Plant Science 159 (2000) 233–242

Enhanced tolerance of transgenic Brassica juncea to cholineconfirms 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 withan aim to introduce glycine betaine biosynthetic pathway, as it lacks any means to synthesize glycine betaine. Western blotanalysis revealed the presence of choline oxidase in the protein extract from the codA transgenic lines, demonstrating that thebacterial codA gene had been successfully transcribed and translated in transgenic lines. Good activity of choline oxidase indicatedits presence in fully functional form in the transformed lines. This was further confirmed by the presence of glycine betaine onlyin the transformed lines of B. juncea. The shoots of both wild type and transformed lines were exposed to various concentrationsof choline in order to evaluate if the introduction of the codA gene in any way enhances the potential of B. juncea to tolerate highlevels of choline. The growth (in terms of fresh weight and dry weight) of the shoots of transformed lines exposed to high levelsof choline was significantly superior to those of wild type. Moreover, the loss in chlorophyll content and the activity ofphotosystem II in shoots of the transformed lines exposed to high concentration of choline were significantly lower than thatobserved in wild type. These results showed that shoots of B. juncea transformed with the codA gene, most probably had thepotential to readily convert choline to glycine betaine. Therefore, choline tolerance can be used as an efficient marker for theidentification 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

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1. Introduction

Glycine betaine, a quaternary ammonium com-pound, is amongst the most effective compatiblesolutes which accumulates in a wide variety oforganisms viz. bacteria, cyanobacteria, algae, ani-mals and higher plants (e.g. members of Chenopo-diaceae and Poaceae) as an adaptive means totolerate abiotic stresses [1,2]. Glycine betaine isknown to protect living systems from salt/osmoticstress by (i) acting as an osmotic solute (in orderto maintain cellular water content by regulating itsosmotic/water potential); (ii) stabilizing functionalunits such as oxygen evolving photosystem IIcomplex; (iii) protecting the structure of proteins

including ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco); (iv) maintaining membraneintegrity; and (v) acting as a counteracting solute[3–9].

Although, a number of higher plants (especiallycertain halotolerant plants) have glycine betainebiosynthetic pathways, there are several importantcrop plants such as tomato, potato, rice and mus-tard which lack any means for the synthesis of thiscompatible solute [10,11]. Having realized the sig-nificance of glycine betaine in enhancing osmotictolerance, several groups are involved in the intro-duction of biosynthetic pathways for synthesis ofglycine betaine into plant species that lack theability [2,11–13] to synthesize glycine betaine.

In angiosperms glycine betaine is synthesizedfrom choline under the combined action of cholinemonooxygenase and betaine aldehyde dehydroge-

* Corresponding author. Tel.: +91-11-6916275.E-mail address: ppsaradhi@hotmail.com (P. Pardha Saradhi).1 E-mail: saradhi@ndf.vsnl.net.in.

0168-9452/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.

PII: S 0 1 68 -9452 (00 )00340 -X

K.V.S.K. Prasad et al. / Plant Science 159 (2000) 233–242234

nase [14,15]. In certain animals and bacteria (Es-cherichia coli ) the synthesis of glycine betainefrom choline is mediated by choline dehydroge-nase and betaine aldehyde dehydrogenase. Bothcholine monooxygenase and choline dehydroge-nase convert choline to betaine aldehyde whereasbetaine aldehyde dehydrogenase converts betainealdehyde to glycine betaine [14,15]. However, incertain bacteria (such as Arthrobacter sp. andAlcaligenes sp.) choline is converted to glycinebetaine in a single step by choline oxidase [16–18].

Choline is one of the important quaternaryammonium compounds that has been shown toplay a role in regulating membrane composition(through synthesis of phosphotidyl choline) andfluidity [19–21]. Exogenous application ofcholine has been shown to (i) promote root in-duction and root growth [22]; (ii) increasegrowth [22,23]; (iii) increase the level of glycinebetaine in tobacco transformed with the CMOgene [2]; and (iv) increase tolerance of certainplants to abiotic stresses [24–26]. However, ap-plication of choline in excess (i.e. in toxic levels)causes deleterious effects on cellular metabolismin plants. In general, high concentration ofcholine has been shown to inhibit the activitiesof 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 andrespiration [27,28].

As choline is a precursor for synthesis ofglycine betaine, we believe that the introductionof gene(s) that are associated with the synthesisof glycine betaine would enhance the potential ofplants (which otherwise do not have any meansto synthesize glycine betaine) to withstand toxiclevels of choline. In this communication, we arereporting that codA transformed lines of B.juncea have the potential to perform well, evenin the presence of choline at levels which areotherwise highly toxic to wild type.

2. Materials and methods

Agrobacterium tumefaciens (EHA 101) with abinary vector designated as pGAH/codA{consisting of transit peptide sequence of thesmall subunit of Rubisco from tobacco, the codA

gene from A. globiformis and nopaline synthaseterminator ligated into HpaI site of pGAH (withgenes for kanamycin and hygromycin resis-tance)} [13] was used for transforming B. juncea.

Shoots selected on kanamycin and hygromycincontaining medium were multiplied indepen-dently and tested through PCR and Southernanalysis in order to ensure the insertion of thecodA gene and to find out number of copies thatgot integrated in genome of B. juncea cv. PusaJaikisan (data not shown). Two of the transgeniclines (viz. 1 and 3) that showed single insert ofthe codA gene were selected and selfed. Theplants obtained by germinating the seeds (fromthese two transgenic lines) on medium withkanamycin and hygromycin were grown ingreenhouse and the seeds obtained by selfingeach individual plant were collected separately.The seed lot which showed 100% germination inthe presence of kanamycin and hygromycin wereconsidered to be obtained from homozygousplants. Seeds obtained from homozygous plantsof transgenic lines (viz. 1 and 3) were used forthe present studies.

2.1. SDS-PAGE and Western analysis

Leaf samples (200–300 mg fresh weight) werehomogenized in a mortar and pestle with 40 mMTris–HCl (pH 7.2), 5 mM EDTA and 10 mMb-mercaptoethanol. The homogenate was cen-trifuged at 18 000×g for 15 min at 4°C. Theclear supernatant thus obtained was used forWestern blot analysis. The protein content wasquantified as described by Bradford [29].

Suitable aliquots equivalent to 20 mg ofprotein 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; MilliporeCorporation, Bedford, MA, USA). These mem-branes were blocked overnight with 3% bovineserum albumin (BSA). Subsequently, immunolog-ical detection was performed according to theprotocol supplied with Vectastain ABC-PO (rab-bit IgG) kit (Vector laboratories, Burlingame,CA) using polyclonal antiserum that was raisedin rabbit against pure choline oxidase from A.globiformis (from Sigma Chemical Co., USA) us-ing standard procedures [31].

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2.2. Determination of choline oxidase acti6ity

For measuring the choline oxidase activityshoots were homogenized in phosphate buffer 40mM (pH 8.0) and the supernatant obtained aftercentrifuging the homogenate at 18 000×g for 15min at 4°C was used for measuring the cholineoxidase activity according to the protocol of Ikutaet al. [16].

2.3. Quantification of glycine betaine and choline

Levels of glycine betaine and choline in leaves ofboth wild type and transformed plants were deter-mined using the 1H-NMR spectroscopy as per theprocedure of Wall et al. [32]. Leaf tissue (4 g freshweight) was powdered in a mortar using liquidnitrogen. The powder was suspended in 25 ml of1.0 N H2SO4 and incubated at 25°C for 2 h. Celldebris was removed by centrifugation at 1000×gfor 10 min and the quaternary ammonium com-pounds (including choline) were recovered fromthe supernatant by periodide precipitation method[32]. The resultant periodide adducts were col-lected by centrifugation at 1000×g for 30 minand dissolved in 0.5 ml of CD3OD (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 NMRtube and 1H-NMR spectrum was recorded at 25°Cwith a NMR spectrometer (DRX 00 Bruker, Karl-sruhe, Germany) with a pulse time of 5 ms and anacquisition time of �4 s. Peak identities wereconfirmed with the help of authentic standards(Sigma Chemical Co., USA). Quantification ofglycine betaine and choline was achieved by com-paring integrated peak intensities against standardcurves. In order to further confirm that the peakscorrespond to these quaternary ammonium com-pounds, spiking experiments were also performedthrough addition of glycine betaine as well ascholine to the periodide adducts. 1H-NMR re-sponse positions (d) for N-methyl protons ofglycine betaine and choline from the plant extractswere 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 chilled80% acetone. The homogenate was centrifuged at

10 000×g at 4°C for 10 min. The absorbance ofthe supernatant was measured at 646, 663 and 750nm, 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 growthTo assess the tolerance to high levels of exoge-

nously applied choline, shoots (measuring 3 cmlong bearing a minimum of four leaves) of wildtype and both transformed lines were transferredto half-strength MS medium [34] supplementedwith choline chloride (filter sterilized) at concen-trations ranging from 0 to 30 mM. The cultureswere 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 16/8-h day/nightcycle for 20 days. Changes in fresh weight, dryweight and chlorophyll content of the shoots weremeasured.

2.5.2. Effect on photosynthesisThe leaves from shoots grown on different con-

centrations of choline chloride for 20 days wereused for isolation of thylakoid membranes.Freshly harvested leaves were dipped in ice coldisolation buffer (pH 7.8) containing 400 mM su-crose, 10 mM NaCl, and 20 mM Tricine andincubated in dark. After 30 min the leaves weretaken out and homogenized in the pre-chilled mor-tar and pestle in ice-cold isolation buffer in dark.The homogenate was filtered through four layersof miracloth and the filtrate was centrifuged at5000×g for 10 min at 4°C. The pellet was washedand resuspended in a small volume of suspensionbuffer (pH 7.5) containing 100 mM sucrose, 10mM NaCl, 2 mM MgCl2 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 aClark type oxygen electrode [35]. For the assay ofPhotosystem (PS) II activity the reaction mixtureconsisted of 500 mM p-benzoquinone along withthe suspension buffer. The thylakoids equivalentto 20 mg chlorophyll was used for each assay. Allthe measurements were carried out at 2592°Cusing saturating white light (�500 mmol m−2

s−1).

K.V.S.K. Prasad et al. / Plant Science 159 (2000) 233–242236

Photosystem II activity was also measured asthe ratio of variable to maximum fluorescence(Fv/Fm) by a fluorometer (PAM 2000, Walz, Effel-trich, Germany) in the pulse amplitude modula-tion mode. The leaves (intact) from the shootsexposed to various treatments were dark adaptedin order to ensure that all the components ofphotosystem II are in an oxidized state (i.e. photo-system II centers are fully open) for 30 min priorto the measurement of Fv/Fm. Original (Fo) andmaximal (Fm) fluorescence yields were measuredwith weak modulated red light (B0.5 mmol m−2

s−1) with 0.8 s pulse of saturating light (\6.8mmol m−2 s−1 PAR), using the data acquisitionsoftware (DA 2000; Walz) attached to the instru-ment. The variable fluorescence yield (Fv) wasdefined as Fm−Fo. The photosystem II activitywas measured as Fv/Fm ratio [36].

3. Results and discussion

Successful transformation of the B. juncea cv.Pusa Jaikisan (an important oil crop of India)with codA gene encoding for choline oxidase wasachieved. The integration and the copy number ofthe codA gene in the transformed lines were deter-mined through PCR and Southern analysis [37].

3.1. Expression of the codA gene

The expression of the integrated codA gene inboth the transgenic lines selected for the presentinvestigations was confirmed through immunoblotanalysis. Western blot analysis with antiserumraised against pure choline oxidase, indicated thepresence of a 60 kDa polypeptide, correspondingto the authentic choline oxidase from A. globi-formis (Sigma Chemical Co.), in the soluble frac-tion from the leaves of both the transgenic lines(Fig. 1). No immunoreactive polypeptide was ob-served in the soluble fraction from wild type plants(Fig. 1). Unlike that observed by Hayashi et al.[13] in Arabidopsis thaliana transformed with thecodA gene, no precursor choline oxidase (corre-sponding to a molecular weight �70 kDa) wasnoted in the transgenic B. juncea indicating thatthe codA gene is not only transcribed and trans-lated but also rapidly processed.

As evident from Fig. 2A, both the transgeniclines showed choline oxidase activity. No cholineoxidase activity was detected in the wild type.However, a significant variation in the cholineoxidase activity was observed between the twotransgenic lines. Similar variation was observed inthe intensities of the bands observed in im-munoblot analysis (Fig. 1). The variation in thecholine oxidase activity could be due to differen-tial expression of the transgene depending on posi-tion of its integration in the genome of B. juncea.Similar differential expression of several transge-nes was noted in other systems [38–41].

3.2. Le6els of glycine betaine and choline

Introduction of the codA gene resulted in thesynthesis of biologically active choline oxidase inthe transformed plants (Fig. 2). Thus, it was im-portant to estimate the level of glycine betaine, asthis provides a direct clue regarding the capacityof this enzyme to convert choline to glycine be-taine under in vivo conditions. 1H-NMR analysisconfirmed the absence of glycine betaine in thewild type genotype of B. juncea, clearly demon-strating that it lacks any means to synthesizeglycine betaine (Figs. 2 and 3A). As expected, boththe transformed lines showed the presence ofglycine betaine (Figs. 2 and 3B) indicating thatthese transgenic lines had potential to synthesizethis compatible solute. However, choline level in

Fig. 1. Immunoblot analysis of choline oxidase in the solublefraction of transformed lines (1and 3) of B. juncea harbouringthe codA gene from A. globiformis. Lane 1: standard cholineoxidase from A. globiformis (S); Lane 2: the soluble fractionfrom wild type (C); Lane 3: soluble fraction from transformedline-1 (1); Lane 4: soluble fraction from transformed line-3(2).

K.V.S.K. Prasad et al. / Plant Science 159 (2000) 233–242 237

Fig. 2. Choline oxidase activity (A) and the levels of glycine betaine and choline (B) in wild type (WT), transgenic line-1 (Line-1)and transgenic line-3 (Line-3) of B. juncea. The values are the average of results of three independent experiments, each with aminimum of three replicates.

the shoots of transgenic lines did not differ signifi-cantly from that of wild type (Fig. 2B).

3.3. Tolerance to exogenous choline

Choline is ubiquitously present in higher organ-isms. However, its presence at higher levels is toxicto the plants [27,28,41]. As choline oxidase readilyconverts choline to glycine betaine, the trans-formed plants expressing the gene for choline ox-idase may be able to survive in the presence ofcholine even at concentrations that are otherwisetoxic to the wild type plant. This hypothesis wastested with the shoots of both wild type andtransgenic lines, as described in materials andmethods (Fig. 4).

Fresh weight and dry weight of the shoots ofwild genotype in the presence of choline at concen-trations up to 10 mM were marginally higher thanthose grown in the absence of choline (Fig. 5Aand B). However, at concentrations above 15 mMa sharp decline in both fresh as well as dry weightof the shoots of wild type was observed. In con-trast, the fresh and dry weight of the shoots ofboth the transgenic lines were significantly higherin the medium with choline at concentration upto15 mM than those grown in the absence ofcholine. Even in the presence of choline at concen-

trations as high as 30 mM, no significant alter-ation in the growth of shoots of transgenic line-1was observed in comparison to those grown in theabsence of choline (Fig. 5). Although, the trans-genic line-3 also showed significant enhancementin tolerance to choline, its shoots showed somesigns of toxicity in the presence of 30 mM choline.

As evident from Figs. 4 and 6A, the chlorophyllcontent in the shoots of wild type rapidly declined(over the control) with an increase in the concen-tration of choline in the medium. In contrast, theshoots of both the transformed lines showed about20% increase in chlorophyll content in the pres-ence of 10 mM choline over those grown in itsabsence. However, a decline in chlorophyll contentin the shoots of transformed lines was observed inmedium supplemented with choline at concentra-tions above 20 mM (Fig. 6A). In contrast, to over90% decline in the chlorophyll content of theshoots of wild genotype, only about 20% declinewas observed in both the transformed lines in thepresence of 30 mM choline. These results sug-gested that the transformation of B. juncea withcodA gene encoding choline oxidase for glycinebetaine synthesis impart tolerance to cholinetoxicity.

Fig. 6B and C shows the photosynthetic effi-ciency measured in terms of photosystem (PS) II

K.V.S.K. Prasad et al. / Plant Science 159 (2000) 233–242238

activity (measured in terms of mmol O2 evolvedmg−1 Chl h−1 as well as Fv/Fm ratio) in theshoots of wild type and transgenic genotypesgrown on medium with various levels of choline.In general, irrespective of the concentration ofcholine in the medium, the PS II activity inshoots of both the transgenic lines was signifi-cantly higher than that of wild genotype.

In spite of choline being an important cellularmetabolite, exogenous application of cholineeven at concentrations as low as 5 mM, causeda significant reduction in the PS II activity in theshoots of wild type. The extent of decline in PSII activity in shoots of wild type plants wasmore prominent with an increase in the concen-tration of choline in the medium. Unlike wildtype, the PS II activity in shoots of both thetransformed lines remained unaffected in thepresence of choline upto 15 mM. However, theshoots of the transformed lines grown on

medium with choline at concentrations above 20mM showed significantly lower PS II activitythan control (0 mM grown shoots). But the de-cline in PS II activity in shoots of transformedlines exposed to higher levels of choline was sig-nificantly less than that in shoots of wild type(Fig. 6B and C). For instance, upon exposure to30 mM choline the loss in PS II activity in thy-lakoids from shoots of wild type was over 96%,in contrast to about 25 and 35% in the trans-formed lines 1 and 3, respectively (Fig. 6B).

These results clearly demonstrated that thetransformed genotypes unlike wild type have in-herent potential to detoxify the toxic levels ofcholine. This is apparently due to the presenceof functional choline oxidase in the transformedlines that is able to readily convert toxic levelsof choline into glycine betaine. Lilius et al. [42]reported that plants transformed with the bacte-rial betA gene (which encodes for choline dehy-drogenase) performed better than wild type inthe presence of 10 mM choline, but higher con-centrations (20 and 30 mM) of choline caused asharp decline in growth of even transformedplants. However, Nuccio et al. [2] studied theeffect of exogenous choline on tobacco plantstransformed with CMO gene encoding spinachcholine monooxygenase only upto concentrationof 5 mM. On the contrary, during the presentinvestigations the transformed lines of B. junceacould tolerate choline even upto 30 mM. Theseresults clearly indicated that the transformedplants of B. juncea have enhanced capacity totolerate toxic levels of choline as compared totheir wild type counterpart. Further, transforma-tion of crop plants with the gene encoding forcholine oxidase seems to be far superior in im-parting choline tolerance than those for cholinedehydrogenase or choline monooxygenase.

In summary, the results presented in this com-munication clearly demonstrated the successfulexpression of the bacterial codA gene in two in-dependent transgenic lines of B. juncea as theyshowed the presence of functional choline ox-idase and glycine betaine. Moreover, the abilityof transgenic lines to photosynthesize and growwell even in the presence of choline at levels thatare highly toxic to wild type is probably becauseof their potential to readily convert choline toglycinbetaine.

Fig. 3. 1H-NMR spectra of quaternary ammonium com-pounds from leaves of wild type (A) and transgenic (B) plantsof B. juncea. Quaternary ammonium compounds were precip-itated as periodides, dissolved in CD3OD and subjected to1H-NMR spectrometer. 1H-NMR response positions (d) forN-methyl protons of glycine betaine and choline were 3.36and 3.23 ppm respectively, with t-butanol at 1.28 ppm (notshown) as an internal standard. The peak at the chemical shiftof about 3.33 ppm is attributed to the CH3OH in CD3OD. Cand GB above the peaks represent choline and glycine be-taine, respectively.

K.V.S.K. Prasad et al. / Plant Science 159 (2000) 233–242 239

Fig. 4. Shoots of wild type, transgenic line-1 and transgenic line-3 of B. juncea exposed to 0, 5, 10, 20 and 30 mM choline for 10days. Note chlorosis and leaf burning in the shoots of wild type in the presence of higher levels of choline.

Acknowledgements

We are grateful to Professor Norio Murata forproviding Agrobacterium strain (EHA 101) bear-ing binary vector plasmid pGAH/codA. We ac-

knowledge the Department of Science andTechnology (Government of India), New Delhi forfinancial assistance through the research grantswith reference No(s). INT/JAP/ JSPS/MBB/P-4/95and SP/SO/A-50/95. One of us (K.V.S.K. Prasad)

K.V.S.K. Prasad et al. / Plant Science 159 (2000) 233–242240

Fig. 5. Change in fresh weight (A), dry weight (B) of shoots of wild type and two transgenic lines (viz. 1 and 3) of B. juncea grownon medium with various concentrations of choline. The initial values of fresh weight, dry weight for wild type and transformedlines were taken as 100%. The initial value of fresh weight of the shoot was 380910 mg and dry weight was 3893 mg in bothwild type as well as transgenic lines. The results are the average of data from three independent experiments. ( ) Wild type; (�)transgenic line-1; () transgenic line -3.

Fig. 6. Change in Chlorophyll content (A) and PS II activity in the leaves of shoots of wild type and transgenic lines of B. junceagrown on medium with various concentrations of choline (B and C). PS II activity was measured in terms of oxygen evolutiondue to the photoreaction H2O� pBQ by the thylakoids isolated from the leaves (B) and Fv/Fm ratio of undetached leaves (after30 min of dark adaptation) (C). The initial values of oxygen evolution and Fv/Fm ratio were taken as 100%. The initial value ofchlorophyll content was 2.1990.21 mg g−1 fresh weight, oxygen evolution by the isolated thylakoids was 210.9918.6 mmol O2

evolved mg−1chl h−1 and the initial Fv/Fm ratio was 0.8390.015 in both wild type as well as transgenic lines. The results arethe average of data obtained from three independent experiments. ( ) Wild type; (�) transgenic line-1 and () transgenic line-3.

is grateful to the University Grants Commissionfor providing fellowship. We are thankful to DrUma Sharma (National NMR Facility, All IndiaInstitute for Medical Sciences, New Delhi) forhelping us in determining the levels of glycinebetaine and choline through 1H-NMRspectrophotometer.

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