enzymes are distributed between plastids and the cytosol [4]. Plastids contain all the Asp-family
enzymes, the complete Cys pathway, CGS and cystathionine b-lyase. Met synthase and SAM syn-
thetase are exclusively cytosolic as are the enzymes for SMM synthesis [5].
Met synthesis is regulated at multiple levels. A general mechanism for control of the Asp-family
amino acids centers on feedback inhibition of Asp kinase AK, the first enzyme of the Asp pathway,
by Lys, Thr, and SAM [6]. Combined treatment with Lys and Thr is herbicidal because they re-
press the Asp pathway blocking the synthesis of the carbon skeleton and causing Met starvation
[7]. A Met-specific control mechanism centers on the competition between CGS and Thr synthase
TS for their common substrate OPH. TS activity is stimulated by SAM and it has a much higher
affinity for OPH than does CGS. Thus, it has been proposed that CGS may compete poorly for OPH
when Met hence SAM is abundant [8,9]. By contrast, when Met is limiting and TS less active
CGS has a greater ability to compete for OPH. There is also evidence that when Met is limiting
CGS expression is induced. For example, com- bined treatment with Thr and Lys causes CGS
activity to increase, whereas Met treatment causes it to decrease [10,11].
With the recent cloning of the CGS cDNA from Arabidopsis thaliana [12] it became possible to
study its function in transgenic plants. Repression of CGS activity was found to limit the ability of
A. thaliana to grow autonomously without exoge- nous application of Met. The CGS-repressed
plants show an abnormal morphology that is in- herited as a recessive trait.
2. Materials and methods
2
.
1
. Preparation of antibodies against recombinant CGS
Recombinant A. thaliana CGS was synthesized as an S-TAG and hexa-His fusion protein ex-
pressed from vector pET30c Novagen. A 1.4 kbp XhoI fragment from the CGS cDNA [12] Gen-
Bank Accession Number U43709 was cloned into pET30c to produce the pET-CGS construct which
was used to transform Escherichia coli strain BL21DE3pLysS Novagen. Transformants were
selected on LB medium with 40 mgml chloram- phenicol and 30 mgml kanamycin. The culture
was grown in liquid LB medium with the antibi- otics at 37°C until an OD at 600 nm of approxi-
mately 0.3 was achieved. Then 2.0 mM IPTG was added and the culture incubated further for 5 h at
30°C. The recombinant enzyme, purified by Ni- affinity chromatography, as described by the pET
protocol from Novagen, showed a characteristic absorption peak at 415 nm associated with pyri-
doxal phosphate enzymes. The ratio between ab- sorbency maxima at 282 and 415 nm was 3.88,
identical to native CGS from spinach [13]. CGS activity was measured as described by Ravanel et
al. [13] using O-succinylhomoserine. The pure en- zyme showed a specific activity of approximately
2.1 mmol cystathionine formedminmg protein at 24°C, pH 7.5. Although plant CGS uses OPH as
the physiological substrate it can also use a variety of homoserine esters including O-succinylhomos-
erine [13] which is commercially available from Sigma.
A New Zealand White rabbit was immunized subcutaneously with purified recombinant CGS
protein 1 mg in Freund’s Complete Adjuvant. The rabbit was boosted with 1 mg CGS in saline
solution at intervals of 1 month. Serum samples were taken 7 days after boost immunization. The
Fig. 1. Met metabolism and regulation in plants. The path- ways for Met synthesis and metabolism are shown along with
their subcellular compartmentation. Enzymes thought to be involved in regulation of Met synthesis are shown in bold
lettering.
sample taken after the third boost was used di- rectly on immunoblots.
2
.
2
. Construction of transgenic plants Antisense repression was used to reduce CGS
activity in A. thaliana. A 1.1 kbp SalI fragment from the 5 end of the CGS cDNA [12] was cloned
into pFF19 [14] placing the CGS gene in the antisense orientation with respect to the 35S pro-
moter. The expression cassette was subcloned into the transformation vector pBI101 Clonetech as
an approximately 2.0 kbp HindIII – EcoRI frag- ment to produce the CGS[ − ] construct. The
CGS[ − ]
construct was
used to
transform Agrobacterium tumefaciens strain pGV2260, and
then A. thaliana C24 by vacuum infiltration [15]. Kan
R
plants were selected on medium with 50 mgml kanamycin.
2
.
3
. Analysis of CGS
[
−
]
plants All soil grown plants were raised in a 23°C
growth chamber with a light intensity of 100 mE m
2
per second and a photoperiod of 14 h light followed 10 h of darkness. The plants were wa-
tered with one-quarter strength Peters™ water-sol- uble 20:20:20 fertilizer Grace-Sierra, Milpitas,
CA prepared in distilled water. Plants requiring exogenously supplied Met for growth in soil were,
in addition, watered daily in the root zone with 1 ml 0.2 mM Met. The nutritional requirements of
the CGS[ − ] plants were tested with axenically grown plants raised on agar solidified MS medium
supplemented as described in the figure or table legends. The plants were incubated in a 21°C
growth chamber with a light intensity of 90 mE m
2
s, 13 h light11 h dark period. Transgenic plants were confirmed to carry the CGS[ − ] con-
struct by a PCR method [16] using a 35S promoter primer 5-TATCTCCACTGACGTAAGGGAT-
GA-3 and a CGS specific primer 5-ATGGC- ATCTGGGATGTGTGC-3
and by
genomic DNA blotting using the CGS cDNA as a probe
[17]. The content of the CGS protein was determined
by immunoblotting carried out as described in Wang et al. [18]. Soluble protein extracts were
prepared from the entire shoot of 40-day-old plants. Total protein 40 mg was analyzed by
SDS-PAGE in a gel containing 10 wv acry- lamide. The protein concentration was measured
using the Bradford dye-binding assay with BSA as a standard BioRad. Antisera against CGS or A.
thaliana serine acetyltransferase SAT [19] were used at a dilution of 1:2000. The SAT antibody
served to control for protein loading. A secondary antibody was horseradish peroxidase-linked goat
anti-rabbit diluted 1:8000 and immune complexes were detected with the Renaissance™ Kit Dupont
NEN.
Soluble amino acids were measured in the shoot of 19- and 40-day-old CGS[ − ] plants and mto
1
plants [20] provided by Dr Satoshi Naito, Hok- kaido University. Amino acids were analyzed by
HPLC after alkylation with phenylisothiocyanate PITC [21]. Plant tissues were extracted and the
amino acids purified by chromatography on AG 50W-8 [20]. After binding to the ion exchange
resin and then washing, the column was eluted with 2 N NH
4
OH. The eluate was dried by evapo- ration under a stream of nitrogen and the residue
dissolved in
a solution
of 7:1:1:1
vvvv ethanol – water – triethanolamine – PITC. Met and
Thr were specifically measured. Their recoveries were estimated by comparing the amount of each
amino acid from a typical tissue sample with that in a spiked tissue sample. The recoveries were, Thr
approximately 81 and Met approximately 88. Total amino acid content was calculated as the
sum of all peak areas from a chromatograph divided by the fresh weight of the plant sample.
SMM was measured as dimethylsulfide DMS released from fresh plant samples after alkaline
treatment at 90°C. The assay was developed for algal samples [22] and was optimized here for A.
thaliana. A similar method has been reported for vascular plant samples [23]. Tissue samples rang-
ing from 25 to 150 mg fresh weight were combined with 400 ml 1 M NaOH in a 2 ml gas tight vial
fitted with double-faced PTFEsilicone septa SU- PELCO, Bellefonte, PA. The vials were heated
for 3 h at 90°C in a heating block. After cooling to room temperature, 200 ml of the gas phase was
removed using a gas tight syringe and injected into a Shimadzu GC-17A gas chromatograph with a
Poraplot Q, 10 m × 0.53 mm fused silica capillary column Chrompack, The Netherlands. DMS was
detected by flame ionization. The chromatograph was resolved with a mixture of hydrogen, nitrogen,
and air at 60, 70, and 50 kPa, respectively. The temperatures of the column, injector, and detector
Fig. 2. Release of DMS from dimethyl sulfonium compounds and A. thaliana. DMSP 100 nmol
, SMM 100 nmol
, or various amounts of A. thaliana tissue were added to a vial containing 400 ml 1 M NaOH. A Incubation
was for the specified time at 23°C
, or 90°C , . B Incubation was for 3 h at 90°C .
The SMM assay is extremely simple and gives reliable measurements. Release of DMS from
SMM requires strongly alkaline conditions and incubation at 90°C for about 2 h Fig. 2A [22].
GC analysis of the volatilized product from pure SMM subjected to these conditions shows a single
peak of DMS with a retention time of 2.5 min. A. thaliana leaf samples produce four volatile prod-
ucts. The peak that migrates with DMS is pro- duced at a rate identical to that from pure SMM
Fig. 2A. Moreover, the DMS peak increases selectively when more sample is added Fig. 2B or
pure SMM is added with the leaf sample. The three additional peaks have retention times of
0.77, 0.95 and 2.2 min. They are released from the leaf sample only after alkaline incubation at 90°C,
but they appear more rapidly than does SMM- derived DMS or the peak area does not correlate
with the amount of tissue added to the assay. Therefore, they are probably unrelated to SMM.
3. Results
