Comparative Proteomics Indicates That Biosynthesis of Pectic Precursors Is Important for Cotton Fiber and Arabidopsis Root Hair Elongation
2010; Elsevier BV; Volume: 9; Issue: 9 Linguagem: Inglês
10.1074/mcp.m110.000349
ISSN1535-9484
AutoresChaoyou Pang, Hui Wang, Yu Pang, Chao Xu, Yue Jiao, Yong‐Mei Qin, Tamara L. Western, Shuxun Yu, Yuxian Zhu,
Tópico(s)Plant nutrient uptake and metabolism
ResumoThe quality of cotton fiber is determined by its final length and strength, which is a function of primary and secondary cell wall deposition. Using a comparative proteomics approach, we identified 104 proteins from cotton ovules 10 days postanthesis with 93 preferentially accumulated in the wild type and 11 accumulated in the fuzzless-lintless mutant. Bioinformatics analysis indicated that nucleotide sugar metabolism was the most significantly up-regulated biochemical process during fiber elongation. Seven protein spots potentially involved in pectic cell wall polysaccharide biosynthesis were specifically accumulated in wild-type samples at both the protein and transcript levels. Protein and mRNA expression of these genes increased when either ethylene or lignoceric acid (C24:0) was added to the culture medium, suggesting that these compounds may promote fiber elongation by modulating the production of cell wall polymers. Quantitative analysis revealed that fiber primary cell walls contained significantly higher amounts of pectin, whereas more hemicellulose was found in ovule samples. Significant fiber growth was observed when UDP-l-rhamnose, UDP-d-galacturonic acid, or UDP-d-glucuronic acid, all of which were readily incorporated into the pectin fraction of cell wall preparations, was added to the ovule culture medium. The short root hairs of Arabidopsis uer1-1 and gae6-1 mutants were complemented either by genetic transformation of the respective cotton cDNA or by adding a specific pectin precursor to the growth medium. When two pectin precursors, produced by either UDP-4-keto-6-deoxy-d-glucose 3,5-epimerase 4-reductase or by UDP-d-glucose dehydrogenase and UDP-d-glucuronic acid 4-epimerase successively, were used in the chemical complementation assay, wild-type root hair lengths were observed in both cut1 and ein2-5 Arabidopsis seedlings, which showed defects in C24:0 biosynthesis or ethylene signaling, respectively. Our results suggest that ethylene and C24:0 may promote cotton fiber and Arabidopsis root hair growth by activating the pectin biosynthesis network, especially UDP-l-rhamnose and UDP-d-galacturonic acid synthesis. The quality of cotton fiber is determined by its final length and strength, which is a function of primary and secondary cell wall deposition. Using a comparative proteomics approach, we identified 104 proteins from cotton ovules 10 days postanthesis with 93 preferentially accumulated in the wild type and 11 accumulated in the fuzzless-lintless mutant. Bioinformatics analysis indicated that nucleotide sugar metabolism was the most significantly up-regulated biochemical process during fiber elongation. Seven protein spots potentially involved in pectic cell wall polysaccharide biosynthesis were specifically accumulated in wild-type samples at both the protein and transcript levels. Protein and mRNA expression of these genes increased when either ethylene or lignoceric acid (C24:0) was added to the culture medium, suggesting that these compounds may promote fiber elongation by modulating the production of cell wall polymers. Quantitative analysis revealed that fiber primary cell walls contained significantly higher amounts of pectin, whereas more hemicellulose was found in ovule samples. Significant fiber growth was observed when UDP-l-rhamnose, UDP-d-galacturonic acid, or UDP-d-glucuronic acid, all of which were readily incorporated into the pectin fraction of cell wall preparations, was added to the ovule culture medium. The short root hairs of Arabidopsis uer1-1 and gae6-1 mutants were complemented either by genetic transformation of the respective cotton cDNA or by adding a specific pectin precursor to the growth medium. When two pectin precursors, produced by either UDP-4-keto-6-deoxy-d-glucose 3,5-epimerase 4-reductase or by UDP-d-glucose dehydrogenase and UDP-d-glucuronic acid 4-epimerase successively, were used in the chemical complementation assay, wild-type root hair lengths were observed in both cut1 and ein2-5 Arabidopsis seedlings, which showed defects in C24:0 biosynthesis or ethylene signaling, respectively. Our results suggest that ethylene and C24:0 may promote cotton fiber and Arabidopsis root hair growth by activating the pectin biosynthesis network, especially UDP-l-rhamnose and UDP-d-galacturonic acid synthesis. Cell elongation and expansion contribute significantly to the growth and morphogenesis of higher plants. Cotton (Gossypium hirsutum) fibers are single cells that differentiate from the outer integuments of the ovule. Cotton lint (the industrial name for fiber) is the most prevalent natural raw material used in the textile industry, so its production plays a significant role in the global economy. The number of fibers present on each ovule (cotton productivity), the final length, and the strength of each fiber (fiber quality) are determined by four separable biological processes: fiber initiation, elongation (primary cell wall synthesis), cell wall thickening (secondary cell wall deposition), and maturation. The fiber initiation stage occurs from 3 days prior to anthesis to 3 days postanthesis (dpa) 1The abbreviations used are:dpadays postanthesisUERUDP-4-keto-6-deoxy-d-glucose 3,5-epimerase 4-reductaseUGPUDP-d-glucose pyrophosphorylaseUGDUDP-d-glucose dehydrogenaseGAEUDP-d-glucuronic acid 4-epimeraseRhal-rhamnoseXyld-xyloseGalAd-galacturonic acidGlcAd-glucuronic acid2-DEtwo-dimensional gel electrophoresisflfuzzless-lintlessS/Nsignal to noise ratioRACErapid amplification of 5′ or 3′ cDNA endsBLASTbasic local alignment search toolFDRfalse discovery rateQRT-PCRquantitative real time RT-PCRAtArabidopsis thalianaGhG. hirsutum4K6DG4-keto-6-deoxyglucoseAVGl-(2-aminoethoxyvinyl)glycine hydrochlorideKEGGKyoto Encyclopedia of Genes and GenomesRHMrhamnose synthaseNCBINational Center for Biotechnology InformationUGEUDP-glucose 4-epimerase. and is characterized by the enlargement and protrusion of epidermal cells from the ovule surface. During the fiber elongation period (5–25 dpa), cells demonstrate vigorous expansion with peak growth rates of >2 mm/day until the fibers reach their final dimensions (1.John M.E. Keller G. Metabolic pathway engineering in cotton: biosynthesis of polyhydroxybutyrate in fiber cells.Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 12768-12773Crossref PubMed Scopus (180) Google Scholar, 2.Ji S. Lu Y. Li J. Wei G. Liang X. Zhu Y. A β-tubulin-like cDNA expressed specifically in elongating cotton fibers induces longitudinal growth of fission yeast.Biochem. Biophys. Res. Commun. 2002; 296: 1245-1250Crossref PubMed Scopus (34) Google Scholar, 3.Ji S.J. Lu Y.C. Feng J.X. Wei G. Li J. Shi Y.H. Fu Q. Liu D. Luo J.C. Zhu Y.X. Isolation and analyses of gene preferentially expressed during early cotton fiber development by subtractive PCR and cDNA array.Nucleic Acids Res. 2003; 31: 2534-2543Crossref PubMed Scopus (199) Google Scholar). In the secondary cell wall deposition phase (20–45 dpa), cellulose biosynthesis predominates until the cells contain ∼90% cellulose. In the final maturation stage (45–50 dpa), fibers undergo dehydration and become mature cotton lint. days postanthesis UDP-4-keto-6-deoxy-d-glucose 3,5-epimerase 4-reductase UDP-d-glucose pyrophosphorylase UDP-d-glucose dehydrogenase UDP-d-glucuronic acid 4-epimerase l-rhamnose d-xylose d-galacturonic acid d-glucuronic acid two-dimensional gel electrophoresis fuzzless-lintless signal to noise ratio rapid amplification of 5′ or 3′ cDNA ends basic local alignment search tool false discovery rate quantitative real time RT-PCR Arabidopsis thaliana G. hirsutum 4-keto-6-deoxyglucose l-(2-aminoethoxyvinyl)glycine hydrochloride Kyoto Encyclopedia of Genes and Genomes rhamnose synthase National Center for Biotechnology Information UDP-glucose 4-epimerase. Cotton fibers also serve as an excellent single celled model for studying fundamental biological processes, including cell elongation and differentiation (4.Kim H.J. Triplett B.A. Cotton fiber growth in planta and in vitro. Models for plant cell elongation and cell wall biogenesis.Plant Physiol. 2001; 127: 1361-1366Crossref PubMed Scopus (475) Google Scholar, 5.Wilkins T.A. Arpat A.B. The cotton fiber transcriptome.Physiol. Plant. 2005; 124: 295-300Crossref Scopus (87) Google Scholar, 6.Singh B. Avci U. EichlerInwood S.E. Grimson M.J. Landgraf J. Mohnen D. Sørensen I. Wilkerson C.G. Willats W.G. Haigler C.H. A specialized outer layer of the primary cell wall joins elongating cotton fibers into tissue-like bundles.Plant Physiol. 2009; 150: 684-699Crossref PubMed Scopus (68) Google Scholar). Using cDNA microarray hybridization data obtained from 11,692 cotton fiber UniESTs, we previously identified 778 cDNAs that are preferentially expressed during the fast fiber elongation period (7.Shi Y.H. Zhu S.W. Mao X.Z. Feng J.X. Qin Y.M. Zhang L. Cheng J. Wei L.P. Wang Z.Y. Zhu Y.X. Transcriptome profiling, molecular biological, and physiological studies reveal a major role for ethylene in cotton fiber cell elongation.Plant Cell. 2006; 18: 651-664Crossref PubMed Scopus (431) Google Scholar). Among them, 162 fiber-preferential genes were mapped to 102 metabolic events with ethylene biosynthesis and fatty acid biosynthesis/chain elongation being the most significantly up-regulated processes. Systematic studies showed that a large number of genes encoding nonspecific lipid transfer proteins and enzymes that are involved in various steps of fatty acid chain elongation are highly up-regulated during early fiber development, indicating that biosynthesis of saturated very-long-chain fatty acids and/or their transport may also be required for fiber cell growth (3.Ji S.J. Lu Y.C. Feng J.X. Wei G. Li J. Shi Y.H. Fu Q. Liu D. Luo J.C. Zhu Y.X. Isolation and analyses of gene preferentially expressed during early cotton fiber development by subtractive PCR and cDNA array.Nucleic Acids Res. 2003; 31: 2534-2543Crossref PubMed Scopus (199) Google Scholar, 7.Shi Y.H. Zhu S.W. Mao X.Z. Feng J.X. Qin Y.M. Zhang L. Cheng J. Wei L.P. Wang Z.Y. Zhu Y.X. Transcriptome profiling, molecular biological, and physiological studies reveal a major role for ethylene in cotton fiber cell elongation.Plant Cell. 2006; 18: 651-664Crossref PubMed Scopus (431) Google Scholar, 8.Xu Y. Li H.B. Zhu Y.X. 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Exogenously applied lignoceric acid (C24:0) in the ovule culture medium promotes significant fiber cell growth, possibly by activating the transcription of several 1-aminocyclopropane-1-carboxylic acid oxidases involved in ethylene biosynthesis (12.Qin Y.M. Hu C.Y. Pang Y. Kastaniotis A.J. Hiltunen J.K. Zhu Y.X. Saturated very-long-chain fatty acids promote cotton fiber and Arabidopsis cell elongation by activating ethylene biosynthesis.Plant Cell. 2007; 19: 3692-3704Crossref PubMed Scopus (192) Google Scholar). To date, biochemical reactions downstream of ethylene signaling that lead to cell elongation have not been reported. Two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF MS has recently been used to study brassinosteroid signal transduction pathways (13.Tang W. Deng Z. Oses-Prieto J.A. Suzuki N. Zhu S. Zhang X. Burlingame A.L. Wang Z.Y. Proteomics studies of brassinosteroid signal transduction using prefractionation and two-dimensional DIGE.Mol. Cell. 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Here we found that the biosynthesis of a specific subset of carbohydrates, including UDP-Rha, UDP-GlcA, and UDP-GalA, required for pectic polymer production, was significantly activated in developing fiber cells. Genetic studies using a series of Arabidopsis mutants with defects in UDP-Rha and UDP-GalA biosynthesis or in control of upstream regulatory components confirmed the importance of these two metabolic steps for both cotton fiber and Arabidopsis root hair growth. Upland cotton (G. hirsutum L. cv. Xuzhou 142) and the fuzzless-lintless (fl) mutant, originally discovered in the Xuzhou 142 cotton field in China (16.Zhang T. Pan J. Genetic analysis of a fuzzless-lintless mutant in Gossypium hirsutum L.Jiangsu J. Agric. Sci. 1992; 7: 13-16Google Scholar), were grown in an artificial soil mixture in fully climate-controlled walk-in growth chambers. Bolls excised from cotton plants at the indicated growth stages were dissected in a laminar flow hood to obtain intact ovules. Cotton materials were frozen and stored in liquid nitrogen immediately after harvest until use for protein and RNA extractions. All Arabidopsis plants, including three mutant lines in the Col genetic background (ein2-5; At uer1-1, SALK_100812; At gae6-1, SALK_104454C) and the cut1 mutant in the Ler genetic background, were grown in fully automated growth chambers as described (17.Feng J.X. Liu D. Pan Y. Gong W. Ma L.G. Luo J.C. Deng X.W. Zhu Y.X. An annotation update via cDNA sequence analysis and comprehensive profiling of developmental, hormonal or environmental responsiveness of the Arabidopsis AP2/EREBP transcription factor gene family.Plant Mol. Biol. 2005; 59: 853-868Crossref PubMed Scopus (135) Google Scholar). Plant tissues were ground in liquid nitrogen using a mortar and pestle. Fine powder was produced at −20 °C with 10% (w/v) trichloroacetic acid in cold acetone containing 0.07% (w/v) 2-mercaptoethanol for at least 2 h. After centrifugation at 20,000 × g for 1 h, the pellet was washed first with cold acetone containing 0.07% (w/v) 2-mercaptoethanol and then with 80% cold acetone and finally suspended in a lysis buffer (7 m urea, 2 m thiourea, 4% CHAPS, 20 mm dithiothreitol), and the soluble fraction was purified using the 2-D Clean-Up kit (GE Healthcare). Protein concentration was determined with a 2-D Quant kit (GE Healthcare). 2-DE was performed as described (18.Li H.B. Qin Y.M. Pang Y. Song W.Q. Mei W.Q. Zhu Y.X. A cotton ascorbate peroxidase is involved in hydrogen peroxide homeostasis during fibre cell development.New Phytol. 2007; 175: 462-471Crossref PubMed Scopus (100) Google Scholar, 19.Fu Q. Wang B.C. Jin X. Li H.B. Han P. Wei K.H. Zhang X.M. Zhu Y.X. Proteomic analysis and extensive protein identification from dry, germinating Arabidopsis seeds and young seedlings.J. Biochem. Mol. Biol. 2005; 38: 650-660PubMed Google Scholar). Total cotton ovule proteins (100 μg or 1.5 mg) were applied for silver- or Coomassie-stained gels, respectively. Isoelectric focusing was performed with the IPGphor system (GE Healthcare). Immobiline pH 4–7 and 3–10, 24-cm linear DryStrips (GE Healthcare) were run at 30 V for 8 h, 50 V for 4 h, 100 V for 1 h, 300 V for 1 h, 500 V for 1 h, 1000 V for 1 h, and 8000 V for 12 h using rehydration buffer (8 m urea, 2% CHAPS, 20 mm DTT) containing 0.5% (v/v) IPG Buffer (GE Healthcare). SDS-PAGE was performed using 12.5% polyacrylamide gels without a stacking gel in the Ettan Daltsix Electrophoresis Unit 230 (GE Healthcare). Gels were stained with 0.04% (w/v) PhastGel Blue R (Coomassie Brilliant Blue R-350; GE Healthcare) in 10% acetic acid and destained with 10% acetic acid or were silver-stained using a Hoefer Automated Gel Stainer apparatus. Images of the gels were scanned by a PowerLook 2100XL (UMAX) and analyzed using ImageMaster 2-DE Elite (version 4.01, Amersham Biosciences). Protein samples were prepared in triplicate using different plant materials for each 2-DE image. Differentially expressed proteins were excised and digested with trypsin essentially as reported (20.Wang B.C. Wang H.X. Feng J.X. Meng D.Z. Qu L.J. Zhu Y.X. Post-translational modifications, but not transcriptional regulation, of major chloroplast RNA-binding proteins are related to Arabidopsis seedling development.Proteomics. 2006; 6: 2555-2563Crossref PubMed Scopus (31) Google Scholar). Mass spectra were recorded on an Ultraflex MALDI-TOF/TOF mass spectrometer (Bruker Daltonik GmbH) using the FlexControl 2.2 software (Bruker Daltonik GmbH). TOF results were analyzed by FlexAnalysis 2.2 (Bruker Daltonik GmbH), peaks with S/N >100 were selected as precursor ions that were accelerated in TOF1 at a voltage of 8 kV and fragmented by lifting the voltage to 19 kV. Both MALDI-TOF and MS/MS spectra were processed by FlexAnalysis 2.2 (Bruker Daltonik GmbH) and were searched using MASCOT 2.1.0 (Matrix Science). All spectra were searched against the in-house National Center for Biotechnology Information non-redundant (NCBInr) database (release date, June 10, 2008; including 6,573,034 sequences, 2,244,863,856 residues) with species restriction to Viridiplantae (green plants) (483,288 sequences) and a cotton EST database downloaded from NCBI "EST others" (release date, January 22, 2009; including 369,596 sequences, 254,288,404 residues) (p < 0.05). We used the following parameters for the search: S/N ≥ 3.0; fixed modification, carbamidomethyl (Cys); variable modification, oxidation (Met); maximum number of missing cleavages, 1; MS tolerance, ±100 ppm; and MS/MS tolerance, ±0.7 Da. The ion cutoff score was 51 (p < 0.01, E < 0.01) following a published protocol (21.Zulak K.G. Khan M.F. Alcantara J. Schriemer D.C. Facchini P.J. Plant defense responses in opium poppy cell cultures revealed by liquid chromatography-tandem mass spectrometry proteomics.Mol. Cell. Proteomics. 2009; 8: 86-98Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Several identified protein spots deemed potentially important were further analyzed using nano-liquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry (nano-LC-FTICR MS) techniques as described (22.Liu Y. He J. Ji S. Wang Q. Pu H. Jiang T. Meng L. Yang X. Ji J. Comparative studies of early liver dysfunction in senescence-accelerated mouse using mitochondrial proteomics approaches.Mol. Cell. Proteomics. 2008; 7: 1737-1747Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Trypsin-digested peptides were dissolved in 0.1% formic acid and separated by a nano-LC system (Micro-Tech Scientific) that was equipped with a C18 reverse-phase column using 0–50% acetonitrile gradient in 0.1% formic acid at a constant flow rate of 400 nl/min in 120 min. Mass spectra were recorded on a 7-tesla FTICR mass spectrometer (Apex-Qe, Bruker Daltonics). Data were acquired in data-dependent mode using ApexControl 1.0 software (Bruker Daltonics). The MS/MS spectra were processed by DataAnalysis 3.4 (Bruker Daltonics) with S/N ≥4.0 and searched against the in-house cotton EST database using the Mascot 2.1.0 search engine (Matrix Science). Fixed and variable modifications were specified as described under "Protein Identification by MALDI-TOF/TOF MS." Maximum number of missing cleavages was set to 1. MS tolerance was ±5 ppm, and MS/MS tolerance was ±15 millimass units. The ion cutoff score was 41 (p < 0.01, E < 0.01). The criteria for positive identification we used result in less than 5% false positives at the protein level as determined by searching a target-decoy database constructed with shuffled sequences in the decoy. The false-positive rate was calculated as follows: 2 × decoy hits/total hits (23.Majeran W. Zybailov B. Ytterberg A.J. Dunsmore J. Sun Q. van Wijk K.J. Consequences of C4 differentiation for chloroplast membrane proteomes in maize mesophyll and bundle sheath cells.Mol. Cell. Proteomics. 2008; 7: 1609-1638Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). To obtain putative full-length cotton cDNAs, all 375,441 cotton ESTs available from NCBI (http://www.ncbi.nlm.nih.gov/Genbank/) as of April 10, 2009 were downloaded. Putative full-length cDNA sequences were obtained on a Linux operating system using the local cotton EST database, the BLAST results, and the CAP3 sequence assembly program (24.Huang X. Madan A. CAP3: A DNA sequence assembly program.Genome Res. 1999; 9: 868-877Crossref PubMed Scopus (4026) Google Scholar). When a putative full-length cDNA was not available in our cDNA collection, we used rapid amplification of 5′ or 3′ cDNA ends (RACE) (17.Feng J.X. Liu D. Pan Y. Gong W. Ma L.G. Luo J.C. Deng X.W. Zhu Y.X. An annotation update via cDNA sequence analysis and comprehensive profiling of developmental, hormonal or environmental responsiveness of the Arabidopsis AP2/EREBP transcription factor gene family.Plant Mol. Biol. 2005; 59: 853-868Crossref PubMed Scopus (135) Google Scholar) to recover the missing sequences. The entire coding region with any available upstream and downstream sequences was amplified again to confirm that the RACE products were assembled correctly from a single gene and not from a chimeric gene sequence of the A and D subgenomes. All full-length cDNAs were verified by sequencing the corresponding clone from a cotton cDNA library that was constructed using RNA extracted with the hot borate method (25.Wan C.Y. Wilkins T.A. A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L.).Anal. Biochem. 1994; 223: 7-12Crossref PubMed Scopus (851) Google Scholar). We used guanidine hydrochloride (final concentration, 6 m) as the denaturant and 1% polyvinylpyrrolidone to remove major phenolic compounds from cotton ovule or fiber cells. The quality of the library was verified because putative open reading frames were found in more than half of the genes related to plant hormone biosynthesis (7.Shi Y.H. Zhu S.W. Mao X.Z. Feng J.X. Qin Y.M. Zhang L. Cheng J. Wei L.P. Wang Z.Y. Zhu Y.X. Transcriptome profiling, molecular biological, and physiological studies reveal a major role for ethylene in cotton fiber cell elongation.Plant Cell. 2006; 18: 651-664Crossref PubMed Scopus (431) Google Scholar). The software KOBAS, which stands for Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology-based Annotation System (26.Mao X. Cai T. Olyarchuk J.G. Wei L. Automated genome annotation and pathway identification using the KEGG orthology (KO) as a controlled vocabulary.Bioinformatics. 2005; 21: 3787-3793Crossref PubMed Scopus (2072) Google Scholar), was used to identify biochemical reactions involved in cotton fiber development and to calculate the statistical significance of each step. This program assigns a given set of genes to pathways by first matching the genes to similar genes (as determined by a BLAST similarity search with cutoff E values <1 × 10−6, rank 55%) in known pathways in the KEGG database. We ranked pathways (or biochemical events) by statistical significance to determine whether a pathway contained a higher ratio of fiber-preferential proteins among all Arabidopsis proteins mapped to the same pathway. Because a large number of pathways were involved, we implemented FDR correction to control the overall Type I error rate of multiple testing using GeneTS (2.8.0) in the R (2.2.0) statistics software package. Pathways with FDR-corrected p values <0.001 were considered statistically significant. Cotton ovules harvested at specific growth stages were first frozen in liquid nitrogen before RNA extraction using a modified hot borate method (25.Wan C.Y. Wilkins T.A. A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L.).Anal. Biochem. 1994; 223: 7-12Crossref PubMed Scopus (851) Google Scholar). Total RNA was extracted from wild-type or fl mutant cotton materials after various treatments, and cDNA was reverse transcribed from 5 μg of total RNA. Primers for QRT-PCR analysis are listed in supplemental Table 1. All PCR experiments were performed in triplicate using independent RNA samples prepared from different cotton or Arabidopsis materials. Cotton UBQ7 (NCBI accession number AY189972) and Arabidopsis UBQ5 (At3g62250) were used as internal controls for PCR experiments using the respective plant materials. Gh UER1-specific antibody was produced from rabbit using a synthesized polypeptide, KESLIKYVFEPNKKT, derived from the C terminus of UER1, which was identified commercially using Peptide-Antigen Finder software (Chinese Peptide Corp.). Western blotting experiments were performed as reported previously (27.Han P. Li Q. Zhu Y.X. Mutation of Arabidopsis BARD1 causes meristem defects by failing to confine WUSCHEL expression to the organizing center.Plant Cell. 2008; 20: 1482-1493Crossref PubMed Scopus (54) Google Scholar). Either 10-dpa cotton fiber cells or ovules (5-g fresh weight) were ground in liquid nitrogen using a mortar and pestle. The fine powders were washed with 70% aqueous ethanol and pelleted by centrifugation at 10,000 × g for 15 min. The resulting pellet was washed with a 1:1 (v/v) mixture of chloroform and methanol and was then washed twice with acetone before drying in a SpeedVac vacuum system (Savant Instruments). Starch contaminants were removed by successive treatments with α-amylase (5 units/mg of cell wall; overnight at room temperature) (Sigma-Aldrich) and dimethyl sulfoxide (1 ml/mg of cell wall; overnight at room temperature). Pectin fractions were obtained by first boiling the cell wall pellets three times in 50 mm EDTA (pH 6.8; 10 min each) and then extracting three times at room temperature for 12 h in 50 mm Na2CO3 containing 1% NaBH4. Hemicelluloses were successively extracted from remnant cell wall pellets in 1 m (three times) and 4 m (three times) KOH containing 1% NaBH4 at room temperature for 12 h each time. The alkali fractions were neutralized with acetic acid. All six pectin and hemicellulose extracts were combined respectively and dialyzed extensively in dialysis tubing (1000-Da cutoff) against water. Both fractions were then concentrated using a Stirred Ultrafiltration Cell (Millipore) equipped with ultrafiltration membranes (1000-Da limit; Millipore), lyophilized to dryness, and weighed. The Updegraff assay (28.Updegraff D.M. Semimicro determination of cellulose in biological materials.Anal. Biochem. 1969; 32: 420-424Crossref PubMed Scopus (1623) Google Scholar) was used to determine relative cellulose content in the remaining cell wall pellets to deduce the amount of "other unidentified cell wall components" (called "others" ). Starch-free total cell wall materials, purified pectin, and hemicellulose were subjected to 2 m TFA at 120 °C for 2 h to produce monosaccharides. The neutral monosaccharides were converted into alditol acetates, whereas uronic acids were derivatized by trimethylsilyl methoxime before GC/MS analysis (29.Usadel B. Kuschinsky A.M. Rosso M.G. Eckermann N. Pauly M. RHM2 is involved in mucilage pectin synthesis and is required for development of the seed coat in Arabidopsis.Plant Physiol. 2004; 134: 286-295Crossref PubMed Scopus (116) Google Scholar, 30.Western T.L. Young D.S. Dean G.H. Tan W.L. Samuels A.L. Haughn G.W. MUCILAGE-MODIFIED4 encodes a putative pectin biosynthetic enzyme developmentally regulated by APETALA2, TRANSPARENT TESTA GLABRA1, and GLABRA2 in the Arabidopsis seed coat.Plant Physiol. 2004; 134: 296-306Crossref PubMed Scopus (148) Google Scholar). Briefly, different fractions were run on a GC/MS instrument (6890N-5975B, Agilent Technologies) with helium as the carrier gas to determine their sugar composition. For alditol acetate derivatives, a J&W HP-5MS column (30 m × 0.25 mm × 0.25 μm; Agilent Technologies) was used with the following program: 2 min at 110 °C, 10 °C/min until 200 °C, 5 min at 200 °C, 10 °C/min until 250 °C, and hold at 250 °C for 10 min. For trimethylsilyl methoxime derivatives, a J&W DB-5MS column (30 m × 0.25 mm × 0.25 μm; Agilent Technologies) was used with the following program: 1 min at 160 °C, 10 °C/min until 172 °C, 5 °C/min to 208 °C, decrease to 200 °C in 10 s, hold at 200 °C for 2 min, decrease to 160 °C in 30 s, and hold at 160 °C for 2 min. Compounds were first confirmed by comparison with the retention time obtained from the individual monosaccharide standard and were further identified through GC/MS coupled to the National Institute of Standards and Technology (NIST) database. Putative full-length cotton UER1, UGD1, UGP1, UGP2, and GAE3 cDNAs were cloned into pET28a to produce pET28a-GhUER1, pET28a-GhUGD1, pET28a-GhUGP1, pET28a-GhUGP2, and pET28a-GhGAE3, respectively. The plasmids were separately transformed into Escherichia coli BL21 (DE3) pLy
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