Results

Plants Have Genes Specifying PabA-PabB Hybrid Proteins. blast searches of GenBank and TIGR databases with the protein sequences of E. coli PabA and PabB detected a single Arabidopsis gene (At 2 g 28880) and a cognate cDNA (RAFL 09-32-D04) encoding a 103-kDa polypeptide with PabA- and PabB-like domains. Truncated tomato expressed sequence tags specifying a similar protein were also found; 5′ RACE was used to obtain the missing sequence, and complete tomato cDNAs encoding a 102-kDa protein were then isolated by RT-PCR. The deduced Arabidopsis protein is shown in Fig. 2 and is aligned with its tomato counterpart in
Fig. 7, which is published as supporting information on the PNAS web site. Both plant proteins comprise tandem domains that share 33-37% identity with E. coli PabA and PabB, and 26% identity with yeast PABA synthase (Figs. 2 A and 7). The plant
proteins share 61% overall identity with each other. As in yeast and other PABA synthases, the PabA and PabB domains of the plant proteins are separated by a long linker (≈90 residues) whose sequence is not conserved but is rich in basic amino acids (Figs. ​(Figs.2B2B and 7). In contrast to yeast PABA synthase, the plant proteins have
a ≈45-residue insertion near the end of the PabA domain. Like the interdomain linker, this extra sequence is not conserved between Arabidopsis and tomato but is basic in character. The plant proteins also have nonconserved N-terminal extensions of ≈85 residues that display the features of chloroplast targeting peptides (Figs. ​(Figs.22 an) fig. 2.

Primary structure of the deduced Arabidopsis PabA-PabB polypeptide. (A) Scheme comparing the organization of the Arabidopsis polypeptide with that of eukaryotic PABA synthases and with E. coli PabA and PabB. Interdomain linker, red; insertion in PabA (more ...)







Plant PabA-PabB cDNAs Complement E. coli and Yeast PABA Synthesis Mutants. To determine whether the Arabidopsis and tomato cDNAs encode functional enzymes, the sequences predicted to specify the mature proteins (Figs. ​(Figs.2B2B and 7) were cloned into E. coli expression vector pLOI707HE and yeast
expression vector pVT103-U. pLOI707HE allows tightly IPTG-controlled gene expression in diverse host strains (18). The resulting constructs were transformed into an E. coli pabA pabB double mutant and a yeast PABA synthase deletant, both of which are PABA auxotrophs (Fig. 3A). The Arabidopsis and tomato constructs
yielded PABA-independent transformants of the E. coli and yeast mutants with high frequency, and their growth was similar to that of wild-type strains (Fig. 3A). No
complementation was seen with vectors alone (Fig. 3A). In E. coli harboring plant
cDNA constructs, complementation depended strictly on addition of the IPTG inducer, indicating that the plant cDNAs were responsible for the effect (Fig. 3A).
Retransformation of the yeast mutant with rescued plasmid containing the Arabidopsis cDNA restored PABA prototrophy, further confirming that complementation was due to the plant cDNA (data not shown). Taken together, these data indicate that plant PabA-PabB proteins have PabA plus PabB activity (i.e., that they are functional ADCSs) but say nothing about their PabC (ADC lyase) activity.

Fig. 3.
Plant PabA-PabB proteins encode functional enzymes. (A) Complementation of a yeast PABA synthase mutant (Upper) and an E. coli pabA pabB double mutant (Lower). Similar numbers of cells of wild-type (WT) yeast (strain 971/6c) or E. coli (strain K12) and (more ...)


Arabidopsis PabA-PabB Has ADC Synthase but Not Lyase Activity. To investigate ADCS and lyase activities directly, the mature Arabidopsis PabA-PabB protein was overexpressed in E. coli by using the pET-28a plasmid and the host strain BL21-CodonPlus (DE3)-RIL (which is wild type with respect to pabA, pabB, and pabC). ADCS activity was first assayed in desalted extracts, by using glutamine as amino donor and adding excess E. coli PabC to convert ADC to PABA, which was quantified by HPLC. In this coupled assay, vector-alone cell extracts produced traces of PABA (Fig. 3B), attributable to the endogenous PabA and PabB activities of the
host. Extracts of cells overexpressing the Arabidopsis PabA-PabB protein gave 20-fold more PABA (Fig. 3B), providing initial biochemical evidence for ADCS activity.
To corroborate this result and to test for ADC lyase activity, we used size-exclusion chromatography to separate the recombinant Arabidopsis PabA-PabB protein from the E. coli host's PabC enzyme, which is smaller (≈50 kDa; ref. 19). By using glutamine as amino donor, the column fractions were assayed for PABA formation, plus or minus addition of E. coli PabC. When PabC was added, there was a large peak of PABA-forming activity at ≈110 kDa (fractions 19-20), which is close to the predicted mass of the monomeric recombinant enzyme (93 kDa) (Fig. 4A). This
peak disappeared when PabC was omitted (Fig. 4A) and was not given by extracts of
cells transformed with vector alone (Fig. 4B). The minor peak of PABA formation in
fractions 23-24 is attributable to endogenous activity from the host cells (Fig. 4 A and B). These data show that the Arabidopsis PabA-PabB protein has
ADCS activity but lacks ADC lyase activity. The Arabidopsis and tomato proteins may thus be termed ADCSs (AtADCS and LeADCS, respectively). The AtADCS peak of Fig. 4A was unstable, losing all activity within 12 h at 4°C.

Fig. 4.
The Arabidopsis PabA-PabB protein has ADCS but not ADC lyase activity. Extracts of E. coli cells overexpressing the Arabidopsis PabA-PabB protein (A) or harboring the vector alone (B) were fractionated by size exclusion chromatography. Fractions were (more ...)





Amino Donors and Inhibitors of Arabidopsis ADC Synthase. When testing amino donors, we compared the Arabidopsis enzyme with the E. coli enzyme, using assay conditions developed for the latter (19). Like PabA, AtADCS did not use asparagine (Table 1). AtADCS was able to use NH3, like PabB, but apparently more efficiently because the NH3-dependent rate was 50% of the glutamine-dependent rate for AtADCS but only 9% for PabB (Table 1). AtADCS was not inhibited by physiological concentrations of PABA (0.5 μM), PABA glucose ester (6 μM), or folates (10 μM tetrahydrofolate, 5-methyltetrahydrofolate, or 5-formyltetrahydrofolate pentaglutamate), nor was it sensitive to the PABA analogs PABA ethyl ester, p-acetamidobenzoate, p-hydroxybenzoate, or anthranilate (50 μM) (data not shown).


Table 1.
Activities of Arabidopsis and E. coli ADC synthases with various amino donors

Subcellular Localization of Arabidopsis ADC Synthase. To analyze the subcellular localization of AtADCS in vivo, its N-terminal region (85 residues) was fused to the GFP marker protein. Expression of this fusion protein in Arabidopsis protoplasts resulted in a punctate pattern of green fluorescence that colocalized with the red autofluorescence of chlorophyll (Fig. 5 E and F). In contrast, control
protoplasts expressing GFP alone showed green fluorescence throughout the cytoplasm and nucleus (Fig. 5C). These data demonstrate that AtADCS contains a
functional chloroplast targeting peptide, suggesting that plant ADCS is a plastidial enzyme.

Fig. 5.
Expression in Arabidopsis protoplasts of GFP fused to the N-terminal region (residues 3-87) of the Arabidopsis PabA-PabB protein and of GFP alone. Images are optical photomicrographs (A and D), chlorophyll fluorescence (B and E, red pseudocolor), and (more ...)

ADC Synthase mRNA Expression During Tomato Fruit Development. Because nothing is known about PABA synthesis in plants, we measured LeADCS transcript expression and total PABA pools in leaves and in fruits harvested at four stages. LeADCS mRNA levels in unripe fruits were initially ≈10% of that in leaves but dropped dramatically to beneath the detection limit after the breaker stage (Fig. 6A). The
detection of the Nr transcript, which is induced in ripening fruit (25), showed that this fall was not due to general mRNA degradation as ripening advanced (Fig. 6A Inset).
Concentrations of PABA in the fruit increased progressively as the fruit ripened from the mature green to the red and red-ripe stages (Fig. 6B). As previously observed
(24), the majority of PABA (80-90%) was present as its glucose ester (data not shown).

Fig. 6.
Levels of LeADCS mRNA, PABA, and folate in tomato fruits and in leaves. (A) LeADCS mRNA was quantified by real-time RT-PCR on 250 ng of total RNA from pericarp tissue of fruits at mature green (MG), breaker (B), ripe (R), and red-ripe (RR) stages, or (more ...)

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.

Abstract

In this experiment, organic chemistry laboratory students synthesized derivatives of their own design of the plant auxins indole-3-acetic acid (IAA) and 1-naphthalene acetic acid (NAA). Their syntheses varied from making ester to amide derivatives. After optimizing their synthesis, the organic chemistry student groups shared their products with a student group from the biochemistry–cell biology lab. The biochemistry–cell biology students compared the development of Ceratopteris richardii gametophytes on three types of sterile medium: medium without hormones, medium with 10-5 M NAA, and medium with 10-4 M of an auxin derivative synthesized in the organic chemistry lab. The organic syntheses were performed over a four-week period, and observations of C. richardii gametophytes were done for a two week period, after which the gametophyte measurements where analyzed quantitatively using Microsoft Excel. The project terminated in a poster session presented by both the organic chemistry and biochemistry–cell biology classes during which the students discussed their results and compared any structure activity relationships that were evident. Results from this bioassay of auxin derivatives typically fall into four groups: (i) there is an effect on gametophyte growth similar to that of known auxins, (ii) there is no effect on the growth of the gametophyte, (iii) there is a novel effect on gametophyte growth, or (iv) the auxin derivative is lethal to the gametophyte.