Tomato plants (Lycopersicon esculentum cv. Alisa Craig) were grown in a greenhouse under natural light and irrigated manually every other day. For cytological, texture, cell wall composition and molecular analysis, fruits of WT and T1 generation transgenic lines were harvested at the immature green (15 DPA), mature green (MG, 35 DPA), Breaker (BR), and Red Ripe (RR, 7 days after BR) stages after tagging of flowers at anthesis.
Amino acid sequence analyses
Signal peptide and GPI modifications were predicted with SignalP Version 3.0 (http://www.cbs.dtu.dk/services/SignalP/)
 and big-PI (http://mendel.imp.ac.at/gpi/gpi_server.html)
, respectively. N-glycosylation site prediction was performed using NetNGlyc 1.0 (http://www.cbs.dtu.dk/services/NetNGlyc/). SUPER-FAMILY 1.69 (http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY/hmm.html)
 was used to predict cellulose-binding domains. Protein sequences were aligned using ClustalX
 and the resulting alignments were used as input to generate a phylogenetic tree using MEGA2.1
. Statistical confidence of the nodes of the tree are based on 10,000 bootstrap replicates.
The full-length SlCOBRA-like cDNA was isolated by reverse transcription (RT)–PCR from tomato seedlings using PrimeSTAR HS DNA Polymerase (TaKaRa) and gene-specific primers (Additional file
2: Table S1). The cDNA was cloned into the modified binary vector pBI121 to generate an overexpression construct driven by the fruit-specific TFM7 promoter (Accession no.X95261)
. Transgenic plants were generated by Agrobacterium tumefaciens-mediated transformation as described by Fillatti et al.
. Transformed lines were selected on medium containing kanamycin (70 mg/L) and further confirmed by PCR for the presence of the NPTII (Kanr) marker gene (Additional file
2: Table S1). After RT-PCR analysis to verify SlCOBRA-like mRNA accumulation in the positive transgenic lines, three independent overexpressing lines (TFM7-OE) and three independent co-suppression lines (TFM7-CS) were identified from the primary transgenic (T0) population.
Gene expression analysis
Total RNAs were extracted using Trizol reagent following the protocol provided by the manufacturer (Invitrogen, Carlsbad, CA) and treated with DNase (TaKaRa, Dalian, China). About 1 μg of total RNA from each sample was used for first-strand cDNA synthesis. For real-time quantitative RT-PCR, the PCR reaction was performed using SyBR Green PCR Master Mix (Applied Biosystems) and gene-specific primers (Additional file
2: Table S1) on the iCycler PCR system (BIO-RAD, Hercules, California, USA). Each sample was amplified in triplicate. REST software
 was used to quantify the mRNA levels of SlCOBRA-like and other selected genes with the UBI3 gene (Accession no.X58253) serving as the internal reference. Normalization was performed by the 2-Ct method. All primers used in this work are listed in Additional file
2: Table S1.
Histochemical staining and cytology
For fruit epidermis analysis, tissue was carefully isolated from the fruit surface with a razor blade, and the tissue-bound slide was rinsed twice in distilled water and mounted in 15% HCl under a cover slip and photographed using a Leica LDM 2500 microscope. Six exocarp slices from three different fruits per plant were isolated from identical positions of fruits at 15 DPA. For each exocarp slice, epidermal cell size (not including cell wall) and cell separation (spacing distance between cells) were measured at three different positions (10 cells each position) using the ImageProPlus software (IPP6.0, Media Cybernetics, Inc.). Values represent the means of 180 (6×30) cells.
For cytological assesment, three fruits per plant were collected at the MG stage. Fresh hand-cut pericarp sections (~0.1 mm thick) were incubated in a 0.005% aqueous solution of calcofluor (fluorescent brightener 28; Sigma) for 2 min
 and visualized with a fluorescent microscope (Leica, Wetzlar, Germany). To examine pericarp cell wall structure, paraffin-embedded transverse sections (10 μm in thickness) were obtained using a Leica microtome (RM 2265, Meyer Instruments, Inc) and stained with safranin O (a cell wall-specific dye), followed by photography using a Leica microscope (LDM 2500).
Cell wall material (CWM) isolation and cell wall component analysis of RR fruit pericarp
Three RR fruits per plant were collected from non-transformed WT and three independent overexpression or co-suppression lines, respectively, and then their pericarp tissues were mixed, rapidly ground into fine powder in liquid nitrogen, and stored at −80°C until use. In each case, approximately 15 g frozen power was incubated with 70% ethanol for 90 min at 70°C to prevent autolytic activity. Insoluble material was washed sequentially with 95% ethanol, chloroform:methanol (1:1, v/v), and acetone. The dried pellets constituted crude cell wall extract/alcohol insoluble solids [AIRs] and were assayed for cellulose content using anthrone as a coloring agent using α-Cellulose (Sigma) as the standard, according to methods described previously
The remaining AIRs were subsequently extracted with 90% (v/v) dimethyl-sulfoxide (DMSO) for 22 h at room temperature to solubilize starch, ending with two washes of the wall pellets with acetone and dessication in a vacuum oven
. The pellets (the cell wall materials, CWM) were stored in a glass desiccator until use. To determine non-cellulosic sugar composition, about 5 mg CWMs was hydrolyzed with 2 M trifluoroacetic acid (TFA) containing 4 mM of myoinositol as an internal standard at 105°C for 3 h, and then the TFA-soluble fraction was converted to alditol acetates and analyzed by gas chromatography as described previously
. Equimolar standards were also converted to alditol acetates to calculate response factors for quantitation of mol% relative to the myoinositol standard.
Pectin fractionation was carried out following the procedure of Rose et al. (1998)
. About 100 mg CWMs was extracted with water, 50 mM CDTA in 50 mM sodium acetate (pH 6.0), 100 mM Na2CO3 containing 0.1% NaBH4, sequentially. The uronic acid (UA) content in the different pectin fractions was estimated colorimetrically using galacturonic acid as a calibration standard
For FTIR spectra analysis, 6 RR fruits per plant from three independent overexpression, co-suppression or WT lines were collected, and the corresponding AIRs of pericarps from 54 (6×9) fruits were extracted as described above. In each case, AIR was spread thinly onto a barium fluoride window, dried on the window at 37°C for 20 min. An area of 50×50 μm was selected for analysis by FTIR microspectroscopy
. All data sets were baseline-corrected and area-normalized before statistical analyses were applied. Exploratory PCA was carried out using PASW statistics software 18 (formerly known as SPSS Statistics, SPSS Inc.). Reference IR absorption spectra of cellulose were used for peak assignments
Textural and shelf-life analysis
Fruit firmness was determined based on compression mass and skin puncture strength of fresh intact fruits collected at MG (35 DPA) and RR (7 days after Break), using TA-XT Plus (Stable Microsystems Texture Analyser, UK). For the compression test, 15 fruits per plant were assayed at each stage. Each fruit was compressed to a 50% strain at the test speed of 2 mm s−1 with a 100 mm compression platen (P/100) and 10 g of applied force. Skin puncture strength and penetration distance of fresh intact fruits were measured by penetration using a 2mm Cylinder Probe (P/2N) with a trigger force of 5 g, loading at 2 mm s-1 to reach a 50% strain. Each fruit was tested three times at equidistant points along the equatorial plane of the fruit. 6 fruits per plant were taken at each stage. Values represent means ±SE (n=18).
For shelf life, fruits at the RR stage were detached and kept at room temperature (23~25°C and 55~60% relative humidity) for approximately 40 days. 6 replicates were taken for each individual plant. Average fresh weight loss was determined every 5 days until they lost their texture and structural integrity.
Statistical analysis was performed using PASW statistics software 18.0 (formerly known as SPSS Statistics, SPSS Inc.). For analyses of epidermal cells, cellulose content, pectin fractions, and sugar content, significance was calculated using the Student’s t test. For gene expression between WT and CS plants, a multiple comparison was performed by the LSD (Least significant difference) method.
Accession numbers for the SlCOBRA-like sequences reported in this article are BT013422 and JN398667. Other SlCOBL sequences were listed in Table
1. Sequence data in Figure
7 were listed in Additional file
2: Table S1. Other sequence data from this article can be found in GenBank under the following accession numbers:
Arabidopsis AtCOB(At5g60920), AtCOBL1 (At3g02210), AtCOBL2 (At3g29810), AtCOBL4 (At5g15630), AtCOBL5 (At5g60950), AtCOBL6 (At1g09790), AtCOBL7 (At4g16120), AtCOBL8 (At3g16860), AtCOBL9 (At5g49270), AtCOBL10 (At3g20580), AtCOBL11 (At4g27110); Zea mays ZmBK2 (ACF79122.1), ZmBK2L3 (NP_001104946), ZmBK2L6 (NP_001105970), ZmBK2L7 (NP_001105971.1), ZmCOBL4 (EU955798.1); Oryza sativa OsBC1 (Os03g0416200), OsCOBL2 (Os03g0416300), OsCOBL3 (Os05g0386800), OsBC1L6 (Os07g0604300); OsCOBL6 (Os07g0604400).