Plant Science 159 2000 233 – 242
Enhanced tolerance of transgenic Brassica juncea to choline confirms successful expression of the bacterial codA gene
K.V.S.K. Prasad, P. Sharmila, P. Pardha Saradhi
,1 Plant Physiology and Biotechnology Laboratory, Department of Biosciences, Jamia Millia Islamia, New Delhi
110025
, India Received 28 March 2000; received in revised form 13 July 2000; accepted 13 July 2000
Abstract
Brassica juncea cv. Pusa Jaikisan was transformed with the codA gene for choline oxidase from Arthrobacter globiformis with an aim to introduce glycine betaine biosynthetic pathway, as it lacks any means to synthesize glycine betaine. Western blot
analysis revealed the presence of choline oxidase in the protein extract from the codA transgenic lines, demonstrating that the bacterial codA gene had been successfully transcribed and translated in transgenic lines. Good activity of choline oxidase indicated
its presence in fully functional form in the transformed lines. This was further confirmed by the presence of glycine betaine only in the transformed lines of B. juncea. The shoots of both wild type and transformed lines were exposed to various concentrations
of choline in order to evaluate if the introduction of the codA gene in any way enhances the potential of B. juncea to tolerate high levels of choline. The growth in terms of fresh weight and dry weight of the shoots of transformed lines exposed to high levels
of choline was significantly superior to those of wild type. Moreover, the loss in chlorophyll content and the activity of photosystem II in shoots of the transformed lines exposed to high concentration of choline were significantly lower than that
observed in wild type. These results showed that shoots of B. juncea transformed with the codA gene, most probably had the potential to readily convert choline to glycine betaine. Therefore, choline tolerance can be used as an efficient marker for the
identification of the lines transformed with the codA gene. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Brassica juncea; Transformation; Choline oxidase; Glycine betaine; Choline toxicity www.elsevier.comlocateplantsci
1. Introduction
Glycine betaine, a quaternary ammonium com- pound, is amongst the most effective compatible
solutes which accumulates in a wide variety of organisms viz. bacteria, cyanobacteria, algae, ani-
mals and higher plants e.g. members of Chenopo- diaceae and Poaceae as an adaptive means to
tolerate abiotic stresses [1,2]. Glycine betaine is known to protect living systems from saltosmotic
stress by i acting as an osmotic solute in order to maintain cellular water content by regulating its
osmoticwater potential; ii stabilizing functional units such as oxygen evolving photosystem II
complex; iii protecting the structure of proteins including ribulose 1,5-bisphosphate carboxylase
oxygenase Rubisco; iv maintaining membrane integrity; and v acting as a counteracting solute
[3 – 9].
Although, a number of higher plants especially certain halotolerant plants have glycine betaine
biosynthetic pathways, there are several important crop plants such as tomato, potato, rice and mus-
tard which lack any means for the synthesis of this compatible solute [10,11]. Having realized the sig-
nificance of glycine betaine in enhancing osmotic tolerance, several groups are involved in the intro-
duction of biosynthetic pathways for synthesis of glycine betaine into plant species that lack the
ability [2,11 – 13] to synthesize glycine betaine.
In angiosperms glycine betaine is synthesized from choline under the combined action of choline
monooxygenase and betaine aldehyde dehydroge-
Corresponding author. Tel.: + 91-11-6916275. E-mail address
:
ppsaradhihotmail.com P. Pardha Saradhi.
1
E-mail: saradhindf.vsnl.net.in. 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 3 4 0 - X
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
