nase [14,15]. In certain animals and bacteria Es- cherichia coli the synthesis of glycine betaine
from choline is mediated by choline dehydroge- nase and betaine aldehyde dehydrogenase. Both
choline monooxygenase and choline dehydroge- nase convert choline to betaine aldehyde whereas
betaine aldehyde dehydrogenase converts betaine aldehyde to glycine betaine [14,15]. However, in
certain bacteria such as Arthrobacter sp. and Alcaligenes sp. choline is converted to glycine
betaine in a single step by choline oxidase [16 – 18].
Choline is one of the important quaternary ammonium compounds that has been shown to
play a role in regulating membrane composition through synthesis of phosphotidyl choline and
fluidity [19 – 21].
Exogenous application
of choline has been shown to i promote root in-
duction and
root growth
[22]; ii
increase growth [22,23]; iii increase the level of glycine
betaine in tobacco transformed with the CMO gene [2]; and iv increase tolerance of certain
plants to abiotic stresses [24 – 26]. However, ap- plication of choline in excess i.e. in toxic levels
causes deleterious effects on cellular metabolism in plants. In general, high concentration of
choline has been shown to inhibit the activities of some of the most vital enzymes such as Ru-
bisco, glyceraldehyde-3-phosphate dehydrogenase, isocitrate dehydrogenase and malate dehydroge-
nase that are associated with photosynthesis and respiration [27,28].
As choline is a precursor for synthesis of glycine betaine, we believe that the introduction
of genes that are associated with the synthesis of glycine betaine would enhance the potential of
plants which otherwise do not have any means to synthesize glycine betaine to withstand toxic
levels of choline. In this communication, we are reporting that codA transformed lines of B.
juncea have the potential to perform well, even in the presence of choline at levels which are
otherwise highly toxic to wild type.
2. Materials and methods
Agrobacterium tumefaciens EHA 101 with a binary
vector designated
as pGAHcodA
{consisting of transit peptide sequence of the small subunit of Rubisco from tobacco, the codA
gene from A. globiformis and nopaline synthase terminator ligated into HpaI site of pGAH with
genes for kanamycin and hygromycin resis- tance} [13] was used for transforming B. juncea.
Shoots selected on kanamycin and hygromycin containing
medium were
multiplied indepen-
dently and tested through PCR and Southern analysis in order to ensure the insertion of the
codA gene and to find out number of copies that got integrated in genome of B. juncea cv. Pusa
Jaikisan data not shown. Two of the transgenic lines viz. 1 and 3 that showed single insert of
the codA gene were selected and selfed. The plants obtained by germinating the seeds from
these two transgenic lines on medium with kanamycin
and hygromycin
were grown
in greenhouse and the seeds obtained by selfing
each individual plant were collected separately. The seed lot which showed 100 germination in
the presence of kanamycin and hygromycin were considered to be obtained from homozygous
plants. Seeds obtained from homozygous plants of transgenic lines viz. 1 and 3 were used for
the present studies.
2
.
1
. SDS-PAGE and Western analysis Leaf samples 200 – 300 mg fresh weight were
homogenized in a mortar and pestle with 40 mM Tris – HCl pH 7.2, 5 mM EDTA and 10 mM
b-mercaptoethanol. The homogenate was cen- trifuged at 18 000 × g for 15 min at 4°C. The
clear supernatant thus obtained was used for Western blot analysis. The protein content was
quantified as described by Bradford [29].
Suitable aliquots
equivalent to
20 mg
of protein were subjected to electrophoresis on 10
polyacrylamide gel that contained 0.1 SDS [30]. For Western analysis the proteins were elec-
trophoretically transferred on to a polyvinyl difl- uoride membrane Immobilon™ – N; Millipore
Corporation, Bedford, MA, USA. These mem- branes were blocked overnight with 3 bovine
serum albumin BSA. Subsequently, immunolog- ical detection was performed according to the
protocol supplied with Vectastain ABC-PO rab- bit IgG kit Vector laboratories, Burlingame,
CA using polyclonal antiserum that was raised in rabbit against pure choline oxidase from A.
globiformis from Sigma Chemical Co., USA us- ing standard procedures [31].
2
.
2
. Determination of choline oxidase acti6ity For measuring the choline oxidase activity
shoots were homogenized in phosphate buffer 40 mM pH 8.0 and the supernatant obtained after
centrifuging the homogenate at 18 000 × g for 15 min at 4°C was used for measuring the choline
oxidase activity according to the protocol of Ikuta et al. [16].
2
.
3
. Quantification of glycine betaine and choline Levels of glycine betaine and choline in leaves of
both wild type and transformed plants were deter- mined using the
1
H-NMR spectroscopy as per the procedure of Wall et al. [32]. Leaf tissue 4 g fresh
weight was powdered in a mortar using liquid nitrogen. The powder was suspended in 25 ml of
1.0 N H
2
SO
4
and incubated at 25°C for 2 h. Cell debris was removed by centrifugation at 1000 × g
for 10 min and the quaternary ammonium com- pounds including choline were recovered from
the supernatant by periodide precipitation method [32]. The resultant periodide adducts were col-
lected by centrifugation at 1000 × g for 30 min and dissolved in 0.5 ml of CD
3
OD E-Merck, Germany
containing 0.5
mM 2-methyl-2-
propanol E-Merck, Germany as an internal stan- dard. This solution was transferred to the NMR
tube and
1
H-NMR spectrum was recorded at 25°C with a NMR spectrometer DRX 00 Bruker, Karl-
sruhe, Germany with a pulse time of 5 ms and an acquisition time of 4 s. Peak identities were
confirmed with the help of authentic standards Sigma Chemical Co., USA. Quantification of
glycine betaine and choline was achieved by com- paring integrated peak intensities against standard
curves. In order to further confirm that the peaks correspond to these quaternary ammonium com-
pounds, spiking experiments were also performed through addition of glycine betaine as well as
choline to the periodide adducts.
1
H-NMR re- sponse positions d for N-methyl protons of
glycine betaine and choline from the plant extracts were compared with that of the standards.
2
.
4
. Measurement of chlorophyll Plant material 1 g fresh weight was homoge-
nized in a mortar and pestle using 5 ml of chilled 80 acetone. The homogenate was centrifuged at
10 000 × g at 4°C for 10 min. The absorbance of the supernatant was measured at 646, 663 and 750
nm, respectively, and chlorophyll content was cal- culated as per the method of Arnon et al. [33].
2
.
5
. Choline tolerance experiments
2
.
5
.
1
. Effect on growth To assess the tolerance to high levels of exoge-
nously applied choline, shoots measuring 3 cm long bearing a minimum of four leaves of wild
type and both transformed lines were transferred to half-strength MS medium [34] supplemented
with choline chloride filter sterilized at concen- trations ranging from 0 to 30 mM. The cultures
were incubated at 25°C under cool white fluores- cent tubes Philips India Ltd. having a light inten-
sity of 120 mmol m
− 2
s
− 1
with 168-h daynight cycle for 20 days. Changes in fresh weight, dry
weight and chlorophyll content of the shoots were measured.
2
.
5
.
2
. Effect on photosynthesis The leaves from shoots grown on different con-
centrations of choline chloride for 20 days were used
for isolation
of thylakoid
membranes. Freshly harvested leaves were dipped in ice cold
isolation buffer pH 7.8 containing 400 mM su- crose, 10 mM NaCl, and 20 mM Tricine and
incubated in dark. After 30 min the leaves were taken out and homogenized in the pre-chilled mor-
tar and pestle in ice-cold isolation buffer in dark. The homogenate was filtered through four layers
of miracloth and the filtrate was centrifuged at 5000 × g for 10 min at 4°C. The pellet was washed
and resuspended in a small volume of suspension buffer pH 7.5 containing 100 mM sucrose, 10
mM NaCl, 2 mM MgCl
2
and 20 mM HEPES. Chlorophyll content of the thylakoid was esti-
mated according to the method of Arnon et al. [33].
Photochemical activities of the isolated thy- lakoids were assayed polarographically with a
Clark type oxygen electrode [35]. For the assay of Photosystem PS II activity the reaction mixture
consisted of 500 mM p-benzoquinone along with the suspension buffer. The thylakoids equivalent
to 20 mg chlorophyll was used for each assay. All the measurements were carried out at 25 9 2°C
using saturating white light 500 mmol m
− 2
s
− 1
.
Photosystem II activity was also measured as the ratio of variable to maximum fluorescence
F
v
F
m
by a fluorometer PAM 2000, Walz, Effel- trich, Germany in the pulse amplitude modula-
tion mode. The leaves intact from the shoots exposed to various treatments were dark adapted
in order to ensure that all the components of photosystem II are in an oxidized state i.e. photo-
system II centers are fully open for 30 min prior to the measurement of F
v
F
m
. Original F
o
and maximal F
m
fluorescence yields were measured with weak modulated red light B 0.5 mmol m
− 2
s
− 1
with 0.8 s pulse of saturating light \ 6.8 mmol m
− 2
s
− 1
PAR, using the data acquisition software DA 2000; Walz attached to the instru-
ment. The variable fluorescence yield F
v
was defined as F
m
− F
o
. The photosystem II activity was measured as F
v
F
m
ratio [36].
3. Results and discussion
