Materials and Methods

Plants Material. Tomato (Lycopersicon esculentum Mill. cv. MicroTom) plants were grown in a greenhouse (maximum temperature 27°C) in potting mix with standard fertilizer and pesticide treatments. Fruit and leaves were harvested in June of 2003, frozen in liquid N2, and stored at -80°C.
cDNA Clones and Expression Constructs. Arabidopsis cDNA clone RAFL09-32-D04 coding for AtADCS was isolated at the RIKEN Genomic Sciences Center (16). 5′-Truncated tomato expressed sequence tags (TIGR contig TC108445) were used to design a primer (5′-CCTTTGGGGTGGAACCACGA-3′), for RACE PCR, which yielded the missing 5′ sequence. Full-length cDNAs encoding LeADCS were then obtained from fruit mRNA by reverse transcription and PCR amplification with the primers 5′-ATTTCTGCACCAAGCGTTTT-3′ (forward) and 5′-AAAATGAAACGTGGAATCATCA-3′ (reverse). To express AtADCS and LeADCS in yeast or E. coli, targeting regions were removed and replaced by initiation codons by using suitable PCR primers; the changes were V85 → M for AtADCS and V84 → M for LeADCS. The vector for yeast expression was pVT103-U (17); the truncated ADCS cDNAs were ligated between its BamHI and PvuII sites and electroporated into E. coli DH10B cells. Yeast transformation and retransformation were carried out as described (6). The E. coli vector for complementation experiments was pLOI707HE, which allows tight control of gene expression by isopropyl β-d-1-thiogalactopyranoside (IPTG) (18). The truncated ADCS cDNAs were inserted between the NotI and SstI sites and introduced into E. coli DH10B. For overexpression, the truncated AtADCS sequence was inserted between the NcoI and XhoI sites of pET-28a (Novagen) and introduced first into E. coli DH10B, then into BL21-CodonPlus (DE3)-RIL cells (Stratagene). E. coli PabC cloned in pJMG30 (10), PabA in pSZD51 (19), and PabB in pSZD52 (19) were likewise introduced into BL21-CodonPlus (DE3)-RIL cells.
Functional Complementation. Yeast strains BY4741 (Mata his3-1 leu2-0 met15-0 ura3-0 YNR033w::kanMX4) and 971/6c (Mata ade2-1 his3-11,15 leu2-3,112 ura3-1 can1) were obtained from EUROSCARF (Frankfurt) and M. L. Agostini Carbone (Università di Milano), respectively. Yeast cells transformed with pVT103-U constructs were cultured at 30°C in appropriately supplemented synthetic minimal medium, prepared as specified in the 1984 Difco manual, except that folic acid was omitted, and PABA was included at 0.2 μg·ml-1or omitted. E. coli BN1163 (pabA1, pabB::Kan, rpsL704, ilvG-, rfb-50, rph-1) harboring pLOI707HE constructs was cultured at 37°C in M9 synthetic minimal medium containing 50 μg·ml-1 kanamycin, 10 μg·ml-1 tetracycline, and 100 μM IPTG, plus or minus 0.5 μg·ml-1 PABA.
Recombinant Protein Production and Analysis. E. coli cells were grown at 37°C in LB medium until A600 was ≈1, at which point IPTG was added (final concentrations were 100 μM for At-ADCS and 500 μM for PabA, -B, and -C), and incubation was continued for 3 h at 30°C (AtADCS) or 37°C (PabA, -B, and -C). Subsequent operations were at 4°C. Pelleted cells from 50- to 500-ml cultures were resuspended in 1-4 ml of 0.1 M Tris·HCl (pH 7.5)/10 mM 2-mercaptoethanol and shaken with 0.1-mm zirconia-silica beads in a MiniBeadbeater (Biospec Products, Bartlesville, OK) at 5,000 rpm for 6 × 20 s. The extracts were centrifuged (15,000 × g, 20 min) and desalted on PD-10 columns (Amersham Biosciences) equilibrated in 0.1 M Tris-HCl (pH 7.5)/10 mM 2-mercaptoethanol/10% (vol/vol) glycerol. Desalted extracts were routinely frozen in liquid N2 and stored at -80°C; this preserved enzyme activity. AtADCS was partially purified, and its molecular mass was estimated by using a Waters 626 HPLC system equipped with a Superdex 200 HR 10/30 column (Amersham Biosciences). Desalted extract (0.2 ml) was applied to the column, which was equilibrated and eluted with 0.1 M Tris·HCl (pH 7.5)/10 mM 2-mercaptoethanol. Carbonic anhydrase, BSA, β-amylase, and apoferritin were used as standards. Protein was estimated by Bradford's method (20), using BSA as the standard.
Enzyme Assays. Assays of PABA synthesis activities were modifications of published procedures (19). Briefly, standard assays (100 μl) contained 50 mM Tris·HCl (pH 7.5), 10 mM MgCl2, 10 mM DTT, 5 mM l-glutamine, 100 μM chorismate (glutamine-dependent assays) or 40 mM triethanolamine (pH 8.5), 26 mM (NH4)2SO4, 8 mM MgCl2, 4 mM DTT, and 80 μM chorismate (NH3-dependent assays) and were run at 37°C for 30-120 min. Desalted PabC extract (7 μg of protein) was added when indicated. Reactions were stopped with 20 μl of 75% (vol/vol) acetic acid, incubated on ice for 1 h, and centrifuged (15,000 × g, 4°C, 20 min). Supernatants (60 μl) were injected onto a Supelco Discovery C18 column (5 μm, 250 × 4.6 mm) and eluted isocratically with 0.5% acetic acid containing 20% (vol/vol) methanol at a flow rate of 1 ml·min-1. The PABA peak was detected by fluorescence (290-nm excitation, 340-nm emission) and quantified relative to standards.
Transient Expression of GFP Fusion Protein in Arabidopsis Protoplasts. The N-terminal region of AtADCS (MNFSFC... GFVRT; residues 3-87) was amplified by PCR with the primers 5′-GAGAGTCGACATGA ATTTT TCGT T T TGT TCA AC-3′ and 5′-GAGACCATGGAAGTCCTCACAAAACCAAGCTTC-3′. [The sequence context of the codon for the methionine at position 3 is closer to the plant translation initiation consensus (21) than that of the first methionine codon.] The amplificate was digested with SalI and NcoI and cloned in frame upstream of the GFP gene in the 35Ω-sGFP(S65T) plasmid (22). Arabidopsis protoplasts were prepared from cell suspension cultures and transformed with the AtADCS construct or the empty vector as described (8, 23). Samples were analyzed by confocal laser scanning microscopy with a Leica TCS-SP2 operating system. GFP and chlorophyll fluorescence were excited (488 and 633 nm, respectively) and collected sequentially. Fluorescence emission was collected from 500 to 535 nm for GFP and 643 to 720 nm for chlorophyll.
PABA Determination. Total PABA (i.e., free PABA plus PABA glucose ester) was isolated from fruit pericarp tissue by methanol extraction, cation exchange chromatography, and ethyl acetate partitioning and quantified by HPLC with fluorescence detection as described (24).
Real-Time Quantitative RT-PCR. Total RNA was isolated from fruit and leaf samples (1-2 g) as described (6) and DNase-treated (DNA-free, Ambion, Austin, TX). Real-time quantitative RTPCR was performed on 250 ng of total RNA in 25-μl reactions by using TaqMan One-Step RT-PCR Master Mix Reagents and a GeneAmp 5700 sequence-detection system (Applied Biosystems). The primers and TaqMan probe were as follows: forward primer, 5′-GGA ATGACCT TGGGCGTGTA-3′; reverse primer, 5′-TGCATAGGATTCAATTTCCATGA-3′; probe, 5′-TGAGACTGGCTCTGTTCATGTCCCACA-3′, with the fluorescent reporter dye 6-carboxyfluorescein and the quencher 6-carboxytetramethylrhodamine. Controls without reverse transcriptase were run to check that the amplifications were not due to contaminating genomic DNA. [3H]UTP standard RNA was prepared from LeADCS cDNA by using the MAXIscript in vitro transcription kit (Ambion). mRNA integrity was checked by semiquantitative RT-PCR analysis of the Never-ripe (Nr) transcript (25). PCR (35 cycles) was conducted on cDNA prepared from 250 ng of total RNA by using the Nr-specific primers 5′-GCAGACGATTTATTCAACTT-3′ and 5′-TTACAGACTTCTTTGATAGC-3′. Controls without reverse transcription gave no amplification product.