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
Six Kan
R
plants were isolated from a transfor- mation with the CGS[ − ] vector. All were confi-
rmed to carry the CGS[ − ] transgene construct using a tissue PCR method not shown. Four of
the transformants, analyzed by genomic DNA blotting with the CGS cDNA as a probe showed a
pattern of hybridization indicative of having arisen through independent single integration events. The
transformants were analyzed with HindIII or BamHI, enzymes that produce, respectively, either
approximately 5 or approximately 15 kbp hy- bridizing fragments in wild type A. thaliana. The
endogenous CGS fragment was observed in each of the transformants, but they showed in addition,
hybridization to a second fragment of variable length, depending upon the transformant, corre-
sponding to the transgene construct Fig. 3.
Analysis of the T2 revealed that without Met feeding all the transgenic plants Kan
R
plants appeared normal up to 10 days after germination.
However, after transfer to soil one-third developed severe growth stunting and where unable to repro-
duce while the other two thirds developed nor- mally and produced viable progeny. Abnormal
and normal siblings are shown in Fig. 4A, com- pare plant A1 with A2. In addition to growth
stunting the abnormal plants developed a cluster of apical shoots and the oldest leaves, which ap-
Fig. 3. Southern blot analysis of CGS[ − ] lines. Total DNA was digested with HindIII lanes 1 – 5 or BamHI lanes
6 – 10. The plant samples were: 415-5 lanes 1 and 7, 415-7 lanes 2 and 8, 415-11 lanes 3 and 9, 415-16 lanes 4 and
10, untransformed A. thaliana ecotype C24 lanes 5 and 6. The blot was probed with a 1.1 kbp fragment from the 5 end
of the CGS1 cDNA. Faint bands are highlighted with aster- isks . The approximate 15 kbp fragment corresponding to
the endogenous CGS gene was probably not detected due to inefficient transfer from the gel to the membrane. All the large
fragments in this experiment show weak hybridization for the same reason.
were 120, 160, and 160°C, respectively. DMS had a retention time of 2.5 min and it was quantitated
by preparing a standard curve with pure SMM Sigma or DMSP Research Plus, Bayonne, NJ.
DMSP is a sulfonium compound that is less stable than SMM, decaying to DMS under alkaline con-
ditions at room temperature.
Fig. 4. Morphology of plants with repressed levels of CGS. A, siblings from CGS[ − ] line 415-7 T2 generation showing abnormal A1 and normal A2 morphology. B, siblings of the plants in A grown for 51 days without Met feeding. The plants
at the top B2 are normal while those at the bottom B1 are of the abnormal type. C, the same plants shown in A were photographed 16 days after initiating the feeding of a 0.2 mM solution of Met. The plants were grown for 35 days at the time
Met feeding was initiated. D, Wild-type A. thaliana 25 days after being watered with a 1 mM solution of propargylglycine. Fig. 5. Suppression of the abnormal morphology in CGS[ − ] plants grown with nutritional supplements. Homozygous plants
from abnormal CGS[ − ] line 415-5 T3 generation were grown axenically for 30 days on unsupplemented agar medium A or with 0.2 mM Met B. The photographs show a typical result that occurs with all the CGS[ − ] lines. Similar changes in
appearance were used to determine whether other nutrient supplements suppress the abnormal phenotype, reported in Table 2.
peared normal early on, became thickened, curled and in some of the CGS[ − ] lines accumulated a
red-brown pigment in the petioles. The changes in morphology became clearly evident within 40 days
after germination. The abnormal plants were un- able to produce flowers and eventually died with-
out reproducing Fig. 4B, observe plants labeled B1. However, when watered with a solution of
Met their growth was restored sufficiently to allow them to flower and to set viable seed. Plant A1
was photographed before Met-feeding Fig. 4A. The stimulation of growth is evident in the same
plant 16 days after initiating Met-feeding Fig. 4C, plant C1. Visible signs of growth became evident
within 48 h after initiating Met-feeding. The pro- liferation of apical shoots observed in abnormal
CGS[ − ] plants became clearly evident after Met was fed and each of the shoots developed into an
independent inflorescence Fig. 4C, plant C1.
The progenies derived from abnormal CGS[ − ] plants were all Kan
R
and developed the abnormal phenotype. By contrast, the morphologically nor-
mal Kan
R
plants produced Kan
R
progeny at a ratio of approximately 3:1. The Kan
R
plants segre- gated into normal and abnormal phenotypes at a
ratio of 2:1 Table 1. The segregation results indicate that the antisense transgene behaves as a
single recessive locus that produces abnormality when in the homozygous state rather than the
hemizygous condition. Similar results were ob- tained with all six independently isolated CGS[ − ]
transgenic lines. The abnormal morphology has been stable over six generations.
The developmental progression of plants from abnormal homozygous CGS[ − ] lines was studied
by recording the time of emergence of new leaves. Homozygous CGS[ − ] plants grew like wild-type
and hemizygous siblings until 24 – 28 days after germination after which new leaves stopped
emerging and the single apical shoot proliferated into a mass of apical shoots. The growth of the
homozygous CGS[ − ] plants arrested prior to conversion to reproductive growth.
Testing a variety of sulfur compounds for the ability to suppress the abnormal phenotype pin-
pointed the lesion in the CGS[ − ] lines. This ex- periment was carried out on agar medium under
axenic conditions in order to eliminate the possi- bility of interference from or metabolism of the
supplements by microorganisms. After growth on unsupplemented agar medium the abnormal phe-
notype was clearly visible after 30 days Fig. 5A. The phenotype differs slightly from plants grown
on soil in that the plants become chlorotic and the apical shoot remains very small Fig. 5A. The
phenotype is suppressed by Met feeding Fig. 5, compare A and B similar to the results with soil
grown plants. Cystathionine and Hcy also sup- pressed the abnormal phenotype but neither Cys
or glutathione, a Cys-containing metabolite, were able to restore the normal phenotype Table 2.
Table 1 Inheritance of the abnormal phenotype in CGS[−] plants
a
Kan
R
Phenotype Line
Abnormal frequency
Abnormal Normal
41 24
415-4
b
17 0.41
0.26 415-5
b
24 69
93 169
415-5
c
169 1.00
442 299
415-7
b
143 0.32
179 415-7
c
179 1.00
0.34 43
83 126
415-11
b
137 415-11
c
137 1.00
65 45
20 415-16
b
0.31 65
48 17
415-18
b
0.26
a
Segregation analysis was performed with self-pollinated CGS[−] plants in the T3 generation. Kan
R
plants were selected, transferred to soil, and the plants scored for mor-
phology after 45 days. The total number of Kan
R
and the number segregating into normal and abnormal plants are
given.
b
Seeds were from morphologically normal Kan
R
plants.
c
Seeds were from abnormal Kan
R
plants. Table 2
Chemical supplements suppress the abnormal morphology of CGS[−] plants
a
Chemical Appearance
B
D
- or
L
-Met Cystathionine
B B
D
,
L
-homocysteine A
L
-Cys A
Glutathione 5-Methylthioadenosine MTA
B B
4-Methylthio-2-oxobutanoic acid MTOB 4-Methylthio-2-hydroxy butyric acid
B MTHB
a
CGS[−] plants were germinated and axenically grown for 45 days on M-S agar medium with the indicated chemical
supplement at 0.2 mM. The ability of the chemical to sup- press the abnormal phenotype was scored. A and B refers to
the plant appearance as shown in Fig. 5A and B.
Table 3 CGS activity in A. thaliana
a
Specific activity nmolmin per mg Line
A. thaliana 0.330 9 0.060
0.040 9 0.010 415-5
0.036 9 0.009 415-7
415-11 0.073 9 0.011
a
CGS enzyme activity was measured in the shoot of 40- day-old plants grown in soil. The transgenic lines analyzed
were homozygous T3 plants. The averages 9 S.D. of three independent experiments are shown.
2 that plants can convert to Met without the aid of CGS see Fig. 1. MTA is an intermediate in the
Yang cycle that functions in recycling of Met from SAM [4]. MTHB and MTOB are hydroxy and oxo
acids that can be converted to Met via ubiquitous amino acid transaminases and dehydrogenases [3].
Yet another indication that low CGS activity may account for the growth abnormalities of CGS[ − ]
plants is that treatment of wild type A. thaliana with the CGS inhibitor, propargylglycine PAG
[10] produces similar growth abnormalities Fig. 4D.
CGS activity is repressed in abnormal CGS[ − ] lines Table 3. Compared with wild-type, the
CGS[ − ] lines 415-5, 415-7 and 415-11 had 5 – 9- fold lower CGS activity and a comparably lower
amount of CGS protein as measured by im- munoblotting Fig. 6. Analysis of the amino acid
content in the transgenic lines revealed that the level of free Met and SMM is similar to wild type
at 19 and 40 days after germination Table 4. The level of Thr and total amino acids was increased
3 – 5-fold in 40-day-old but not in 19-day-old plants. Therefore, the development of abnormality
is correlated with the increase in total amino acids. However, Thr is not specifically increased. By
comparison the mto
1
mutant of A. thaliana shows 19 – fold higher Met and 14-fold higher SMM in
19-day-old plants but no higher level of total amino acids. By 40 days there was no greater Met
than in the wild type. Similar results with mto
1
, an ethionine-resistant mutant,
which accumulates Met in the early vegetative stage, have been previ-
ously published [20].
4. Discussion
