 plants
were grown in the field in Beijing, China. The cul-
tural practices, including irrigation and application of
fertilisers and insecticides, were conducted on the
usual basis. On the day of anthesis, individual flow-
ers were tagged, and healthy bolls were harvested for
the analysis of cellulose content, as well as the levels
of IAA, GA S, ABA and iP + iPA in the fibres. To min-
imise the effect of environmental variations, data
were collected from flowers that had bloomed during
as narrow a period as possible.
Freshly harvested bolls were opened with a scapel.
Intact ovules were taken at 2 DPA. At 9 DPA and
subsequently, the fibres were separated manually
from the ovule. Both freshly-separated fibres and in-
tact ovules were weighed and then frozen in liquid
nitrogen and stored at 20 C for later analysis.
For in vitro studies, fertilised ovules at 2 DPA were
excised from bolls collected when the wilting corolla
became dried-up and the stigma began turning brown.
A set of 20 ovules derived from the same boll were
cultured in a modified medium of Beasley and Ting
(1973), which contained the basal medium, but su-
crose was used instead of fructose. It was also sup-
plemented with 5 molL 1 NAA and 1 molL 1 centage recovery of each hormone was calculated by
GA 3. Ovules were cultured for 14 days and, at 16
DPA, were transferred to a subculture medium modi-
fied by adding 5 gL 1 activated charcoal. At 30 DPA,
ovules were transferred to further subculture media
modified by adding one of a series of concentrations
of NAA, GA 3, kinetin or ABA instead of 5 molL 1
NAA and 1 molL 1 GA 3. Each treatment consisted
of at least ten flasks. At 44 DPA, six flasks of each
treatment were examined and ten seeds from every
flask were selected at random. The seeds were washed
several times with distilled water, then the fibres were
manually removed from the seeds. The fibres were
weighed after being oven-dried to a constant weight
at 70 C.The weight of fibres was expressed in terms
of dry weight per seed. Each data point represented
the mean SD.
The cellulose content of the fibres was determined
as described by Updegraff (1969).
The extraction, purification and determination of
endogenous levels of IAA, GA S, ABA and iP + iPA
by an indirect ELISA technique were performed as
described by He (1993). The samples were homoge-
nised in liquid nitrogen and extracted in cold 80%
(v/v) methanol with butylated hydroxytoluene (1
mmolL 1) overnight at 4 C. The extracts were col-
lected after centrifugation at 10000 g (4 C) for 20
min, the extracts were passed through a C 18 Sep-Pak
catridge (Waters, Milford, MA) and dried in N 2. The
residues were dissolved in PBS (0.01 molL 1, pH
7.4) in order to determine the levels of IAA, GA S,
ABA and iP + iPA. Microtitration plates (Nunc) were
coated with synthetic IAA, GA S, iP + iPA or ABA-
ovalbumin conjugates in NaHCO 3 buffer (50
mmolL 1,pH 9.6) and left overnight at 37 C. Oval-
bumin solution (10 mg/ml) was added to each well in
order to block nonspecific binding. After incubation
for 30 min at 37 C, standard IAA, GA S, ABA,
iP + iPA, samples and antibodies were added and in-
cubated for a further 45 min at 37 C. The antibodies
against IAA, GA S, ABA and iP + iPA were obtained
as described by Weiler et al. (1981). Then horserad-
ish peroxidase-labelled goat antirabbit immunoglobu-
lin was added to each well and incubated for 1 h at
37 C. Finally, the buffered enzyme substrate (ortho-
phenylenediamino) was added, and the enzyme reac-
tion was carried out in the dark at 37 C for 15 min,
then terminated using 3 molL 1 H 2SO 4. The absor-
bance was recorded at 490 nm. Calculations of the
enzyme-immunoassay data were performed as de-
scribed by Weiler et al. (1981). In this study the per-
adding known amounts of standard hormone to a split
extract. Percentage recoveries were all above 90%,
and all sample extract dilution curves paralleled the
standard curves, indicating the absence of nonspecific
inhibitors in the extracts.
 235

Figure 1. Fibre length vs. Boll age. Each data point is the mean of Figure 2. Changes in cellulose content of fibres against boll age.
three replicates. Vertical bars representSD. The curve of the fi- Each data point is the mean of three replicates. Vertical bars
bres grown on cotton plants is defined by representSD. The curve of the fibres grown on cotton plants is
Y= 0.0001X 4 0.0107X 3 + 0.266X 2 0.5563X + 0.0535. The r 2 co- defined by Y=0.0005X 4+0.0351X 30.6045X 2+3.5161X+1.621.
efficient is 0.9991. The curve of the fibres cultured in vitro is de- The r 2 coefficient is 0.9885. The curve of the fibres cultured in vitro
fined by Y= 0.0002X 4 0.0133X 3 + 0.2815X 2 0.5612X 0.0027. is defined by
The r 2 coefficient is 0.9981. Y=0.0002X 4+0.0158X 30.1674X 2+0.8778X+1.1916. The r 2 co-
efficient is 0.9944.
Results

It is well documented that cell walls from fibres cul-

tured in vitro are remarkably similar to those derived
from the fibre of field-grown cotton plant, both in
terms of composition and in terms of relative changes
in composition during development (Meinert and
Delmer 1977). However, in our study, it was neces-
sary to compare the entire developmental sequence of
the fibre on the plant with that of the cultured fibre.
The length and cellulose content of the fibre were
fitted to polynomial equations of different degrees,
and the best-fit equation was determined statistically
by performing a t-test for different regression coeffi-
Figure 3. Changes in levels of plant hormones of field-grown fi-
cients (r 2 coefficient). The data on fibre length and bres against boll age. Each data point is the mean of three repli-
cellulose content in fibres were explained adequately cates estimated by an indirect ELISA. Vertical bars represent SD.
by a forth degree polynomial equation (Figures 1 and
2). Although the final length of the cultured fibre was velopment, whereas the IAA and ABA levels changed
shorter than that on the plant, the entire developmen- obviously as the fibre developed (Figure 3). No rela-
tal sequences of both were quite similar. Fibres both tionship was observed between the level of either
grown on the plant and cultured in vitro continued to IAA or ABA and the rate of cellulose accumulation.
elongate up to 30 DPA when the maximum length However, when the ratio of ABA to IAA against boll
was attained. The rate of fibre elongation per day age was considered, it could be seen that the ratio in-
reached a maximum at about 15 DPA. The change in creased rapidly after 30 DPA (Figure 4). The ratio
cellulose content of fresh fibres showed a sigmoid changed in correspondence with the rate of cellulose
pattern with a definite lag phase during the early pe- accumulation during 30 44 DPA (Figure 2).
riod of fibre growth and a linear phase during 18 DPA In order to confirm the role of the ratio of ABA to
to 44 DPA. It was shown that the secondary wall IAA in the process of secondary wall thickening, the
thickening overlapped with fibre elongation. effect of a series of NAA, GA 3, ABA and kinetin on
It was observed that the GA S and iP + iPA levels secondary wall thickening was in vitro investigated
in the fibre remained almost constant during fibre de- during the period 30 to 44 DPA. Compared with the
236
Figure 4. Changes in the ratio of ABA to IAA of field-grown fi-
bres against boll age
Figure 5. The effect of ABA on the dry matter and cellulose ac-
cumulation in fibres cultured in vitro during 30 to 44 DPA. Vertical
bars represent SD.
control, the dry matter accumulation in the fibre was
markedly enhanced by 5 50 molL 1 ABA and the
cellulose content markedly increased in the 50
molL 1 ABA treatment. The dry matter and cellu-
lose accumulation in the fibre were not markedly en-
hanced by other treatments of ABA (Figure 5), nor by
those of NAA (0.5 10 molL 1), GA 3 (0.5 10
molL 1) and kinetin (0.1 5 molL 1) (data not
shown).
Previous literature indicated that the number of fi-
bres on each ovule was fixed before 10 DPA (Basra
and Malik 1984; DeLanghe 1986). In our study, when
cotton ovules were cultured under the same condi-
tions before 30 DPA, the length and dry weight of fi-
bres on each ovule were not significantly different at
30 DPA. Since the fibre did not elongate after 30
DPA, any differences of dry matter accumulation ob-
served during the period 30 44 DPA could only be
regarded as the result of different secondary wall
thickness caused by treatments with NAA, GA 3,
ABA and kinetin.
Discussion
Previous literature indicated that the change of ABA
content of the fibre against boll age and the first peak
of ABA content of the fibre occurred during fibre
elongation and the second during secondary wall
thickening (Davis and Addicott 1972; Gokani et al.
1998). Similar results were observed in the present
study. The experimental results showed that the ABA
content of the fibre reached the second peak at 37
DPA and seemed to have no evident relationship with
the rate of cellulose accumulation (Figures 2 and 3).
However, the ratio of ABA to IAA of fibres increased
rapidly after 30 DPA (Figure 4), and the accumula-
tion of cellulose in fibres was almost linear (Figure 2).
This suggests that the ratio of ABA to IAA in fibres
could have a regulating role on cellulose accumula-
tion and secondary wall thickening. This possibility
was supported by our in vitro data. The dry matter and
cellulose accumulation in fibres were markedly en-
hanced by ABA (Figure 5), but not by NAA, kinetin
or GA 3 (data not shown). This implies that ABA
might have a unique role in regulating the endoge-
nous hormones in favour of secondary wall thicken-
ing of the cotton fibre.
Considerable evidence indicates that ABA inhibits
fibre elongation, i.e., ABA has an opposite role to that
of IAA and GA S (Beasley and Ting 1973; Dhindsa et
al. 1976; Holt and Stewart 1994; Kosmidou-Dim-
itropoulou 1986; Nayyar et al. 1989). Based on our
observation, the ABA content of fibres reached a peak
at 16 DPA (Figure 3). The fibre continued to elongate
up to 30 DPA and the rate of fibre elongation per day
reached a peak about 15 DPA (Figure 1). The fibre
entered the linear phase of cellulose accumulation
around 18 DPA (Figure 2). The fact that ABA content
of fibres reached a peak during fibre elongation im-
plies that elevated levels of ABA in fibres might be a
signal for the onset of secondary wall thickening.
Subsequently, the ratio of ABA to IAA decreased so
that the fibre could slower the process of wall-tight-
ening caused by secondary wall thickening, and main-
tain the wall extensibility, the precondition of fiber
elongation. Once elongation completed, the relatively
higher ratio of ABA to IAA might be beneficial for
secondary wall thickening.

Acknowledgements
We thank Professor He Zhong-Pei for the use of her
Laboratory of Chemical Control Technology on Crop,
College of Crop Science, China Agricultural Univer-
sity, and for a generous gift of ELISA kits. We are
grateful to Professor Cheng Xu, Paula Brook and
Yang Zuo-Min for critically reading the manuscript.
This work was supported by the National Natural Sci-
ence Foundation of China (Grand No. 39800097).
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