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Plant Growth Regulation 35 : 233 – 237 , 200 1 . © 200 1 Kluwer

Plant Growth Regulation 35: 233237, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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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; 2 College 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 mol·L 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 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

cotton fibre properties (such as length, strength and 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-

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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 bres of eld-grown cotton plants and also explored the effect of NAA, GA 3 , kinetin and ABA on secondary wall thickening of the cultured - bre.

Materials and methods

Gossypium hirsutum L.cv Zhongmiansuo-12 plants were grown in the eld 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 ow- 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 bres. To min- imise the effect of environmental variations, data were collected from owers 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 bres were separated manually from the ovule. Both freshly-separated bres 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 modied 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 mol·L 1 NAA and 1 mol·L 1 GA 3 . Ovules were cultured for 14 days and, at 16 DPA, were transferred to a subculture medium modi- ed by adding 5 g·L 1 activated charcoal. At 30 DPA, ovules were transferred to further subculture media modied by adding one of a series of concentrations of NAA, GA 3 , kinetin or ABA instead of 5 mol·L 1 NAA and 1 mol·L 1 GA 3 . Each treatment consisted of at least ten asks. At 44 DPA, six asks of each

treatment were examined and ten seeds from every ask were selected at random. The seeds were washed several times with distilled water, then the bres were manually removed from the seeds. The bres were weighed after being oven-dried to a constant weight at 70 °C.The weight of bres was expressed in terms of dry weight per seed. Each data point represented the mean ± SD. The cellulose content of the bres was determined as described by Updegraff (1969). The extraction, purication 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 mmol·L 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 mol·L 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 mmol·L 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 nonspecic 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 mol·L 1 H 2 SO 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- centage recovery of each hormone was calculated by 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 nonspecic inhibitors in the extracts.

Figure 1. Fibre length vs. Boll age. Each data point is the mean of three replicates.

Figure 1. Fibre length vs. Boll age. Each data point is the mean of three replicates. Vertical bars represent±SD. The curve of the - bres grown on cotton plants is dened by Y= 0.0001X 4 0.0107X 3 + 0.266X 2 0.5563X + 0.0535. The r 2 co- efficient is 0.9991. The curve of the bres cultured in vitro is de- ned by Y= 0.0002X 4 0.0133X 3 + 0.2815X 2 0.5612X 0.0027. The r 2 coefficient is 0.9981.

Results

It is well documented that cell walls from bres cul- tured in vitro are remarkably similar to those derived from the bre of eld-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 bre on the plant with that of the cultured bre. The length and cellulose content of the bre were tted to polynomial equations of different degrees, and the best-t equation was determined statistically by performing a t-test for different regression coeffi- cients (r 2 coefficient). The data on bre length and cellulose content in bres were explained adequately by a forth degree polynomial equation (Figures 1 and 2). Although the nal length of the cultured bre was shorter than that on the plant, the entire developmen- tal sequences of both were quite similar. Fibres both grown on the plant and cultured in vitro continued to elongate up to 30 DPA when the maximum length was attained. The rate of bre elongation per day reached a maximum at about 15 DPA. The change in cellulose content of fresh bres showed a sigmoid pattern with a denite lag phase during the early pe- riod of bre growth and a linear phase during 18 DPA to 44 DPA. It was shown that the secondary wall thickening overlapped with bre elongation. It was observed that the GA S and iP + iPA levels in the bre remained almost constant during bre de-

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Figure 1. Fibre length vs. Boll age. Each data point is the mean of three replicates.

Figure 2. Changes in cellulose content of bres against boll age.

Each data point is the mean of three replicates. Vertical bars represent±SD. The curve of the
Each
data
point is
the mean of three replicates. Vertical bars
represent±SD. The curve of the fibres grown on cotton plants is
defined
by Y=−0.0005X 4 +0.0351X 3 −0.6045X 2 +3.5161X+1.621.
The r 2 coefficient is 0.9885. The curve of the fibres cultured in vitro
is
defined
by
Y=−0.0002X 4 +0.0158X 3 −0.1674X 2 +0.8778X+1.1916. The r 2 co-
efficient is 0.9944.

Figure 3. Changes in levels of plant hormones of eld-grown - bres against boll age. Each data point is the mean of three repli- cates estimated by an indirect ELISA. Vertical bars represent ± SD.

velopment, whereas the IAA and ABA levels changed obviously as the bre developed (Figure 3). No rela- tionship was observed between the level of either IAA or ABA and the rate of cellulose accumulation. However, when the ratio of ABA to IAA against boll age was considered, it could be seen that the ratio in- creased rapidly after 30 DPA (Figure 4). The ratio changed in correspondence with the rate of cellulose accumulation during 30 44 DPA (Figure 2). In order to conrm the role of the ratio of ABA to IAA in the process of secondary wall thickening, the effect of a series of NAA, GA 3 , ABA and kinetin on secondary wall thickening was in vitro investigated during the period 30 to 44 DPA. Compared with the

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236 Figure 4. Changes in the ratio of ABA to IAA of fi eld-grown fi -

Figure 4. Changes in the ratio of ABA to IAA of eld-grown - bres against boll age

236 Figure 4. Changes in the ratio of ABA to IAA of fi eld-grown fi -

Figure 5. The effect of ABA on the dry matter and cellulose ac- cumulation in bres cultured in vitro during 30 to 44 DPA. Vertical bars represent ± SD.

control, the dry matter accumulation in the bre was

markedly enhanced by 5 50 mol·L 1 ABA and the cellulose content markedly increased in the 50 mol·L 1 ABA treatment. The dry matter and cellu- lose accumulation in the bre were not markedly en- hanced by other treatments of ABA (Figure 5), nor by

those of NAA

(0.5 10 mol·L 1 ), GA 3 (0.5 10

mol·L 1 ) and kinetin (0.1 5 mol·L 1 ) (data not shown). Previous literature indicated that the number of - bres on each ovule was xed 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 - bres on each ovule were not signicantly different at 30 DPA. Since the bre 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 bre against boll age and the rst peak of ABA content of the bre occurred during bre 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 bre 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 bres increased rapidly after 30 DPA (Figure 4), and the accumula- tion of cellulose in bres was almost linear (Figure 2). This suggests that the ratio of ABA to IAA in bres 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 bres 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 bre. Considerable evidence indicates that ABA inhibits bre 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 bres reached a peak at 16 DPA (Figure 3). The bre continued to elongate up to 30 DPA and the rate of bre elongation per day reached a peak about 15 DPA (Figure 1). The bre entered the linear phase of cellulose accumulation around 18 DPA (Figure 2). The fact that ABA content of bres reached a peak during bre elongation im- plies that elevated levels of ABA in bres might be a signal for the onset of secondary wall thickening. Subsequently, the ratio of ABA to IAA decreased so that the bre could slower the process of wall-tight- ening caused by secondary wall thickening, and main- tain the wall extensibility, the precondition of ber elongation. Once elongation completed, the relatively higher ratio of ABA to IAA might be benecial 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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