i::ul'h.l'tic(J 85: 209 216. 1995. 209 © 1995 K/I/H'C/" AU/tlemi" Pl/lJlishers. Primetl in the Ne/her/antls.

Different 5/ leader sequences modulate ,3-glucuronidase accumulation levels in transgenie Nicotiana tabacum plants

Marc De Loosel, Xavier Danthinne2, Erik Van Bockstaele l , Marc Van Montagu 2 &

Ann Depicker2

I RijksslC/tion voor Plantenveredeling, Cenlrum voor Landbouwkundig Onderzoek Gent, Burg. Van

Gansherghe/aan /09, B-9820 Mere/beke, Belgium; 2 Laboralorium voor Genetica, Universiteil Gent, KL Ledeganckslraat 35, B-9000 Gent, Be/giunl

Key \Yards: ,U-glucuronidase, plant, silencing, translational control, 5'-untranslated region, variation of gene expressIon

Summary

Three random synthetic leaders and three naturally-occurringleaders, the lObaceo mosaic virus (TMV) coat protein, the satellite tobacco necrosis virus (STNV) and the plant chlorophyll a/b-binding protein (Cab22L), were shown 10 modulate the U-glucuronidase reporter protein accumulation levels in transient expression experiments. The same chimcric constructs also confer differential distribution patlems of reporter protein accumuiation in stably­transformed tobacco calli or regenerated transgenic plants. When the highest expression levels with a given leader are compared, the 31-nucleotide random leader stimulates trans lat ion 20- and 100-fold reiative to the 9- and 4­nucleotide synthetic leaders respectivel y. However, this 3 I-nucleotide random leader is approx. 2 to 3-fold wcaker than the 30-nucleotide STNV leader and even S-fold weaker than both the 79-nuclcotide TMV leader and the 66-nuclcotide Cab22L leader. These results confirm the findings in transient expression experiments and stress the import~lncC of the S' -untranslated reg ion for the production of heterologous proteins in transgenic plants.

Introduction effect on translational enhancement (Gallie & Walbot. J992). The satellitt: tobacco necrosis virus (STNV)

Of all the regions of messenger RNA that can be (Danthinne et al., 1991) RNA lacks a conventional cap involved in translational efficiency, the leader sequence structure at the 5' terminus, but neverthe1ess thc viral is probably the most important (Kozak, 1989; Hershey, coat protein is very efficiently translated (Danthinne 1991). Firstly, this region is undoubtedly implicated in et al., 1993). The Cab22L leader also has a stimula­thc process of translational initiation and this is usually tory effect upon the trans-protein accumulation after consiclcred to be the rate-limiting step of translation. transient expression compared with the effect of the Sccondly, the 5' leader can be directly or indircctly cauliAower mosaic virus (CaMV) 3SS 5' leader (Harp­involved in mRNA stability. ster et al., 1988).

Plant viral RNA leader sequences, like those of In an in vitro translation system derived from wheat tobacco mosaic virus (TM V), alfalfa mosaic virus germ, thc STNV leader sequence only weakly stimu­(A!MV) and plant potyvirus, have been shown to act lates translation relative to the leaders of TMV and in cis as dlicient enhancers of translation at least in in of Cab22L origin (Danthinne & Van Emmelo, 1990). vitro systems or during transient expression in pro!O­ In transient expression the STNV-I, the TMV and the pla:;ts (Gallie et al., 1987a. 1987b; Jobling & Gehrke. Cab22L leaders exhibit a 4-, 12-. and 8-fold enhance­1987; Sleat ct al., 1987; CalTington & Frecd, 1990, ment respectively, relative to a 31 nucleotide random Nicoiaisen et al., 1992). Motil's in the TMV lead­ leader (Danthinne & Van EmmeJo, 1990). er of 77 nucleotides were studicd in detail for their

210

This work analyzes whether the effect of the leader sequence on heterologous protein accumulation seen after transient expression (Danthinne & Van Emmelo, J990) can be extrapolated to that which is found in stably-transfonned plant tissues. The work also aims to determine whether the leader affects the distribu­tion of protein accumulation levels beyond the vari­ation of expression levels due to position and silenc­ing effects. Therefore, the expression of the different chimeric gene constructions was studied first of all by comparing the values for the GUS activities in two series of 30 different calli. Subsequently the expres­sion of the same chimeric constructs was evaluated in regenerated transgenie plants and their progeny.

Materials and methods

Plasmid constructions

The cauliftower mosaic virus promoter (P35S) was mutagenized to create a PstI restriction site (Danthinne & Van Emmelo, 1990) immediately downstream from the three putative transcript ion start sites (Ow et al., 1987; Sanders et al., 1987; Harpster et al., 1988). All leader sequences were fused to the ,8-g1ucuronidase (gus)-coding sequence at the Ncol site positioned at the first codon. Downstream, the gus-coding sequence was fused to the 3' region of the nopaline synthase gene (Depieker et al., J982; Ingelbrecht et al., 1989) at the EcoRI site located 6 nucleotides downstrcam from the stop codon (Jefferson, 1987). The chimeric gus genes were put under the control of the mutagenized 35S promoter in such a way that only three heterologous nucleotides GAC are present at the 5' end of all the transcripts. The chimeric genes were introduced into the XbaI site of the binary T-DNA vector pDEIOOI (Denecke el al., 1992) in both orientations relative to the right border. The resulting plasmids (pXD601 10

pXD612) were given an odd or an even number when the gus caSsette pointed to the inside or to the outside of the T-DNA, respectively (see Figs IA and 1B).

Conjup,ation and plant cell transformations

All the T-DNA vectors were mobilized from Escherichia coli MC 1061 (Casadaban & Cohen, 1980) to the Ap,robacrerium strain C58C I, containing plas­rnid pGV2260 (Deblaere et al., 1987). Transconju­gants harbouring the correct T-DNA constructs werc co-cultivated with Nicotiana rabacul11 SR I protoplasts

(Depieker et al., 1985) or with leaf discs (De Block et al., 1987). Kanamycin-resistant calli were grown on B5 medium (Gamborg et al., 1968) supplemented with honnones and 50 lig/mi kanamycin. Each T-DNA COn­struct was transformed twice. From both independent­Iy performed co-cultivations, 30 calli wcre analyzccl for GUS activity. The GUS-negative calli were no\ considered. The calli are indicated by CXD followed by the number of the T-DNA plasmid construct and a number to designate the individual clone. Kanamycin­resistant shoots were cut from leaf discs and grown in glass containers. The independently obtained plant lines are indicated by LXD followed by the number of the corresponding T-DNA construct and a number indicating each individual shoot. For each construcl (except XD603), between 4 and 20 axenically-grown transgenics were analyzed at the 6-1eaf stage approxi­mately 3 months after their isolation from the leaf disc. Subsequently, 2 to 5 of the highest expressing trans­genics were transferred to soil, grown in the green­house and allowed to set seed. About 50 secds of the RI generation were sown on Murashige and Skooi' medium containing 50 pg/ml kanamycin. The r lil between kanamycin-resist<mt and kanamycin-sensitive seedlings was detennined. FIOm lines with a single T­DNA locus, eight to twelve kanamycin-resistant Rl plants were grown in vitro up to the 6-leaf stage anel analyzed for their GUS content.

GUS assays

Fig.!.The enzyme activities (Jefferson, 1987) were detel P35S-1 mined by means of continuous kinetic reactions per­ indic31

Pnos.formed spectrophotometrically (340 ATTe, SLT or the noiEMS, Labsystems Inc.) or ftuorimetrically (Flu­B. NUl

oroscan, Labsystems Inc.) in a computer-directed the Val

,G-gluc

(1993). The detection limit (values two-fold abov· background) was 0.1 U GUS/mg protein. The pr<

microtitre plate reader as described by Breyne et al

mant:tein concentrations were determined by the method ol casse'Bradford (1976) using the kit supplied by Bio-Rad. positi the si nurnb

Results

DisfnT-DNA construcrs

ealli .'

The basic structure of the set of constructed T-DNA Distri

vectors is presented in Fig. J. All T-DNAs carry the genic

same neomycin phosphotransferase hybrid gene allo\.\!­els. F

ing the select ion for kanamycin-resistant transfor­

:Iock 'n on with Con­jent­yzed

not lwed nd a Icin­'own )Iant nber nber truct 'own 'oxi­iisc. 'ans­een-f the :oog ratio itive Ie T­

RI and

~ter-

per­I or Flu­cted t al. love pro­dof

'NA the

ow­for-

LB RBA. 211 odd number

3' oes np til P nos 3'nas gus P 355

LB RB

even mumber

3' Des np ril P nos P 355 gus 3' nos

B. Plasmid constructionsLeaders

pXD601 pXD6024nt random leader

(i.ce.uei-

pXD603 pXD6049nt random leader

G .. CUCGACC ... uCi -

30nt STNV leader pXD605 pXD606

ei ...cu ........G... c ... GG ........ Cuuu .. CUG ACU -cc ~

pXD607 pXD60B31 nt random leader

GACGU ...CCCGGGG ....ucc:JC'.l ...G....GucO..ccc~

79nt TMV leader pXD609 pXD610I G"'C G"U cc Gu"'uuuu IJ AC ..... c ....uIJ"C CAC" ..CA ..... .a.c ...... c .. ..C..... "C'" AC"U IJ AC ......VVV "'CU ...uu Cu"'O ......UIJ ...cc~

66nt Cab22L leader pXD611 pXD612

I co ",cu CG "'Geve.u IJ IJ =veu "uu ..euve ..Gc .....u .....c...... .toG ....CUcuuvucuCUUCuv ..uu "',. "'cc "'UG

Fig. I. Description of the T·DNA consUlJcts. A. Schematic represenlalion of the T-DNA construcLs used. The veclors containing the cassene P35S-leader-gus-3' nos oriented to the insiJe or the outside of the T-DNA are indicated with odd and even numbers respeclively. The star symbol Ifldicatcs the position where the various leaders, described in Fig. IB. were cloned. Abbrevialions: P35S. caulillower mosaic virus promoter; Pnos. nopaline synthase promoter; SliS. O-glucuronidase gene; nptll. neomycin phosphouansferase ll-coding sequenee; 3' nns. 3' region of !he nopaline synthase gene; 3'ocs. 3' region of the octopinc synthase gene; RB. T-DNA right border region; LB. T-DNA lef! border region. Il. Nucleotide sequence of the leaders tesled for uanslational enhancement and the nomenclature of the vee tors earrying the T-DNAs with 'he various leader fusions. The tirst residues are derivcd from the uanscription start of the CaMV 35S promoter. The initialion codon of the ./-glucuronidasecoding sequenee is underlined.

mams. The different chimeric P35S-leader-gus-3'nos cassettes were all insened into one vector at the same position in two orientations (Fig. I A). Figure 18 shows lhe six different leaders analyzed in this work and the number of the constructs in which they OCCUr.

Dislribulion ofCUS aClivilies in dijferenllransgenic calLi series

Distributions of the GUS activities of the CXD trans­genie ealli were obtained from GUS accumulation lev­'1:'. figurc 2A shows the GUS amounts in 16 calli

of all transgenic series plotted on a Jogarithmic scale. To emphasize the large differences in absolute GUS amounts among the different series as weil as with­in one series, the same results were pJotted on a Jin­ear scale (Fig. 28). Each series contained calli with low, intennediate and high GUS accumulation levels. The same range of variation with the same absolute amounts for the lowest and highest expressing cal­li was confirmed in a second experiment in which a funher 30 calli were analyzed. Thc main ditlerence bet ween the results of the two consecutive experiments was the number of calli with low and high GUS activ­

212

U GUS/mg protein

A. 10,000,

I I

1,000 I I

100

0.1

1000 U GUS/mg protein

B. 10

8

6

4

2

0 c)'

C:) C)'l,.

C:) c:::,"IQ dJ<

IQ

'~I C)0

IQ c:::,re,

IQ c)/\..

IQ

ii c:::,'ó

IQ C)~

IQ ,c:::,

re, ,,'re, ,,'l,.

re, 1-<:) 1-<:) 1-<:) 1-<:) 1-<:)1-<:) 1-<:) 1-<:) 1-<:) 1-<:) 1-<:) 1-<:)

G G G G G G G G G G G G I I I I I I I I I I I I 4 nt RL 9 nt RL 30 nt 31 nt RL 79 nt 66 nt

STNV L TMV L Cab22L L

CXD calli Fig. 2. DislIihulion of GUS aClivilics in lransgenic calli series. GUS aClivilies in lIansgenic calli series were plolIed on a. logarithmic (Al anti a linear (B) scaIe. The G US aClivilics delermined in (ach callus numher of lhe different scrics arc ft'prcsc/lted by bars. Within each scrics. t.hc numbers of calli arc arranged accorJing 10 their increasing GUS conlent.

ity. A comparison shows lhat lhe distributions of GUS 1l1e 4-nllcleotide random leader (RL) yielded thc accumuJation levels in series containing an identical lowest expression but was significantly above bad chil1leric f!,/IS gene but in opposite orientations. were ground. Higher GUS levels \\'ere fOllnd in calli witl: similar. the 9-nucleolide RL transcript (series CXD60] ;(IHI

However. the GUS activity dislributions of series CXD604) and still higher levels were found in tran~

containing J;US COnSlrlJCIS wilh different leaders dis­ genic calli with a 31-nllcleolide RL in the RUS triU1scrip\ play a pronounced difference (Figs 2A and 28). (series CXD607 and CXD608). The highest aCClll1lll

lati pat I

ST]

nllc and and

Infl pril

For wel ma in t

gro GU wel 3A p]al ry/ GU pro teir the nllc for the mll sho trar

cu

Onl lint owi RI ber plal DN seg sen 3: I mal ana of tI mu and

the in t

nel h~

Je

k­th Id s­pt J-

lat ion levels and more or less comparabie distribution pattems were found in thescries with the 30-nuc\eotide STNV leader (series CX 605 and CXD606), the 77­nucleotide TMV leader (series CXD609 and CXD61 0), and the 66-nucleotide Cab22L leader (series CXD611 and CXD612).

Infiuence oflhe leader on GUS accumu/alion in primary Iransgenie planls

For each of the T-DNA constructs transgenie plants were obtained and analyzed for their GUS expression in a leafofan in vilro-grown plant (Figs3A and 38). As in the transgenie calli series, the primary axenically­grown transformants also showed a high variation in GUS units per mg of tot al protein. The obtaincd reslJlts were plotted on a logarithmic and a linear scale in Figs 3A and 38 respectively. As the number of transgenie plants analyzed was limited, it is not possible to car­ry out a statistical analysis of the data. The highest GUS values per leader construct are 6150 U GUS/rng protein for the Cab22L leader, 3536 U GUS/mg pro­tein for the TMV leader, 1167 U GUS/rng protein for the STNV leader, 309 U GUS/mg protein for the 31­nucleotide random leader (RL), 36 U GUS/mg protein for the 9-nucleotide RL and 20 U GUS/mg protein for the 4-nucleotide RL. Thus a comparison of GUS accu­mulation in leaf material of primary transgenie plants shows the same hierarchy of leader effects as in the transgenie calli series.

GUS expression in progeny planls

One or more of the highest-expressing, transgenie lines per T-DNA construct, indicated by a dark shad­owing in Fig. 38, were allowed to f10wer and the RI seeds were collected. Subsequently the num­ber of kanamycin-resistant versus kanamycin-sensitive plants was determined to estimate the number of T­DNA loci. Most of the RI progenies show a 3 to I segregation of kanamycin-resistant versus kanamycin­sensitive seedlings. Of the transgenics with c\ear 3: I segregation ratias for the T-DNA-linked selection marker, 8 or J 2 R I in vilro-grown progeny plants were analyzed for GUS accumulation levels (Fig. 4). Most of the progeny shows two distinct classes of GUS accu­mulation probably corresponding to the heterozygotes and the homozygotes for the T-DNA locus.

We can derive from the RI population analysis that the same hierarchy of expression levels is maintained in the progeny of the primary transgcnics: narnely the

213

9-nucleotide RL < 31-nucleotide RL < STNV leader < TMV and Cab22L leaders. Although the TMV-gu.\ and Cab22L-gus constructs gave rise to similar GUS distributions, it may not be a coincidence that the high­est accumulation levels were always found in trans­genie material with the Cab22L leader sequence.in the transcript.

Discussion

The results on GUS accumulation levels in different transgenie lines with a particular T-DNA construct illustrate the commonly-observed variation in trans­gene expression. Clearly, every series contains a num­ber of transgenie calli that express the P35S-gus trans­gene to a very low extent in comparison with a number of calli with high (normal) expression levels. Howev­er, the resulting low accumulation levels were always approximately 0.0 1ofthe highest accumulation level in the respcctive series. The variation in gene expression might be the result of position effects such as f1anking enhancing or silencing DNA sequences, the surround­ing chromatin structure or the methylation status of the insert locus. However, other processes not under­stood such as gene silencing and co-suppression may contribute to the observed variation in gene expression in primary transgenic populations (Hobbs et al., 1990, 1993; Ingelbrecht et al., 1994).

Nevertheless, the results demonstrate the impor­tanee of the leader sequence upon heterologous gene expression. Very often chimeric leaders contain link­ers and adaptors which can lower substantially the flnal recombinant protein accumulation (Jones et al., 1985).

The 5'-untrarJslated leader might directly influence the accu mulat ion of the gene product as it is undoubted­Iy involved in the initiationoftranslation. This isexem­plifled by the pOOI' translation of second cistrons in dicistronic transcript ion units (Angenon et al., 1989). However, the 5' -untranslated leader is also involved in mRNA accumulation levels (Harpster et al., 1988). The leaders may increase the mRNA stability because enhanced translation proteets the transcript from more rapid degradation (Vancanneyt et al., 1990). The anal­ysis and correlation of GUS accumulation levels with transcript accumulation levels wil! allow us to c1arify events.

We conclude from the experiments that both the 5'-untranslated region of the Cab22L gene (Harpster et al., 1988) and the 5' leader of the TMV viral coat

214

Ä. U GUS/mg prolein

10,000

1,000

1000 U GUS/mg prolein

B. 7

4

5

6

3

2

4 nl RL 9 nl RL 30nl 31 nl RL 79 nl 66 nl STNV L TMVL L Cab22 L L

Primary Iransgenie planls

Fig. 3. Distribution of GUS aClivitics in young Icaf matcri,,1 of prilll"ry rransgcnic pianIs conIaining mc various IcmJcr-gu5 conslrUCls. plotll',­on a logarithmic scale (Fig 3A) and a Iinear scak (Fig. 38). The GUS activities detcmlincd are reprcsenled by bars plollcd on me ordinale. 1111' are grouped per T-DNA construct. Wimin each group. mc GüS arnounts are arranged according 10 incrcasing alllounis. The blacK bars indiellt' me transgenic plants of which me RI progeny was rurther analy7.cd in detail: LXD6ü4-4. LXD6üS-II. LXD6ü7-20, LXD609-17. LXD610 ,', LXD61O-2.and LXD6II-7.

protein (Gall ie et al., 1987a) enhéUlce the expression éUld Cab22L leaders resulting in a Jack of seconda.rv of chimeric gus genes in transgenic pJants. Both lead­ structure is nolable. This may allow easy scanning 1'01'

ers show similarities In length and nucleotide compo­ the first AUG in a favourable context (Kozak, 1989) sition. They are bet ween 50 and 70 nucleotides long, Functional analysis of the TMVleader idenlified 1111

which corresponds to the length of the majority of plant poJy(CAA) region as the primary element responsl­5' leaders (Joshi, 1987; Hershey, 1991). The under­ bie 1'01' enhancement in vivu (Gallie & Walbot, 1992) representation of guanosine nuclcotides in the TMV Therefore, it is possible that the analogous CA-rich

Fig. !he !caf

reg but 01'1

Ac:

We Sta Em disl

215

1000 U GUS/mg protein

5

4

3

:ed ICY

ale -4,

ry or I),

le

;1­

~ ),

:h

2

1

605-11 607-20 609-17 610-2 610-4 610-17 611-7

30nt 31 nt RL 79 nt TMV L 66 nt STNV L Cab22L L

R1 plants

Fig. 4. GUS amOUnIS in R I progeny plants of transgen ics containing various high ex pressing chimeric gus constrllCIS. The nrs! number indicalc, the T-DNA construct; the second number indicatcs the idenlily of me transgcnic isolale. Thc bars show the GUS accumulation levels in young leaf tissue of me dil'l'crellt RI progeny plants tested.

regions in the Cab22L leader have the same function, of the work, and MaI1ine De Cock for help with the but this has to be tested by rigorous mutation analysis manuscript. This research was suppoI1ed by grants of the Cab22L leader. from the Belgian Program me on Interuniversity Poles

of Allraction (Prime Minister's Office, Science Policy Programming, # 38),

Acknowledgements

We thank Anni Jacobs, Isabellc Strobbe and Annique References Staelens for excellent technical assistance, John Van

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216

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a plalll-mRNA strongly inllllcnces its accumulation in transgcnic plants. Nucl. Acids Res. 18: 2917-2921,

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