Plant Science 225 (2014) 15–23

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The effect of mepiquat chloride on elongation of cotton (Gossypium hirsutum L.) internode is associated with low concentration of gibberellic acid Li Wang a,b , Chun Mu a , Mingwei Du a , Yin Chen a , Xiaoli Tian a , Mingcai Zhang a,∗ , Zhaohu Li a,∗ a b

State Key Laboratory of Plant Physiology and Biochemistry, College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China College of Life Sciences, Henan Normal University, Xinxiang, Henan 453007, China

a r t i c l e

i n f o

Article history: Received 25 February 2014 Received in revised form 5 May 2014 Accepted 12 May 2014 Available online 20 May 2014 Keywords: Mepiquat chloride GA GA biosynthesis Cell elongation Internode elongation Cotton

a b s t r a c t The growth regulator mepiquat chloride (MC) is globally used in cotton (Gossypium hirsutum L.) canopy manipulation to avoid excess growth and yield loss. However, little information is available as to whether the modification of plant architecture by MC is related to alterations in gibberellic acid (GA) metabolism and signaling. Here, the role of GA metabolism and signaling was investigated in cotton seedlings treated with MC. The MC significantly decreased endogenous GA3 and GA4 levels in the elongating internode, which inhibited cell elongation by downregulating GhEXP and GhXTH2, and then reducing plant height. Biosynthetic and metabolic genes of GA were markedly suppressed within 2–10 d of MC treatment, which also downregulated the expression of DELLA-like genes. A remarkable feedback regulation was observed at the early stage of MC treatment when GA biosynthetic and metabolic genes expression was evidently upregulated. Mepiquat chloride action was controlled by temporal translocation and spatial accumulation which regulated GA biosynthesis and signal expression for maintaining GA homeostasis. The results suggested that MC application could reduce endogenous GA levels in cotton through controlled GA biosynthetic and metabolic genes expression, which might inhibit cell elongation, thereby shortening the internode and reducing plant height. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Cotton (Gossypium hirsutum L.) is a worldwide grown and important fiber crop. It is cultivated annually on about 4 million ha and 20% of global cotton production is in China. Cotton plant has a perennial and indeterminate growth habit which is very sensitive to environmental changes and management. Excessive vegetative growth can lead to undesirable shade within the plant canopy, fruit abscission and yield reductions [1]. Much labor is required to cut the top buds of main stem and branches to control excessive growth

Abbreviations: MC, mepiquat chloride; GA, gibberellic acid; GA20ox, gibberellin 20-oxidase; GA3ox, gibberellin 3-oxidase; GA2ox, gibberellin 2-oxidase; PGR, plant growth regulator; EXP, expansin; XTH, xyloglucan endotransglucosylase/endohydrolases; 2ODD, 2-oxoglutarate-dependentdioxygenase; PAC, paclobutrazol; Pro-Ca, prohexadione-Ca; LC/MS, liquid chromatography–mass spectrometry; SEM, scanning electron microscope. ∗ Corresponding authors. Tel.: +86 10 6273 3049; fax: +86 10 6273 3427. E-mail addresses: [email protected] (M. Zhang), [email protected] (Z. Li). http://dx.doi.org/10.1016/j.plantsci.2014.05.005 0168-9452/© 2014 Elsevier Ireland Ltd. All rights reserved.

in China. The use of plant growth regulators is one of the strategies routinely used in cotton production for manipulating plant canopy, adjusting plant vegetative and reproductive growth and improving yield by decreasing auto-shading [2–4]. The plant growth regulator mepiquat chloride (MC), N,Ndimethylpiperidinium chloride, is a water soluble organic molecule favorably absorbed by the green parts and redistributed throughout the plant, and has been most successful and worldwide used to control plant canopy size in cotton production [4]. Application of MC almost leads to a more compact plant [5] resulting from shortened internode elongation, reduced main stem nodes, and decreased leaf expansion and leaf area in cotton [2,3,6]. Several literatures hypothesize that MC inhibits gibberellic acid (GA) biosynthesis to decrease cell elongation which can eventually result in lower cotton leaf area and shorter internodes [7–9]. The GA synthesis inhibitors are divided into onium compounds, nitrogen-containing heterocyclic compounds and acylcyclohexanediones, which control specific for the enzymes catalyzing the steps in GA synthesis [9]. Onium compounds, such as chlorocholine chloride (CCC), chlorphonium and AMO-1618, block the cyclases

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ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) involved in the early steps of GA biosynthesis [8,10]. Because MC belongs to onium compound growth regulators, it was proposed that MC may have the same site of inhibition as CCC and AMO-1618 based on their similar chemical structures [8,11]. However, little is known of the effects of MC on the bioactive GA levels and how to control GA biosynthesis in plants. It is well known that GA is a key regulator of shoot growth in plants. Bioactive GAs are present predominantly in actively growing and elongating tissues, such as shoot apices, young, developing internodes and expanding leaves [12], suggesting that bioactive GAs are primarily synthesized at the their site of action. GA regulates cell elongation and cell wall relaxation by inducing the expression of expansins (EXP) and xyloglucan endotransglucosylase/hydrolases (XTHs), which cause cell wall changes that allows turgor-driven cell expansion [13–15]. Terpenecyclases, cytochrome P450 monooxygenases and 2-oxoglutarate-dependentdioxygenases are the key three-type GA biosynthetic enzymes. Bioactive GAs is deactivated by GA2-oxidase (GA2ox), a member of the 2-oxoglutarate-dependentdioxygenase (2ODD) family. All dioxygenases are encoded by a small multigene family. Bioactive GA levels are maintained by feedback and feedforward regulation of genes encoding 2ODDs that catalyze the late steps of the GA pathway [15,16]. In addition to the active GA level, DELLAs as the component of GA signaling has been found to affect cell elongation modulated by light in Arabidopsis [17,18] and Sorghum bicolor [19]. However, it is unclear whether MC-induced growth inhibition is associated with GA metabolism and signaling. This study was aimed to evaluate the roles of GA metabolism and signaling in MC-induced inhibition of internode elongation in cotton. It was hypothesized that MC inhibited the elongation of internode and cell by downregulating GA biosynthesis or upregulating GA sensitivity repressor, DELLAs. Therefore, we would have determined the effect of MC on the levels of endogenous GA and expression pattern of genes encoding GA metabolism and signaling pathway during internode elongation. We also analyzed the effect of MC on the internode cell morphology and the expression pattern of cell wall loosening genes.

2. Materials and methods 2.1. Plant materials and treatments Seeds of cotton (Gossypium hirsutum L., Xinkang 4) were sown in pots (13 cm diameter and 13 cm in height) filled with a mixture of vermiculite and peat (1:1) in a growth chamber with a 14 h photoperiod at a 28/20 ◦ C day/night temperature cycle, with a light intensity of 550 ␮mol m−2 s−1 and at 60% relative humidity. Four seeds were sown per pot. After the seedlings reached the first true leaf stage, they were thinned to one plant per pot. Seventy-five seedlings were randomly divided into five treatments comprising four concentrations of MC (40, 80, 160 and 320 mg/l) and deionized water as control. There were 15 seedlings per treatment. The MC standard (purity, 97.0%) was supplied by Hebei Guoxin ahadzi-nonon Biological Technology Co., Ltd. (Hejian, Heibei, China). At the three-leaf stage, deionized water and the four concentrations of MC were separately applied to the seedlings by foliar spray. Plant height was measured at 10 d after treatment, as the distance from the cotyledonary node to the tip of the shoot apex. The first, second and third internode lengths were also measured at 10 d after treatment. The experiment was repeated three times. Based on the results of the concentration experiment, 80 mg/l MC was selected for studying the effect on elongation and cell morphology of the internode, endogenous GA content and

gene expression levels. Two-hundred and eighty seedlings were randomly divided into 2 treatments of deionized water as control and MC application. At the three-leaf stage, the two treatments were applied by foliar spray. Only the second internode, an elongating internode, was labeled. Plant height and internode length were measured before plants were subjected to MC, and then at 2day intervals for 10 days. The upper halves of the internodes were sampled at 0, 3, 6 h, 1, 2, 4, 6, 8 and 10 d after treatment for RNA extraction, and at 0, 2, 6 and 10 d after treatment for analysis of endogenous GA content. All the samples were immediately frozen in liquid nitrogen and then stored at −80 ◦ C. The experiment was repeated three times. To better understand the time course of MC’s effect on the internode elongation, the seedlings were selected at the sevenleaf stage to examine the translocation of MC. One hundred and sixty seedlings were randomly assigned to the two treatments (deionized water as control and MC at 80 mg/l). There were 60 seedlings per treatment, and each treatment had three replications (20 seedlings per replication). At the seven-leaf stage, 400 ␮l MC was evenly dripped onto the surface of the fifth leaf several times with a drop of approximately 5 ␮l each time. The third leaf, treated leaf, seventh leaf, upper stem (above the treated leaf), lower stem (below the treated leaf) and roots were sampled at 6, 12, 24, 48 and 96 h after treatment, rinsed several times with distilled water, and then stored at −20 ◦ C until analyzed. 2.2. Microscopy analysis For light microscopy, samples taken from upper zones of the second internode (approximately 5 mm each) at 10 d after treatment were fixed with FAA solution (5% acetic acid, 45% ethanol, and 5% formaldehyde) under a vacuum [20] embedded in paraffin and microtome sectioned with a Leitz 1515 microtome. Samples on slides were deparaffinised, stained with safranin-fast green [21] and fixed with Eukitt (O. Kindler GmbH, Freiburg, Germany). Microscopy was conducted with a Zeiss Axioplan microscope (Carl Zeiss AG, Oberkochen, Germany). For scanning electron microscope (SEM), samples taken from upper zones of the second internode (approximately 5 mm each) at 10 d after treatment were fixed with 2.5% glutaraldehyde solution at 4 ◦ C overnight. The fixed samples were dehydrated with a gradual ethanol series, dried by a critical point drying method using liquid carbon dioxide (Model HCP-2, Hitachi, Tokyo, Japan), gold-coated with an IB-3 ion sputter coater (EIKO, Tokyo, Japan) and then observed under a S-34000N variable pressure scanning electron microscope (Hitachi, Tokyo, Japan). 2.3. RNA extraction and real-time PCR analysis Total RNA was extracted from the second internode with the Plant RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. Two microgram of total RNA was DNase I treated and used for cDNA synthesis with oligo(dT) primers and reverse transcriptase (Promeaga). The synthesized cDNAs were diluted 10 times in H2 O and their concentrations were normalized based on the amplification of GhUBQ7. Quantitative RT-PCR was carried out in an Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems). The reaction volume was 15 ␮l which contained 1.5 ␮l of diluted cDNA, 0.3 ␮l of ROX reference dye, 0.3 ␮l of each 10 ␮M forward primer and reverse primer, and 7.5 ␮l SYBR Premier Ex Taq mix (Takara, Japan). PCR amplification was performed using two-step cycling conditions of 95 ◦ C for 30 s, followed by 40 cycles of 95 ◦ C for 5 s and 60 ◦ C for 35 s. Primers were listed in Supplementary Table S1. Melt curve analysis of qPCR samples showed only one product for each gene primer reaction, confirming specific amplification. The levels of each gene

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transcript were calculated relative to its corresponding untreated control. Fold-changes of RNA transcripts were calculated by the 2−Ct method [22]. Values reported represent the average of two biological repeats with three independent trials.

2.4. Quantitative analysis of endogenous GA3 and GA4 To determine whether a decrease in internode cell elongation was involved in GA biosynthesis following MC application, the endogenous GAs such as GA3 and GA4 content were determined. For quantitative analysis of GAs, samples taken from upper zones of the second internode (0.2 g) were homogenized in 80% (v/v) aqueous methanol. After adding 1 ng 2 H-labeled GA3 and GA4 (OlChemIm, Ltd., Olomouc, Czech Republic) as internal standards, the filtered extracts were evaporated. Purification of GAs was done according to Xiao et al. [23]. The residues were redissolved in NH4 Ac (0.1 M, pH 9.0) and then centrifuged at 27 000 × g for 20 min. The supernatant was purified by passage through a Bondesil DEA column (5 g, Varian Associates, Palo Alto, CA, USA) and then further purified using a Sep-Pak C18 column (Waters Associates, Milford, MA, USA). The column was eluted with 50% methanol. The effluent was evaporated, dissolved in 50% methanol, and then analyzed with a liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) system (TSQ Quantum Ultra, Thermo-Finnigan, Ringoes, NJ, USA). MS/MS data were then analyzed using the software Xcalibur 2.0 (Thermo-Finnigan) and quantified by reference to ratios of specific ions of GAs in natural samples over those of internal standards using equations for isotope dilution analysis.

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3. Results 3.1. MC decreased plant height and internode length The effects of different concentrations of MC on plant height and internode length of cotton seedlings varied in a dose-dependent manner (Fig. 1). At concentrations increasing from 40 to 320 mg/l, the plant height decreased by 26 to 68%, the second internode length by 73 to 188% and the third internode length by 21 to 286% (Fig. 1A and B). The time course of MC’s action on the second internode length showed that MC at 80 mg/l significantly inhibited the elongation, and the length of second internode was reduced by 23, 44, 101, 147 and 143% in MC-treated plants at 2, 4, 6, 8 and 10 d after treatment (Fig. 1D), respectively. Thus, the height of MC-treated plants was decreased by 5, 16, 28, 38 and 49%, respectively (Fig. 1E).

3.2. MC inhibited internode cell transverse and longitudinal growth The morphology of the internode cells was investigated to determine whether cell elongation was repressed by MC application to shorten the internode (Fig. 2). MC-treated plants had significantly smaller stem diameter than control plants at the 10th day after treatment (Fig. 2A and B). In stem transverse sections, the transverse size of the pith cell and vessel was markedly decreased by MC (Fig. 2C and D). The internode epidermal cells showed shorter longitudinal lengths in MC-treated plants than control plants (Fig. 2E and F). In stem longitudinal sections, the longitudinal size of the cortex and pith cells was significantly inhibited by MC (Fig. 2G and H).

2.5. Extraction and assay of MC content The MC was extracted according to the slightly modified method of Li et al. [24]. Leaf (1 g), stem (1 g) and root (2 g) samples were weighed into a 50 ml centrifuge tube. The leaf and stem samples were extracted with 10 ml methanol–ammonium acetate solution (10 mM, pH 3.0, 10:90, v/v). For root samples, 20 ml extract solvent was used. The centrifuge tube was shaken in an air-bath mechanical shaker for 2 h at room temperature and then extracted in an ultrasonic bath for 20 min. Then the mixture was centrifuged at 2500 × g for 10 min. After the extracts were centrifuged, 5 ml of the supernatant was passed through C-18 SPE cartridge (3 ml/200 mg, AGT CleanertTM , Agela) preactivated with 5 ml methanol and 5 ml ultrapure water. The SPE was washed and eluted with 5 ml ultrapure water, and the eluent solution was dried under vacuum. The extracts were redissolved in 2 ml ultrapure water. The treated leaf extracts were diluted 10 times with ultrapure water before analysis. One milliliter extract solution was transferred to a HPLC sample vial for instrumental analysis after filtration through a 0.22 ␮m polypropylene filter. A standard stock solution of MC (1000 mg/l) was prepared in ultrapure water and stored at −20 ◦ C. Working standard solutions were prepared by dilution of the corresponding stock standard solution with ultrapure water and stored at −20 ◦ C. Quantification of MC using LC–MS/MS (Agilent Technologies, USA) system was done as described previously [24].

2.6. Statistical analysis Data for plant height and internode length among treatments at different concentrations of MC were statistically analyzed by one-way analysis of variance (ANOVA) and treatment means were compared using the LSD at P < 0.05. Other data were subjected to independent Student’s t-test.

3.3. Translocation and accumulation of MC in cotton seedlings Except for the 3rd leaf, MC was detected in other organs such as leaves, stem and root at 6 h of treatment (Table 1). Its accumulation increased constantly with time, and the peak accumulation across organs was observed at 96 h (except for the 5th leaf at 24 h and lower stem at 48 h). The highest accumulation was in the upper stem at 96 h after treatment, except for the 5th leaf.

3.4. MC reduced GA3 and GA4 accumulation There were obvious decreases in GA3 and GA4 contents in the MC-treated plants during internode elongation (Fig. 3). On the first 2 d of treatment, there were no significant differences in GA3 and GA4 levels between MC-treated and control plants (Fig. 3A and B). However, the values of GA3 and GA4 in MC-treated plant decreased significantly by 304 and 43% on the 6th day and 111 and 26% on the 10th day after treatment.

3.5. MC decreased the expression levels of GhEXP and GhXTH2 Expression of the cell wall loosening genes GhEXP and GhXTH2 was markedly affected in cotton seedlings exposed to MC treatment during internode elongation (Fig. 4). The expression of GhEXP in MC-treated plants was evidently downregulated within 2–10 d of treatment (Fig. 4A). The expression of GhEXP decreased 67–155% within 2–10 d after MC treatment. A similar variation was observed for the expression of GhXTH2 in seedlings treated with MC (Fig. 4B). The expression level of GhXTH2 in MC-treated plants was only 30–47% of that in control plants at 2–10 d after treatment.

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Fig. 1. Effect of mepiquat chloride (MC) on plant height and internode length of cotton seedlings. (A) Different concentrations of MC affected plant height at 10th day after treatment. (B) Changes in internode length of cotton seedlings at different concentrations of MC on the 10th day after treatment. (C) Different concentrations of MC affected morphological changes in internodes on the 10th day after treatment. Nodes were numbered from bottom to top of the stem and node 0 is the cotyledonary node. Bar = 2 cm. (D) Changes in the second internode elongation treated with 80 mg/l MC within 10 d. (E) Changes in height of cotton seedlings exposed to 80 mg/l MC within 10 d. Values are the means ± SD (n = 15); bars with the same letter are not significantly different at P < 0.05. Asterisks indicate a significant difference (P < 0.05) compared with the corresponding control.

3.6. MC regulated the expression levels of GAs biosynthetic genes The expression levels of genes encoding GA biosynthesis during internode elongation of cotton seedlings exposed to MC are shown in Fig. 5. The expression levels of GhCPS and GhKS in MCtreated plants were significantly increased by 63–148% and by 35–84% at 3–24 h of treatment with MC (Fig. 5A and B). However,

the expression levels of GhCPS and GhKS in MC-treated plants were only 16–48% and 26–75% of that in control plants at 2–10 d after treatment, respectively. Similar trends were observed for the expression levels of GA20ox and GA3ox. In MC-treated seedlings, the expression levels of GhGA20ox1, GhGA20ox2 and GhGA3ox1 were considerably upregulated at 3–24 h and then significantly downregulated at 2–10 d after

Table 1 Translocation and accumulation of MC from the treated leaf in cotton seedlings within 96 h of MC treatment.a MC content (ng g−1 FW)

7th leaf 5th leaf 3rd leaf Upper stem Lower stem Root

6h

12 h

39.6 ± 11.9 3938.7 ± 110.8 ND 161.6 ± 2.9 309.1 ± 3.0 2.4 ± 1.2

63.0 4794.3 11.3 413.7 616.9 17.4

24 h ± ± ± ± ± ±

3.3 223.4 1.3 11.0 18.0 0.2

140.1 4994.6 14.7 685.2 1401.7 36.1

48 h ± ± ± ± ± ±

8.1 458.6 0.9 22.0 62.1 5.7

265.1 4773.1 20.4 1387.6 1470.0 100.7

96 h ± ± ± ± ± ±

66.8 519.8 0.9 98.6 112.2 20.1

1377.5 4400.8 31.1 2452.8 1360.8 161.1

± ± ± ± ± ±

85.6 268.7 2.3 158.9 99.9 7.2

a Each value represents means ± SE (n = 3). Leaf was numbered from the base of stem. ND, not detected. 5th leaf, MC-treated leaf in cotton seedling. Upper stem, stem above the treated leaf. Lower stem, stem below the treated leaf.

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Fig. 2. Morphology of the internode cell of cotton seedlings 10 d after treatment with mepiquat chloride (MC). Transversal sections of the second internode in control (A, C) and MC-treated (B, D) cotton plants observed by SEM and light microscope; bar = 1 mm and 500 ␮m, respectively. Epidermal cells of the second internode in control (E) and MC-treated (F) cotton plants at the axial direction observed by SEM; bar = 100 ␮m. Longitudinal sections of second internode in control (G) and MC-treated (H) cotton plants observed by light microscope; bar = 500 ␮m. Ve, vessel; Pi, pith; Co, cortex.

treatment compared with control (Fig. 5C–E). The largest reductions in GhGA20ox1, GhGA20ox2 and GhGA3ox1 expression levels by MC were observed 6 d after treatment, when the expression levels of these genes were reduced to 13%, 17% and 20% compared with that in control plants, respectively. 3.7. MC modulated the expression level of GA2-oxidases and DELLA genes The GA2-oxidases and DELLA genes are key regulatory genes that decrease bioactive GA and its signaling in plant. In MC-treated

seedlings, the expression of GA catabolism genes (GhGA2ox1, GhGA2ox3, GhGA2ox4 and GhGA2ox6) showed a similar expression pattern as GA biosynthesis genes such as GhCPS and GhKS (Fig. 6A–D). At 3–24 h, MC treatment markedly improved the expression levels of GhGA2ox1, GhGA2ox3, GhGA2ox4 and GhGA2ox6, but significantly decreased those expression levels within 2–10 d. The largest reductions in GhGA2ox1, GhGA2ox3, GhGA2ox4 and GhGA2ox6 expression levels by MC occurred 6 d after treatment, when the expression levels of these genes were reduced to 33%, 18%, 16% and 13% of that in control plants, respectively. The expression levels of GhGAI4a and GhGAI4b in MC-treated

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Fig. 3. Changes in endogenous GA3 (A) and GA4 (B) contents in the second internode of cotton seedlings due to mepiquat chloride. Values are the means ± SD (n = 4); and asterisks indicate a significant difference (P < 0.01) compared with the corresponding control.

seedlings were increased by 10–45% and by 23–80% after 3–24 h treatment with MC (Fig. 6E and F). However, the expression levels of GhGAI4a and GhGAI4b were markedly decreased by 83 to 290% and by 151–279% in MC-treated plants within 2–10 d after MC application. 4. Discussion It has been shown that MC can induce more compact plants through shortening the internode and decreasing leaf expansion in cotton [2–5]. This phenomenon was confirmed by our data, which showed an inhibition of plant height, stem diameter and internode length by different concentrations of MC. Moreover, the size of vessel and epidermal and inner cells was significantly reduced by MC treatment, suggesting that the shorter internode was largely the result of decreased cell transverse and longitudinal growth in cotton seedlings exposed to MC. Similar results were obtained for the epidermal cells of internodes in tobacco treated with uniconazole, an inhibitor of GA biosynthesis [25]. It is essential for cell growth to selectively loosen and rearrange cell wall to promote a turgor-driven expansion [26]. EXP and XTH, the important protein classes involved in this process, are regulated by GA to induce the elongation of leaf and stem [13,14]. In

the present study, we found that MC treatment significantly downregulated GhEXP and GhXTH2 within 2–10 d after treatment. In particular, the contents of bioactive GAs such GA3 and GA4 were markedly decreased by MC application during internode elongation. These indicated that MC inhibited internode cell growth by reducing bioactive GAs leading to decreased expression of GhEXP and GhXTH2. Meanwhile, these also supported the hypothesis by Rademacher [8] and Srivastava [9] that MC inhibits cell elongation involved in GA biosynthesis based on its molecular and chemical character. The bioactive GAs, such as GA3 and GA4 , are synthesized from trans-geranylgeranyl diphosphate (GGDP) and mainly involve seven kinds of metabolic enzymes such as CPS, KS, ent-kaurene oxidase (KO), ent-kaurenoic acid oxidase (KAO), GA20ox, GA3ox, and GA2ox whose genes are isolated from various plants by their related mutants [15,16]. MC significantly suppressed the expression of GhCPS, GhKS, GhGA20ox and GhGA3ox within 2–10 d of treatment during internode elongation, and consistently with the changes in GA3 and GA4 levels in the internode of seedlings treated with MC. Similarly, paclobutrazol (PAC), an inhibitor of KO, leads to a decrease of GA levels with a growth reduction [8], and prohexadione-Ca (Pro-Ca) which inhibited GA biosynthesis at the later stages significantly decreased the endogenous bioactive GA1 contents [27].

Fig. 4. Regulation of the relative expression of GhEXP (A) and GhXTH2 (B) during the internode elongation of cotton seedlings by mepiquat chloride. Expression was detected by RT-PCR by employing gene-specific primer pairs and UBQ7 as a reference gene, as described in Experimental procedures. Transcript levels of the control seedling at 0 h after treatment were set at 1. Values are the means ± SD (n = 4); and asterisks indicate a significant difference (P < 0.05) compared with the corresponding control.

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Fig. 5. The relative expression of GhCPS (A), GhKS (B), GhGA20ox1 (C), GhGA20ox2 (D) and GhGA3ox1 (E) in the internode of MC-treated and control cotton seedlings. Values are the means ± SD (n = 4), and asterisks indicate a significant difference (P < 0.05) compared with the corresponding control.

Bioactive GAs are mostly maintained at a stable level through a homeostatic mechanism based on the feedback regulation by GA metabolism [15]. Inhibition of GA production, by treatment with GA biosynthesis inhibitors, PAC, uniconazole and Pro-Ca, causes upregulation of some of the GA biosynthesis genes (GA20ox and GA3ox) and downregulation of GA catabolism genes (GA2ox) in some plant species [28–31]. In this study, an evident feedback regulation of GA biosynthesis was observed at 3–24 h after MC application, which showed an increase in the expression of GhCPS, GhKS, GhGA20ox and GhGA3ox. However, the expression of GhGA2ox1, GhGA2ox3, GhGA2ox4 and GhGA2ox6 was also significantly upregulated by MC. These results suggested that GhGA2ox was more sensitive to MC and followed a different response mechanism compared to that of other plant species. This finding also indicated that inhibition of GA biosynthesis by MC might be carried out by promotion of the expression of GhGA2ox. Similar results were observed for OsGA2ox6 [32] and OsGA2ox1 [33] in rice and PsGA2ox1 and PsGA2ox2 in pea [34]. Several studies showed that regulation of GA biosynthesis occurs mainly at the later stages of pathway such as GA20ox and GA3ox [16,35,36]. Our results showed that GhCPS and GhKS participated in the feedback regulation of GA biosynthesis by MC. Huang

et al. [37] showed that the expression levels of TaCPS-B, TaCPS-D, TaKO-D and TaKAO-D were increased by PAC in wheat, but GA3 decreased the expression levels of TaCPS-B and TaCPS-D. In addition, the expression of KO, KAO1 and KAO2 was also subject to negative feedback regulation. Bioactive GA levels are effectively controlled within a regular range by feedback/feedforward mechanisms. However, various feedback loops in the GA signaling network may be strongly dependent on the changes in the dosage of regulators due to environmental variation or through transport from other tissues [16,38–40]. From the translocation and accumulation of MC in cotton seedlings, the accumulation of MC in elongating internode (upper stem) showed a steady increase within 4 d of MC treatment. MC accumulation occurred at a lower level during the first 24 h which could result in upregulation of GA biosynthetic and metabolic genes for maintaining GA homeostasis (GA3 and GA4 levels showed no evident changes). However, MC accumulation was at a higher level within 2 to 4 d, and the GA biosynthetic and metabolic genes were significantly repressed, leading to lower GA3 and GA4 levels. This finding showed that the translocation and accumulation of MC controlled GA biosynthesis for maintaining GA homeostasis.

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Fig. 6. Expression patterns of metabolic genes (GhGA2ox1, -3, -4 and -6 shown in A, B, C and D, respectively) and DELLA-like genes (GhGAI4a and GhGAI4b shown in E and F) in the internode of MC-treated and control cotton seedlings. Values are the means ± SD (n = 4), and asterisks indicate a significant difference (P < 0.05) compared with the corresponding control.

In addition to the active level of GAs, DELLAs have been shown to repress GA-dependent growth processes [41,42]. Being consistent with the inhibition of mesocotyl and cell elongation in Sorghum bicolor, PAC significantly increased the DELLAs expression level. Similar results were found in this study as the expression levels of DELLA-like genes (GhGAI4a and GhGAI4b) were markedly increased in cotton seedlings treated with MC within the first 24 h. However, the expression level of DELLA-like genes decreased upon application of MC within 2–10 d. Band et al. [43] reported that DELLA mRNA levels is repressed by accumulated DELLA protein when GA level is reduced. Therefore, the downregulation of DELLA-like genes by MC may be as a result of negative feedback regulation. Similarly, the application of Pro-Ca caused a downregulation of both DELLA genes (GAI and RGA) in strawberry runner samples [30]. In conclusion, the application of MC decreased the endogenous bioactive GAs (GA3 and GA4 ), leading to inhibition of cell

elongation, lowering of plant height and shortening of internode length. The MC reduced endogenous GAs contents by downregulating GA biosynthetic (GhCPS, GhKS, GhGA20ox and GhGA3ox) and metabolic (GhGA2ox) genes, and could invoke DELLA-like genes (GhGAI4a and GhGAI4b) expression. Moreover, the translocation and accumulation of MC was involved in the regulation of GA biosynthesis and signaling for maintaining GA homeostasis. These findings provide novel insights into molecular understanding of shortening cotton internode by MC. This is essential for satisfactory vegetative growth management on cotton and lays the theoretical foundation for the application frequency and doses of MC on cotton. Acknowledgements This work was supported by National Natural Science Foundation of China (Grant No. 31271628). We thank Dr. Langtao Xiao

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(Hunan Agricultural University, China) for his excellent technical assistance in quantitative analysis of GAs. The authors also thank Dr. Calvin G. Messersmith (North Dakota State University, USA) and Dr. A. Egrinya Eneji (University of Calabar, Nigeria) for technical and linguistical improvement of the manuscript. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.plantsci. 2014.05.005. References [1] D. Zhao, D.M. Oosterhuis, Pix plus and mepiquat chloride effects on physiology, growth, and yield of field-grown cotton, J. Plant Growth Regul. 19 (4) (2000) 415–422. [2] J.D. Siebert, A.M. Stewartb, Influence of plant density on cotton response to mepiquat chloride application, Agron. J. 98 (6) (2006) 1634–1639. [3] X. Ren, L. Zhang, M. Du, J.B. Evers, W. van der Werf, X. Tian, Z. Li, Managing mepiquat chloride and plant density for optimal yield and quality of cotton, Field Crops Res. 149 (2013) 1–10. [4] C.A. Rosolem, D.M. Oosterhuis, F.S. de Souza, Cotton response to mepiquat chloride and temperature, Sci. Agric. 70 (2) (2013) 82–87. [5] V.R. Reddy, A. Trent, B. Acock, Mepiquat chloride and irrigation versus cotton growth and development, Agron. J. 84 (6) (1992) 930–933. [6] V.R. Reddy, D.N. Baker, H.F. Hodges, Temperature and mepiquat chloride effects on cotton canopy architecture, Agron. J. 82 (2) (1990) 190–195. [7] T.A. Kerby, Cotton response to mepiquat chloride, Agron. J. 77 (4) (1985) 515–518. [8] W. Rademacher, Growth retardants: effects on gibberellin biosynthesis and other metabolic pathways, Annu. Rev. Plant Physiol. Plant Mol. Biol. 51 (2000) 501–531. [9] L.M. Srivastava, Gibberellins, in: L.M. Srivastava (Ed.), Plant Growth and Development, Academic Press, New York, NY, USA, 2002, pp. 172–181. [10] I. Shechter, C.A. West, Biosynthesis of Gibberellins. IV. Biosynthesis of cyclic diterpenes from trans-geranylgeranyl pyrophosphate, J. Biol. Chem. 244 (12) (1969) 3200–3209. [11] D.T. Dennis, C.D. Upper, C.A. West, An enzymic site of inhibition of gibberellin biosynthesis by Amo 1618 and other plant growth retardants, Plant Physiol. 40 (5) (1965) 948–952. [12] P. Hedden, A.L. Phillips, Gibberellin metabolism: new insights revealed by the genes, Trends Plant Sci. 5 (12) (2000) 523–530. [13] Y. Lee, H. Kende, Expression of ␣-expansin and expansin-like genes in deepwater rice, Plant Physiol. 130 (3) (2002) 1396–1405. [14] A. Jan, G. Yang, H. Nakamura, H. Ichikawa, H. Kitano, M. Matsuoka, H. Matsumoto, S. Komatsu, Characterization of a xyloglucan endotransglucosylase gene that is up-regulated by gibberellin in rice, Plant Physiol. 136 (3) (2004) 3670–3681. [15] D.M. Ribeiro, W.L. Araújo, A.R. Fernie, J.H. Schippers, B. Mueller-Roeber, Translatome and metabolome effects triggered by gibberellins during rosette growth in Arabidopsis, J. Exp. Bot. 63 (7) (2012) 2769–2786. [16] S. Yamaguchi, Gibberellin metabolism and its regulation, Annu. Rev. Plant Biol. 59 (2008) 225–251. [17] P. Achard, L. Liao, C. Jiang, T. Desnos, J. Bartlett, X. Fu, N.P. Harberd, DELLAs contribute to plant photomorphogenesis, Plant Physiol. 143 (3) (2007) 1163–1172. [18] M. de Lucas, J.M. Daviere, M. Rodriguez-Falcon, M. Pontin, J.M. Iglesias-Pedraz, S. Lorrain, C. Fankhauser, M.A. Blazquez, E. Titarenko, S. Prat, A molecular framework for light and gibberellin control of cell elongation, Nature 451 (2008) 480–484. [19] S. Gao, X. Xie, S. Yang, Z. Chen, X. Wang, The changes of GA level and signaling are involved in the regulation of mesocotyl elongation during blue light mediated de-etiolation in Sorghum bicolor, Mol. Biol. Rep. 39 (4) (2012) 4091–4100. [20] L. Pan, M. Kawai, A. Yano, H. Uchimiya, Nucleoside diphosphate kinase required for coleoptile elongation in rice, Plant Physiol. 122 (2) (2000) 447–452.

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The effect of mepiquat chloride on elongation of cotton (Gossypium hirsutum L.) internode is associated with low concentration of gibberellic acid.

The growth regulator mepiquat chloride (MC) is globally used in cotton (Gossypium hirsutum L.) canopy manipulation to avoid excess growth and yield lo...
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