Plant Cell, Tissue and Organ Culture 36: 53-58, 1994. © 1994 Kluwer Academic Publishers. Printed in the Netherlands.

Transient expression in leaf mesophyll protoplasts of Arabidopsis thaliana

Andrea Hoffman, Ursula Halfter & Peter-Christian Morris 1'* Institut fiir Genbiologische Forschung Berlin GmbH, Ihnestrafle 63, 1000 Berlin 33, Germany, (~present address: CNRS, Institute des Sciences V~gdtales, Avenue de la Terrasse, 91198 Gif sur Yvette Cedex, France) (*requests for offprints)

Received 23 February 1993; accepted in revised form 6 August 1993

Key words: Arabidopsis thaliana, GUS, protoplast, transient expression

Abstract

Conditions for maximising transient expression of GUS in leaf mesophyll protoplasts of Arabidopsis thaliana ecotype C24 were investigated. It was found that the factors most influencing expsression levels, with optimum levels in parenthesis, were plasmid DNA quantity (100~g per 5 x 10 protoplasts), inclusion of carrier DNA (50 Ixg), PEG pH and amount (pH above 6, and total PEG concentration at least 9% w/w) and the topological form of the DNA. Linearised plasmid DNA with long flanking sequences 3' and 5' to the marker gene yielded the highest levels of GUS expression.

Abbreviations: 2,4-D - 2,4-dichlorophenoxyacetic acid, GUS - B-glucuronidase, MU - methylumbel- liferone, P E G - polyethylene glycol, X-gluc- 5-bromo-4-chloro-3-indolyl-13-glucuronic acid

Introduction

Transient gene expression in plant protoplasts is a quick and convenient manner in which to monitor structure-function relations of gene pro- moter constructs coupled to such easily detect- able marker genes as chloramphenicol acetyl transferase or GUS. There are several different techniques to transfer genes to protoplasts; a frequently used method is to use PEG-stimulated gene transfer as this is simple to carry out and requires no elaborate equipment (Negrutiu et al. 1987). Nevertheless, protoplasts from every species have their own characteristics and it is important to optimise conditions for PEG-stimu- lated transformation for each species, especially if low levels of expression are anticipated (Neg- rutiu et al. 1990).

The mechanics of transformation for transient expression are in principle the same as those for stable transformation; foreign DNA is induced to be taken up in the nucleus of the recipient

protoplast. Thus improvement and maximisation of transient expression may also be a step in the direction of maximising stable transformations. Here we present an analysis of factors influenc- ing transient expression for the important model organism Arabidopsis thaliana.

Materials and methods

Protoplast preparation and transformation

Protoplasts were prepared from axenically grown four week old plants of Arabidopsis thaliana ecotype C24 as described by Damm & Wil- lmitzer (1988), and checked for viability by staining with Evans Blue (Kanai & Edwards 1973). Protoplast preparations with a viability of under 80% were not used. The protoplasts were taken up at a concentration of 1.6 million per ml in MaMg buffer (15 mM MgCI 2, 0.5 M mannitol, 5 mM MES pH 5.6, Negrutiu et al. 1987). 330 Ixl

54

aliquots (500,000 protoplasts) were added to 12 ml glass tubes and DNA for transformation added into the protoplast suspension. After a five min room temperature incubation period, PEG solution (40% w/w polyethylene glycol 6000 (Merck) in 0.4M mannitol, 0.1M Ca(NO3) 2. Negrutiu et al. 1987) was slowly added with gentle mixing. After a further 20 min incubation at room temperature, 8 ml W5 solu- tion (145 mM NaCI, 125 mM CaCI 2, 5 mM KCI, 5 mM glucose, pH 5.8, Negrutiu et al. 1987) was added to each transformation in 2 ml steps over 10 min. The protoplasts were centrifuged at 60 g for five min, and the pellet taken up in 1 ml B5 medium (Gamborg et al. 1968) containing 72.6gl -1 glucose, l m g 1-1, 2,4-D and 0.15mg 1-1 benzylaminopurine, and incubated at 25°C.

The basic transformation protocol employed was to add to replicate 330p,1 protoplast aliquots, 50 p,g supercoiled pRT102GUS DNA together with 50 I~g sonicated salmon sperm DNA as carrier, followed by 300 Ixl PEG solu- tion. After transformation the protoplasts were incubated for 48 h prior to assay in replicate. Variations in the transformation procedure are described in the results section. The experiments were each performed a minimum of three times with independent batches of protoplasts.

GUS assay

After the incubation period, the protoplasts were centrifuged at 60 g for five min and the pellet taken up in 500 p~l extraction buffer (50mM phosphate buffer pH 7, 10mM B-Mercap- toethanol, 10 mM EDTA, 0.1% v/v Triton X- 100), transferred to an Eppendorf tube, and subjected to three cycles of freezing in liquid nitrogen and thawing at room temperature to solubilise GUS enzyme activity. After centrifu- gation at 13 K g for 10 min, the supernatant was assayed in replicate for protein content (Brad- ford 1976) and for GUS activity by fluorimetry as described by Jefferson (1987). Protoplasts were stained in situ for GUS activity by filtering onto the membrane from a PV 050/3 filtration unit (Schleicher and Schiill) followed by incubation in X-Gluc (Jefferson 1987; Houba-H6rin et al. 1990).

DN A preparation

The DNA used for transformation was the plasmid pRT102GUS (T6pfer et al. 1987; Damm et al. 1989) in the supercoiled form after caesium chloride gradient purification and two sequential ethanol precipitations to remove excess salt (Fig. 1). In some instances the plasmid was linearised by digesting with BamHI or ScaI, or the GUS gene was excised by digestion with HindIII. After the restriction digests the DNA was purified by phenolchloroform extraction followed by ethanol precipitation. To obtain single-stranded DNA, the HindIII fragment of pRT102GUS, containing a CaMV 35S promoter, GUS gene and CaMV 35S polyadenylation site was cloned in both orientations into the HindIII site of the M13mp18 vector (Yanisch-Peron et al. 1985). M13GUS 5 contained the coding strand, and M13GUS 6 the non-coding strand when in the single stranded form. DNA from both the double-stranded replicative form and single- stranded form of these constructs was prepared as described in Sambrook et al. (1989). Carrier DNA was salmon sperm DNA (Sigma) sonicated to below 10kb in size, phenol-chloroform ex- tracted and ethanol precipitated. All DNA prep- arations were dissolved in sterile water.

~ indlll

S c a _ ~ Amp ~r . . . .

\ V \ . . /

Hindlll BamHI

Fig. 1. Structure of the plasmid pRT102GUS, showing the restriction sites employed for linearisation.

55

Results

As a basic procedure, 5 x 105 protoplasts were transformed with 50 Ixg supercoiled pRT102GUS DNA and 50 Ixg carrier DNA by the addition of 300 ixl PEG solution. Using this protocol, the duration of incubation at 25°C after transforma- tion was varied; the results obtained differed from one batch of protoplasts to another but in general, maximum GUS activity was seen after 48 to 72 h, with no change in enzyme activity thereafter for a period of up to one week (Fig. 2). In general, within-experiment error was relatively low, but between-experiment error was much greater. Results and errors are shown from one representative experiment in each case, but each experiment was repeated a minimum of three times with the same result.

~ n

"7

Z~

120

100

80

60

40

20

0 l [ l I 0 50 100 150 200

lag plasmid DNA Fig. 3. GUS activity of a function of pRT102GUS plasmid DNA added. 0 , without carrier DNA, II, with 50 ~g carrier DNA.

Influence of DNA and PEG amounts

The amount of pRT102GUS DNA added, plus or minus 50 Ixg carrier DNA, was varied. In the absence of carrier DNA, GUS activity plateaued with the addition of 100 Ixg plasmid. Enzyme

300

i 250

200 . ~ 1 5 0 - •

100

0 0 20 40 60 80 100

Hours Incubation

Fig. 2. GUS activity as a function of incubation time after transformation. Error bars in all figures are 95% probability limits of the mean. The curve fit is in all figures a 3rd degree polynomial.

activity was higher when 50 Ixg carrier DNA was included; this also reached a maximum at 100 ixg DNA, and then fell off (Fig. 3). The amount of carrier DNA added together with 50 Ixg plasmid DNA was varied; amounts over 50 Ixg of carrier led to a decrease in GUS enzyme activity (data not shown).

To investigate whether the increase in GUS enzyme activity observed when more DNA was added was due to increased numbers of trans- formed protoplasts or to enhanced activity per individual transformed protoplast, GUS activity was determined by direct staining of the proto- plasts with X-Gluc. With no added DNA no blue protoplasts were seen, whereas ~<1% of the protoplasts were visibly blue when transformed with 10jxg plasmid. When 1001xg DNA was added, approximately 10% of the protoplasts were stained blue, and the intensity of staining of the individual protoplasts appeared greater than when 10 Ixg was employed (data not shown).

The amount of PEG added was varied; maxi- mum GUS activity was seen after the addition of 150 ~1 PEG solution (9% final concentration), with no significant change up to 300 txl PEG added (18% final concentration) (Fig. 4). The influence of PEG pH was also investigated; slightly lower activity was seen with PEG of pH 6 but above this value, expression was not

56

50

"- 45 I

E, 35 e~

30

, 25 , I . .~

20

-6 10

0

! I I I ,,I I I

0 3 6 9 12 15 18 % final PEG concentration

Fig. 4. GUS activity as a function of PEG concentration.

changed (Fig. 5). This held true if the pH was adjusted by formulating the PEG with the inclu- sion of 50 IxM buffer solutions, or simply by titration of the PEG with NaOH and HCI.

Influence of topological form of the DNA

400

~ 350

8 3oo Q ~. 25o

~ 2o0

.~ 15o

~ 10o

~ 50

~ 0 A

/ B C D E F G ~H I J K

Topological form of DNA

Fig. 6. GUS activity as a function of DNA topology., Equimolar DNA amounts were added in each case. (A) no added DNA. (B) supercoiled pRT102GUS. (C) replicative form M13GUS 5. (D) replicative form M13GUS 6. (E) single stranded form M13GUS 5. (F) single stranded form M13GUS 6. (G) BamHI digested pRT102GUS. (H) HindlII digested pRT102GUS. (I) ScaI digested pRT102GUS. (J) heat denatured ScaI digested pRT102GUS. (K) heat dena- tured supercoiled pRT102GUS. Activity is relative to super- coiled pRT102GUS (100%), so that comparison can be made between transformations carried out with different protoplast batches.

The topological form of the DNA added was varied (Fig. 6). Approximately equimolar amounts (50 ixg) of single stranded M13GUS DNA, both as the coding (M13GUS 5) (E) and non-coding strand (M13GUS 6) (F), gave ap- proximately half the GUS enzyme activity as compared to double stranded circular DNA,

120

.•100 8 0

"7 60

20

0

i 6 7 8 9

PEG pH Fig. 5. GUS activity as a function of PEG pH.

either as 50 p.g plasmid pRT102GUS (B) or as 100 ~g of the replicative forms of M13GUS 5 (C) and M13GUS 6 (D). Digestion of pRT102GUS with BamH1 to separate the promoter from the structural gene (Fig. 1) reduced activity to al- most background levels (G), whereas a HindlII digest, which excises as one contiguous sequence the promoter, the coding region of GUS and the polyadenylation site from the rest of the plasmid results in GUS activity of about one half of that of the supercoiled control (H). Linearisation with ScaI results in a molecule with long 5' and 3' ends flanking the GUS gene and this configu- ration resulted in expression 3.5 times that of the supercoiled control (I). Heat-denaturing ScaI- linearised pRT102GUS resulted in almost zero activity (J), whereas heat denaturing supercoiled plasmid had little effect (K).

D i s c u s s i o n

In this study we have attempted to optimise some of the conditions for transient expression in Arabidopsis leaf mesophyll protoplasts. Previous

studies have utilised protoplasts obtained from Arabidopsis cell suspension cultures (Axelos et al. 1992; Doelling & Pikaard 1993). However, leaf mesophyll protoplasts may be preferable material for many gene expression studies since they are cells derived from normal, differen- tiated plant tissues rather than undifferentiated callus. Furthermore, suspension cultures may accumulate changes in ploidy and mutations due to somaclonal variation (Larkin & Scowcroft 1981).

From the results presented here it is clear that many factors can have a bearing on the mag- nitude of transient gene expression in Arabidop- sis protoplasts. One such factor is the length of time of incubation after transformation of the protoplasts; this varies from preparation to prep- aration of protoplasts and is thus probably a function of protoplast viability together with messenger RNA and enzyme stability. The studies of Prtls et al. (1988) would indicate that the transformed DNA has a relatively short half life and that the bulk of RNA synthesis occurs essentially within a few hours after protoplast transformation. In this study we chose 48 h as a convenient time point at which to assay enzyme activity, but expression levels were sometimes higher with extended incubations (Fig. 2).

The other factors which most influenced ex- pression levels were amount of plasmid and carrier DNA added, amount of PEG added, and the structure of the plasmid DNA. GUS enzyme activity was saturated when 100 Ixg plasmid DNA was added to the protoplasts, indicating that DNA entering the protoplasts is not a limiting factor. Histochemical staining of the protoplasts for GUS activity showed that higher levels of activity were associated with an increased num- ber of stained protoplasts, also that the intensity of staining of individual protoplasts was greater. It is difficult to state what the actual percentage of the protoplasts that were expressing GUS activity was, since it is probable that the visual threshold of detectability is below that of the actual activity, nevertheless it appears that the actual number of transformed protoplasts was relatively low even under optimised conditions. Axelos et al. (1990) found that protoplasts derived from Arabidopsis cell suspension cul- tures gave up to 44% staining for GUS activity

57

after transformation, furthermore, the expres- sion levels of these protoplasts were some 5-fold higher. This might indicate that Arabidopsis leaf mesophyll protoplasts are intrinsically less competent for transformation than suspension culture derived protoplasts, in contrast to the situation in Nicotiana plurnbaginifolia, maize and carrot (Negrutiu et al. 1990).

The plasmid DNA did not appear to be toxic in the high amounts added, but high DNA purity was found to be critical for high levels of expres- sion, additionally it was found necessary to store the DNA in small aliquots; repeated freezing and thawing led to reduced activity, even though no linearisation or breakdown of the DNA could be observed by gel electrophoretic examination.

Inclusion of 50 Ixg carrier DNA enhanced expression levels by between 50 and 100% up to a maximum of 100 jxg plasmid DNA added, but had an adverse effect thereafter. Increasing amounts of carrier DNA had a negative effect on expression levels. The carrier DNA had not been extensively purified by caesium chloride gradient centrifugation and thus it may be that impurities led to the decrease in expression. The role of carrier DNA is not clear, it may act as an alternate substrate for nucleases, or alternatively it may aid in the uptake of the plasmid DNA.

The pH of the PEG solution, so long as it was above 6, did not have a significant effect on GUS expression, in contrast to the findings of Maas & Werr (1989). These latter authors were however using protoplasts from monocotyledonous plants and media based on seawater, so comparison is not easy. The amount of PEG added clearly had an influence on GUS expression, with an op- timum being reached with 150 p~l (9%) added. With higher levels of PEG added, there was a marked effect on protoplast viability as checked by staining with Evans Blue, thus there is proba- bly a balance between increased expression levels and protoplast viability.

The form of the transforming DNA also has a marked influence on GUS expression; highest levels of expression were seen when double stranded linearised DNA was added with rela- tively long flanking sequences 5' and 3' to the GUS gene. DNA in the linearised form may give better access to RNA polymerase and the flank- ing sequences may protect the GUS gene from

58

the action of nucleases. As shown by Furner et al. (1989), single stranded DNA is also capable of directing transient expression from both the coding and non-coding strand, arguing that the DNA is rapidly converted to the double-stranded form. However, it appears that this is only efficiently achieved with circular single stranded DNA since denatured linearised plasmid gave no GUS expression, and thus might be rapidly degraded.

In summary, the conditions best suited to transient expression with Arabidopsis leaf meso- phyll protoplasts are to use high amounts of highly purified plasmid DNA (100 txg per 5 x 105 protoplasts) linearised in such a way as to leave long flanking sequences 5' and 3' to the gene of interest, together with 50 ~g carrier DNA. The PEG should be above pH 6 and should be added to a final concentration of at least 9% (w/w). However, it is relatively difficult to obtain con- sistent marker gene expression levels from differ- ent batches of protoplasts; two approaches that could be adopted would be to grow the Arabidopsis donor plants under conditions which appear to stabilise the protoplasts (Masson & Paszkowski 1992), and to incorporate, by co- transformation, an internal standard to enable standardisation between experiments (Lepetit et al. 1991).

Acknowledgement

We would like to thank Professor Willmitzer for his support during this work.

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