• Reactive oxygen species (ROS) play important roles in multiple physiological processes such as cellular signalling and stress responses, whereas, the hydrogen peroxide (H2O2) scavenging enzyme ascorbate peroxidase (APX) participates in the regulation of intracellular ROS levels.
• Here, a cotton (Gossypium hirsutum) cytosolic APX1 (GhAPX1) was identified to be highly accumulated during cotton fibre elongation by proteomic analysis. GhAPX1 cDNA contained an open reading frame of 753-bp encoding a protein of 250 amino acid residues. When GhAPX1 was expressed in Escherichia coli, the purified GhAPX1 was a dimer consisting of two identical subunits with a molecular mass of 28 kDa. GhAPX1 showed the highest substrate specificity for ascorbate.
• Quantitative real-time polymerase chain reaction (PCR) analyses showed that GhAPX1 was highly expressed in wild-type 5-d postanthesis fibres with much lower transcript levels in the fuzzless-lintless mutant ovules. Treating in vitro cultured wild-type cotton ovules with exogenous H2O2 or ethylene induced the expression of GhAPX1 and hence increased total APX activity proportionally, followed by extended fibre cell elongation.
• These data suggest that GhAPX1 expression is upregulated in response to an increase in cellular H2O2 and ethylene. GhAPX1 encodes a functional enzyme that is involved in hydrogen peroxide homeostasis during cotton fibre development.
Cotton fibres, the most important materials in the textile industry, are single-cell trichomes and develop from the ovule epidermis. Fibre quality is determined by its final length and strength. Cell elongation or expansion is regarded as a central process in plant morphogenesis. Cell wall loosening is not only catalysed by expansins (Cosgrove, 2000), reactive oxygen species (ROS) such as superoxide radical, hydrogen peroxide (H2O2) and hydroxyl radical were found to participate in plant cell expansion (Schopfer, 2001; Rodriguez et al., 2002; Liszkay et al., 2004). Interestingly, H2O2 acts as a developmental signal in the differentiation of secondary walls in cotton fibres (Potikha et al., 1999). Studies on an Arabidopsis mutant in which root and root hair elongation is impaired indicate that the mutant is defective in the generation of ROS because of disruption of a NAD(P)H oxidase gene and ROS regulated cell growth through the activation of Ca2+ channels (Foreman et al., 2003).
Reactive oxygen species were also shown to be signalling molecules involved in stress perception, pathogen response, programmed cell death and regulation of photosynthesis; control of intracellular ROS level is thought to be essential to maintain cellular redox homeostasis (Mittler et al., 2004). Ascorbate peroxidase (APX, EC 188.8.131.52) uses ascorbate as electron donor to reduce H2O2 to water. Therefore, APX represents one of the most important enzymes scavenging H2O2. It has been identified in many higher plants, with eight isozymes distributed in at least four cellular compartments, including cytosol, chloroplasts, mitochondria and peroxisomes (Shigeoka et al., 2002). Cytosolic APX1 plays an essential role in cross-compartment protection and maintenance of the cellular reactive oxygen network, demonstrated by the collapse of the entire chloroplastic H2O2-scavenging system, an increase in H2O2 concentrations and oxidation of proteins in the absence of APX1 (Davletova et al., 2004). Cytosolic APX2 in Arabidopsis is regulated by several stresses such as ozone, sulphur dioxide, excessive light, heat and heavy metals (Karpinski et al., 1997; Fryer et al., 2003). However, the specific functions of the APX genes in the context of plant growth and development remain to be determined.
Previously, a number of cotton cDNAs were identified that were preferentially expressed during fast fibre expansion period by microarray transcriptome analysis (Ji et al., 2003; Shi et al., 2006). Since two-dimensional electrophoresis (2-DE) coupled with mass spectrometry (MS) is considered an essential method to interpret gene expression at the protein level (Aebersold & Mann, 2003), here we took a proteomic approach to identify proteins that are potentially involved in cotton fibre cell elongation. Four cotton protein spots annotated as cytosolic APX encoded by the GhAPX1 gene, among a total of 49 protein spots that were accumulated specifically in elongating fibre cells, were obtained by matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF MS) and MALDI-TOF MS/MS with a total of more than 20-fold increase compared with that of a fuzzless-lintless mutant (fl), recessive mutant which does not produce fuzz and lint fibres. Functional analysis of GhAPX1 suggested that it might participate in the regulation of fibre cell development by modulating cellular H2O2 content.
Materials and Methods
Upland cotton (Gossypium hirsutum L. cv. Xuzhou 142) and fl mutant cotton ovules were grown in a fully automated glasshouse. Fresh ovules at 1-d postanthesis (dpa) were picked from the plants and used for in vitro ovule cultures. The various materials used for experiments were harvested immediately at indicated times and frozen in liquid nitrogen until use.
A series of 2-DE gels were produced as previously described (Wang et al., 2006). Approximately 1 g of different tissues were ground in liquid nitrogen in a mortar and pestle. The fine powder was precipitated at −20°C with 10% (w : v) trichloroacetic acid (TCA) in cold acetone containing 0.07% (w : v) 2-mercaptoethanol for at least 2 h or overnight. After centrifugation at 20000 g for 1 h, the pellet was washed first with cold acetone containing 0.07% (w : v) 2-mercaptoethanol, then with 80% cold acetone, and finally suspended in a lysis buffer (7 m urea, 2 m thiourea, 4% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulphonate (CHAPS), 20 mm dithiothreitol (DTT)) and the soluble fraction was purified using 2-DE clean up kit (Amersham Pharmacia Biotech, Cambridge, UK). Protein concentration was determined with 2-D Quant kit (Amersham Pharmacia Biotech). Two-dimensional electrophoresis was performed as described in Gorg et al. (1988). Briefly, total protein of 100 µg and 1.5 mg were applied for silver- and Coomassie-stained gels, respectively. Isoelectric focusing (IEF) was performed with pH range 4–7 using the Immobiline 4-7 linear DryStrip and the IPGphor system (Amersham Pharmacia Biotech). Denaturing 12.5% polyacrylamide gel was used for sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Silver- and Coomassie-stained gels were achieved using the method described by Shevchenko et al. (1996) and 0.1% (w : v) Coomassie Blue R-350 (Amersham Pharmacia Biotech) in 10% acetic acid plus continuous destaining with 10% acetic acid, respectively. Silver-stained gels were used for image analysis processed with 2-D imagemaster elite software (version 4.01; Amersham Pharmacia Biotech) according to the manufacturer's instructions, and Coomassie-stained gels were used for in-gel digestion and successive protein identification by MALDI-TOF MS and MS/MS methods. All mass spectra from MALDI-TOF MS and MS/MS were obtained on an Ultraflex TOF/TOF (Bruker Daltonics, Bremen, Germany) and used to search the NCBInr database using the MASCOT search engine (http://www.matrixscience.com) with a tolerance of ± 0.1 Da and one missed cleavage site. All gels were performed in three triplicates from independent protein samples.
Expression and purification of GhAPX1 in E. coli
The GhAPX1 cDNA was amplified by PCR with primers, 5′-CGGGATCCATGACCAAGTGTTACCCAACTGTG-3′ (sense) and 5′-CCCAAGCTTGTGCATCAGCAAATCCTAGCTCAGA-3′ (antisense) containing BamHI and HindIII restriction endonuclease sites (underlined). A PCR amplification reaction was performed using Taq DNA polymerase and the purified product was cloned into plasmid pGEM-T (Promega, Madison, WI, USA). The cDNA of GhAPX1 was verified by sequencing and was cloned into pET28a, resulting in the pET28a-GhAPX1 plasmid containing 6 × His-Tag. The pET28a-GhAPX1 plasmid was transformed into E. coli BL21 (DE3) pLysS cells. The transformed cells were cultured at 37°C in liquid Luria–Bertani (LB) medium (1% Bacto-tryptone, 0.5% yeast extract, and 1% NaCl, pH 7.5) containing 50 µg ml−1 kanamycin with vigorous shaking. Isopropyl-1-thio-β-d-galactoside (IPTG) was added to the culture to a final concentration of 0.4 mm when the cell density reached an optical density at 600 nm (OD600) of 0.6–0.8. After 4 h of additional incubation at 37°C, the cells were harvested by centrifuging at 5000 g for 20 min at 4°C. The pelleted cells were suspended in a binding buffer (50 mm Tris–HCl, 0.5 m NaCl and 1% Triton X-100, pH 8.0). The suspension was sonicated and the lysate was centrifuged at 10 000 g for 10 min at 4°C. The supernatant was loaded on Ni-charged His-Bind column according to the instructions provided by Novagen (Madison, WI, USA). The fractions eluted from the column containing the recombinant GhAPX1 protein were collected and load on a HiLoad 16/60 Superdex 200 (GE Healthcare, Pollards Wood, UK) equilibrated with the buffer containing 20 mm Tris-HCl, 150 mm NaCl, pH 7.5. Bio-Rad (Hercules, CA, USA) Gel Filtration Standard containing mixture of five proteins with known molecular weight: vitamin B12 (1.35 kDa), equine myoglobin (17.5 kDa), chicken ovalbumin (44 kDa), bovine gamma globulin (158 kDa) and thyroglobulin (670 kDa), was used for molecular mass calibration. The resulted peak fractions were analysed by SDS-PAGE and used for further optimum pH, enzyme kinetics and substrate specificity analyses. Protein concentration was determined by the Bradford method using bovine serum albumin as standard.
Ascorbate peroxidase activity measurement
The APX activity was assayed using the spectrophotometric method (Nakano & Asada, 1987). Different tissues (200 mg) were ground in liquid nitrogen, mixed with 1 ml of an extraction buffer containing 50 mm potassium-phosphate (pH 7.0), 1 mm ethylenediaminetetraacetic acid (EDTA), 2% (w : v) polyvinylpyrrolidone-40, 10% (w : v) glycerol, and 1 mm ascorbate (AsA), and centrifuged at 14000 g for 10 min at 4°C. The supernatant was used for the APX activity assay in a reaction mixture that contained 50 mm potassium-phosphate (pH 7.0), 0.1 mm EDTA, 0.1 mm H2O2, 0.5 mm AsA. The reaction was initiated with the addition of H2O2 in a total volume 1 ml. Enzyme activity was performed by measuring the decrease in absorbance of ascorbate at 290 nm (ɛ290/2.8 mm−1 cm−1). One unit of enzyme activity was defined with the oxidization of 1 µmol AsA at 25°C in 1 min. The electron donors except AsA were 10 mm guaiacol (ɛ470/26.6 mm−1 cm−1), 0.15 mm NADH (ɛ340/6.2 mm−1 cm−1), 0.15 mm NADPH (ɛ340/6.2 mm−1 cm−1), 50 µm cytochrome c (ɛ550/19 mm –1 cm−1), 20 mm 3,3′-diaminobenzidine (DAB) (ɛ460/3.73 mm−1 cm−1) and 1 mm glutathione. The rate of hydrogen peroxide-dependent oxidation of the electron donors was determined using the absorption coefficients cited in parentheses. When glutathione was used as substrate, the enzyme activity was determined by the method coupling with glutathione reductase and 0.12 mm NADPH (Little et al., 1970). The pH-dependent APX activity of recombinant GhAPX1 was determined in the following buffers: mixture of 0.1 m Na2HPO4 and 0.05 m citric acid for pH 3–6, 0.05 m sodium-phosphate buffer for pH 6–8 and 0.05 m Tris–HCl buffer for pH 8–10.
RNA extraction and quantitative real-time RT-PCR (QRT-PCR) analysis
Total RNA was prepared using a modified hot borate method as described (Shi et al., 2006). Total RNA (5 µg) was used to synthesize first-strand cDNA with the Superscript first-strand synthesis system for RT-PCR (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendations. Gene-specific primers of GhAPX1 for RT- and quantitative RT-PCR analyses were synthesized commercially with 5′-TCCCTAACCTCACCTACGCT-3′ (sense) and 5′-CCTTCCTTCTCTCCCGACA-3′ (antisense). Cotton UBQ7 primers were used as control to adjust the amount of the template cDNA for each amplification reaction. The QRT-PCR was performed using the SYBR Green PCR kit (Applied Biosystems) and a DNA Engine Opticon continuous fluorescence detection system (MJ Research) as previously described (Qin et al., 2005). Real time-PCR and quantitative RT-PCR results were obtained from three experiments using independent RNA samples.
In vitro ovule culture and treatment with exogenous H2O2 and ethylene
Cotton ovules were collected at 1 dpa, soaked in 70% ethanol for 1 min, rinsed in distilled and deionized water and soaked again in 0.1% HgCl solution containing 0.05% Tween-80 for 20 min to sterilize. Ovules (c. 0.02 g) were placed in liquid media formulated as described (Beasley & Ting, 1973) in 50-ml flasks under aseptic conditions. The ovules generally floated on the surface of 20 ml of the liquid media with or without (CK) exogenous 50 µm H2O2 and 0.1 µm ethylene and were cultured at 30°C in darkness without agitation. The lengths of fibres were measured manually under a bright-field microscope after combing the cells to upright positions, with three independent ovule culture experiments and a total of 90 fibre cells measured on three individual ovules each time (Shi et al., 2006).
Determination of H2O2 content in vivo
Flowers were marked at anthesis and tissues at indicated times were collected immediately for H2O2 measurement. H2O2 content was determined by the method of titanium oxidation with hydroperoxide-titanium complex formed (Brennan & Frenkel, 1977). Tissues (0.5 g) were ground in 1 ml acetone, and 75 µl freshly prepared 20% TiCl4 (v : v in 11 m HCl) and 150 µl NH4OH were added to the acetone extract forming a hydroperoxide–titanium complex. The complex pellet was obtained after centrifugation at 11000 g for 5 min and suspended in 400 µl 1 m H2SO4 with addition of 180 µl acetone. The supernatant was collected by removing the insoluble material after centrifugation at 11000 g for 5 min and the absorbance at 405 nm was measured against a blank. Hydrogen peroxide concentration was determined from the standard curve with 0.01 mm to 1 mm H2O2 added to the titanium solution.
Identification of a cotton APX by proteomic analysis during cotton fibre development
Proteomic analysis was performed on 10 dpa wild-type cotton ovules associated with fibres. For control purpose, 10 dpa fl mutant cotton ovules were also analysed. Proteins extracted from the cotton ovules were analysed by 2-D gel electrophoresis. A total of 1240 ± 20 protein spots were detected on both gels in three independent experiments (see the Supplementary Material, Fig. S1). The numbered spots were excised from gels, in-gel digested with trypsin and subjected to MALDI-TOF MS (Fig. S1). Among them, 61 proteins showing highly up-regulated (see the Supplementary Material, Table S1), downregulated (Table S2) were identified, compared with intensity of the relatively unchanged or housekeeping cotton proteins (Table S3). Spots 1–4, highly accumulated in 10 dpa wild-type ovules associated with fibres (Fig. 1), were all identified as ascorbate peroxidase encoded by the same APX gene (Table S1) through NCBInr database searches using the MASCOT search engine (http://www.matrixscience.com), and the peptides obtained from these four spots were matched perfectly with the deduced amino acid sequence of GhAPX1 (Table S4). Four unique peptides from each of intensive spots 1–4 (Fig. 1) had monocharged ions mass peaks at m/z (mass/charge) m/z 1331.1, m/z 2076.9, m/z 1612 and m/z 2320.1, indicating that the spots were GhAPX1 analyzed by MALDI-TOF MS (Table S4) and MALDI-TOF MS/MS (Fig. S2).
Characterization of GhAPX1
GhAPX1 is encoded by a cDNA containing a 753-bp open reading frame (ORF) with a predicted molecular mass of 28 kDa. The cDNA sequence was submitted to GenBank with accession number EF432582. The deduced amino acid sequence of GhAPX1 was shown in Fig. 2, in alignments with homologous sequences from species including Capsicum annuum, Arabidopsis thaliana, Oryza sativa and Glycine max. The amino acid residues Arg-38 essential for binding and orientation of the ligand, His-42 and Arg-172 critical for Compound I and II formation were found to be conserved in all the APX1 proteins shown in Fig. 2a. A neighbour-joining bootstrap tree constructed based on the homologous APX proteins revealed that they could be divided into four groups and GhAPX1 belongs to cytosolic APX proteins (Fig. 2b; see the Supplementary Material, Fig. S3). At the amino acid level, the GhAPX1 showed pronounced 47–93% similarity and 27–84% identity with other APX proteins (Fig. S3). GhAPX1 did not possess mitochondrial or peroxisomal targeting signal or chloroplast transit peptides, implying it is a cytosolic protein. We propose that GhAPX1 probably encodes for a cytosolic APX: a definitive conclusion may be drawn later by a localization study of the protein.
When pET28a-GhAPX1 was expressed in Escherichia coli and induced by IPTG, soluble extract of the cells were able to convert ascorbate to the compound II at a rate of 2.88 ± 0.21 µmol min−1 mg−1 protein. No activity was obtained from protein extracts using cells without IPTG induction or cells transformed with the empty vector. The expressed recombinant GhPAX1 was purified from the cell extract to apparent homogeneity by two chromatographic steps on a Ni-charged His-Bind column (Novagen) and gel filtration on a Superdex 200 column (GE Healthcare). By SDS/PAGE analysis, the molecular mass of the recombinant protein was 31.5 kDa (data not shown), which agrees with the theoretical molecular mass of 28 kDa plus 3.5 kDa His-tag fused with GhAPX1. Size-exclusion chromatography on Superdex 200 gave an experimental native molecular mass of 64.2 kDa (Fig. 3a), suggesting that the recombinant protein was a homodimer. Steady-state data for oxidation of ascorbate by GhAPX1 are shown in Table 1. The dependence of the rate of substrate oxidation vs pH yields a pH optimum for GhAPX1 at 6.5 for oxidation of ascorbate (Fig. 3b). The dependence of the specific activity of GhAPX1 on the concentration of ascorbate (Fig. 3c) or H2O2 (Fig. 3d) was linear (r2 = 0.9956 and r2 = 0.9327, respectively), suggesting that the both reactions are first order. Figure 3e shows the relative activities of GhAPX1 with different reducing substrates when the activity of GhAPX1 towards ascorbate is defined as 100%. Guaiacol, glutathione, NADH, NADPH, cytochrome c were poorer substrates of GhAPX1 with 0.02- to 0.28-fold of the activity of ascorbate and no oxidation was observed when DAB was used as the substrate (Fig. 3e).
Table 1. Enzymatic properties of recombinant GhAPX1 protein expressed in Escherichia coli
Molecular weight (kDa)
0.43 ± 0.031
0.12 ± 0.008
2.60 × 102 ± 21
46.71 ± 3.92
Kcat/km (s−1 mm−1)
6.04 × 102 ± 5.66
3.89 × 102 ± 2.13
Both GhAPX1 mRNA level and total APX activity reach to peak value during fast fibre cell elongation period
The expression levels for GhAPX1 gene in various developmental stages were examined by QRT-PCR and RT-PCR analyses (Fig. 4a). GhAPX1 mRNA increased to greater than fivefold at 5 dpa (the point of early fibre-cell fast elongating) ovules in wild-type associated with fibres compared with that of –3 dpa ovules and expression of GhAPX1 was found to remain at low level at 10 dpa fl mutant ovules, suggesting that GhAPX1 was specifically upregulated during cotton fibre development. The oxidation rate of ascorbate was measured using protein extracts from the fibre developmental stages indicated. The total APX activity reached a peak value at 5 dpa (Fig. 4b), which agreed with the transcriptional levels of the gene.
Exogenous H2O2 and ethylene treatments promoted fibre elongation as well as GhAPX1 transcription and total APX enzyme activity in cotton ovules
The QRT-PCR analyses showed that GhAPX1 expression was significantly upregulated in wild-type, but not mutant ovules, shortly after exogenous H2O2 or ethylene treatments (Fig. 5a). Total APX activity from in vitro cultured ovules was also found to increase significantly after H2O2 or ethylene supplementation (Fig. 5b). When 50 µm H2O2 or 0.1 µm ethylene were added in the ovule culture media, significant promotion of fibre cell growth was observed over a longer period of time (Fig. 5c). Application of 0.1 µm ethylene to cultured cotton ovules was able to stimulate significant H2O2 production as early as 6 h after the treatment (Fig. 5d). Our results suggest that GhAPX1 may play an important role during early cotton fibre cell development by mediating the endogenous H2O2 homeostasis that was controlled by the plant hormone ethylene.
Recent studies using knockout and antisense plants reveal a strong connection between ROS overproduction in response to environmental stresses and ROS-removal enzymes (Mittler et al., 2004). Reactive oxygen species may act as second messengers in plants, which is supported by the presence of redox-sensitive regulation of gene expression (Mou et al., 2003). Cytosolic APX genes were found to be induced under variable environmental conditions such as high light and high salt, wounding, pathogen infection, fruit ripening and application of paraquat (Mittler & Zilinskas, 1992; Schantz et al., 1995; Donahue et al., 1997; Karpinski et al., 1997, 1999; Storozhenko et al., 1998; Morita et al., 1999; Yoshimura et al., 2000), implying that the high expression of APX genes induced by oxidative stress is essential to remove H2O2 and minimize the oxidative damage. Although ascorbate peroxidases have been identified in many higher plants and comprise a family of isozymes with different characters, their functions in the regulation of plant growth remain unclear.
Our results show that GhAPX1 identified by proteomic analysis (Fig. 1) is highly upregulated during an early stage of the developing cotton fibres (Fig. 4a). Analysis of the amino acid sequence of GhAPX1 suggests that GhAPX1 belongs to the group of cytosolic APX (Fig. 2). Characterization of the purified recombinant GhAPX1 showed that the protein was active with ascorbate and H2O2, verifying that the identified putative GhAPX1 was an ascorbate peroxidase. The GhAPX1 obeyed Michaelis–Menten kinetics toward ascorbate and H2O2 (Fig. 3c,d). From the double-reciprocal plots, the apparent Km values of the enzyme for ascorbate and H2O2 were 0.43 ± 0.031 mm and 0.12 ± 0.008 mm. The kcat value for GhAPX1 was 2.60 × 102 ± 21 s−1 and the kcat/Km value for ascorbate was 6.04 × 102 ± 5.66 s−1 mm−1; these values are comparable to other characterized plant APXs (Mittler & Zilinskas, 1991; Yoshimura et al., 1998; Takeda et al., 2000). GhAPX1 displays much lower or no catalytic activity with the electron donors, including guaiacol, glutathione, NADH, NADPH, cytochrome c and DAB (Fig. 3e).
Plant cells in culture are considered to be subject to high levels of oxidative stress, providing a good model for the study of antioxidative mechanisms (Lee et al., 1999). To study the early response of GhAPX to ROS in the early stage of cotton fibre development, the profiles of GhAPX1 expression and the total APX activities from the wild-type cotton ovules treated with H2O2 were analysed (Fig. 5a,b). The GhAPX1 transcript level reached a maximum at 5 dpa, which is the time point when the APX activity increased at a peak value, implying that there is a direct correlation between the transcription and the activity of GhAPX1. Both the transcript levels of GhAPX1 gene and the APX activity from cotton fibres increased owing to induction by H2O2 (Fig. 5a,b), supporting the hypothesis that GhAPX1 functions to immediately detoxify H2O2 generated during the fibre fast-elongation period. This transient increase in H2O2 followed by cytosolic APX expression was also observed in high-light-exposed Arabidopsis (Karpinski et al., 1997). Ethylene was shown to play an important role in the development of fibre cells (Shi et al., 2006). With regard to the question of whether ethylene can regulate H2O2 production, leading to fibre elongation, treatment of cotton ovules by exogenous ethylene was found to stimulate H2O2 production (Fig. 5d) and increase the transcript level of the GhAPX1 gene (Fig. 5a), leading to enhancement of the total APX activity in cotton ovules (Fig. 5b). Our data are consistent with the expression pattern of the soybean ascorbate peroxidase gene that was induced by ethylene and hydrogen peroxide (Lee et al., 1999). Our data are also supported by the finding that the plant hormone auxin can stimulate the generation of H2O2, which plays a role in root gravitropism (Joo et al., 2001). We propose that cotton APX1 gene participates in hydrogen peroxide homeostasis. Our results indicate that ROS-mediated cell expansion may be one important mechanism that regulates cotton fibre elongation.
This work was supported by grants from China National Basic Research Program (Grant 2004CB117302). We thank Dr Xiao-Dong Su and MSc Cong Liu for determination of native molecular weight of GhAPX1.