This file was created by scanning the printed publication.
Errors identified by the software have been corrected;
however, some errors may remain.
Chapter 10
Differential Expression of aroA Gene in Transgenic Poplar:
Influence of Promoter and Ozone Stress1
David F. Karnosky, Gopi K. Podila, Dongill Shin, and Don E. Riemenschneider
Introduction
Fillatti et al. (1986, 1987a) first demonstrated Agrobacterium-mediated transformation and regeneration of whole
plants with hybrid poplar clone 'NC5339' (Populus alba x
P. grandidentata cv. 'Crandon'). They inserted a mutant bacterial aroA gene with a mannopine synthetase (MAS) promoter using a binary vector plasmid (pPMG 85/587) of
Agrobacterium tumefaciens. Riemenschneider et al. (1988)
conducted glyphosate spray trials that suggested that 5 of
6 transgenic hybrid poplars had improved glyphosate tolerance compared with non transformed controls. However,
the maximum glyphosate concentration tolerated (0.28 kg/
ha) was still much less than that used in commercial applications. Riemenschneider et al. (1988) attributed the low
glyphosate tolerance to arr ineffective MAS promoter and
the lack of transit peptide coding sequences that would
target the modified EPSP synthase to the chloroplasts
where native EPSP synthase is localized (Mousdale and
Coggins 1985).
To determine if herbicide tolerance could be increased
in hybrid poplar by a more efficient promoter and to target aroA gene product activity to the chloroplasts,
Riemenschneider and Haissig (1991) cocultivated hybrid
poplar 'NC5339' with a second binary vector (pCGN 1107).
The second binary vector (pCGN 1107) contained the mutant aroA gene, which was fused to the cauliflower mosaic
virus promoter (CaMV 35S), and RUBISCO small subunit
1
Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds.
Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997.
Micropropagation, genetic engineering, and molecular biology
of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO:
U.S. Department of Agriculture, Forest Service, Rocky Mountain
Research Station. 326 p.
70
transit peptide coding sequences from Pisum sativum and
Glycine max (Fillatti et al. 1987b). Transformed plants from
this experiment were tested for their growth, photosynthetic physiology, and herbicide tolerance (Donahue et al.
1994). As expected, glyphosate tolerance was markedly
higher for transgenic plants containing the 35S promoter
and the RUBISCO transit peptide sequences.
In this study, we sought a molecular explanation of the
Donahue et al. (1994) report that suggested that the 35S promoter and the RUBISCO transit peptide sequences had conveyed increased glyphosate tolerance. We examined: 1)
transcription and translation of the aroA gene by transformed
hybrid poplar 'NC5339' using both MAS and 35S promoters; and 2) intracellular translocation of modified EPSP synthase (via cell fractionization). In addition, we examined the
stability of transgene expression under stress by assessing
the effects of acute oxidative stress (200 ppb ozone [03] for 4
h per day for 1 to 5 days) on aroA gene expression.
Methods
We used Agrobacterium tumefaciens strain C58-based binary
vectors. One strain contained the engineered plasmid pPMG
85/587 with the aroA gene under control of the mannopine
synthase (MAS) promoter. A second strain contained plasmid pCGN 1107 with the aroA gene under control of the 35S
(CaMV 35S) promoter and also ligated to RUBISCO small
subunit transit peptide leader sequences. The leader sequences should target intracellular translocation of EPSP
synthase from the cytosol to chloroplasts. T-DNA vectors
were separately inserted into hybrid poplar 'NC5339' using
the tobacco feeder layer, leaf disc transformation method
(Fillatti et al. 1986, 1987a, 1987b).
Shoots were regenerated via organogenesis from cocultivated leaf discs on selective media (Riemenschneider and
Haissig 1991 ). Shoot cuttings from stabilized shoot cultures
were collected from 3 different transgenic hybrid poplar
clones and 1 untransformed control and rooted ex vitro
DiHerential Expression of a roA Gene in Transgenic Poplar: Influence of Promoter and Ozone Stress
(Riemenschneide r and H aissig 1991). Clone PT-1 and PT7 contained the aroA gene controlled by the 35S promoter
fused to the RUBISCO leade r sequence fo r chloroplas t ta rgeting. Clone 12182 contained the aroA gene con trolled by
the MAS p romote r.
RNA was isola ted (Bugos et al. 1995) and a nalyzed v ia
northern blots using an aroA gene probe (Shin e t a!. 1994).
Protein extraction and weste rn blot analyses of aroA p rotein (EPSP synthase) were pe rformed (Shin et al. 1994).
Cell fractionization a nd isolation of chloroplasts we re performed (Shah e t al. 1986). Protein quantification was perform ed (Brad ford 1976) . Scannin g dens ito m e tr y o f
northern and wes tern blot a utoradiogra ms was done using a mirror scanner and scananalysis softwa re.
Ozone (03 ) fumiga tions were conducted in a continuously sti rred ta nk reactor (CSTR) in a g reenhouse at a concentra tion of 200 p pb fo r 4 h per day from 10 AM to 2 PM
for 1, 3, or 5 d ays . Ozone was gene rated a nd monitored
(Ka rnosky e t a!. 1996). Four-month-old plants (40 to 60 em
in height) were mainta ined under standa rd g reenhouse
condi tions before and d uring fumigations. Immediately
afte r the 1-, 3-, or 5-day exposures, all leaves were removed
and stored in -80 oc after being frozen in liquid nitroge n.
Densitometer readings (da ta not shown) suggest that abou t
5 times more messenger R 'A (mRNA) a nd more protein
was p roduced in transgenic hybrid poplar plants w ith the
35S p romoter than wi th the MAS promoter. These resu lts
are consistent with those of Donahue et al. (1994) and exp lain w hy the 35S promote r conveyed a higher level of
glyphosa te tolerance than the MA S p romote r in previous
studies (Donahue e t al. 1994).
EPSP synthase was found in chloroplasts of popla rs
trans formed with construct p CGN 1107 containing the 35S
p romoter and transit peptide coding sequences (figure 1c).
In contrast, EPSP synthase produced by poplars transformed with cons truct pPMG 85 / 587 was not found in
chloroplas ts (fig ure 1c). We conclude tha t he terologous
RUBISCO small subunit leade r sequences we re e ffective
in ta rgeting the translocation of the modi fied EPSP synthase from the cytosol to chlorop las ts. Size differences between the chloroplastic and cytoso lic frac tions (figure 1c)
suggest that the cytosolic EPSP syn thase possessed an attached leader sequence, w hereas the chloroplastic EPSP
synthase is smaller, ind icating effecti ve post-translocation
excision of the leader sequences.
For genetic engineering to be successful in the long term,
the tra nsge nes mus t be stably expressed , even in the presence of commonly occurring p lant stresses. To exa mine
the effects of acute oxidative stress on aroA gene expression in transgenic hybrid popla r plants, we exposed plan ts
lu 0 3 fur 4 h per day fur either 1, 3, ur 5 d ays in a cuntrolled environment cha mbe r. As is shown in figu re 2a,
0 3 s tress resulted in a d own regu lation of the aroA gene
under control of the MAS p romote r. However, tra nscription o f the aroA gene unde r the influence o f the 35S pro-
Results and Discussion
The 35S promoter was more .efficient than the MA S p romoter for tra nscrip tion o f the aroA gene (figu re la) a nd
transla tion of the aroA gene to EPSP synthase (fig u re 1b).
_,
La--------------
2
3
3
4
5
6
- 1--4~
! ~
_____________.
~
b
2
4
c
Figure 1. Transcriptional and translational expression of the aroA gene in transgenic hybrid poplar and subcellular localization of the EPSP synthase protein. a) Northern blot analysis was performed using 10 J.lg each of total RNA from
untransformed control (lane 1), clone 12182 (lane 2), clone PT-1 (lane 3) , and clone PT-7 (l~ne 4) . Blots were .
probed with Jzp labeled aroA gene probe. Arrow points to aroA RNA. b) Western blot analysis was performed us1ng
20 J.l9 each of total cellular proteins from untransformed control (ian~ 1) •.clone 1 2 1~2 (lane 2), clone PT-1 (ian~ 3),
and clone PT-7 (lane 4). Blots were probed with EPSP synthase antibodies. Detection of EPSP synthase ~rotem
was done using HAP-conjugated, secondary antibodies and ECL detecti o~ method (Amersham Corp ... Arhngton
Heights, IL). c) Western blot analysis of EPSP synthase was pe~ormed usmg 20 J.lg each from cytosohc and
chloroplastic protein fractions. Lanes 1 through 4 are chloroplastlc EPSP synthase from control, clone 12182,
clone PT-1 and clone PT-7, respectively. Lanes 5 through 8 are cytopl asmic EPSP synthase from control, clone
12182, clo~e PT-1, and clone PT-7. Arrow points to mature chloroplastic form of EPSP synthase and arrowhead
points to cytosolic precursor form of EPSP synthase.
USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.
71
Section 11 Transformation a nd Foreign Gene Expression
6
5
r""
r-,
r""
,...,
4
"'CJ
>
CJ
.....l
<
3
I"
z
C!:
< 2
E:
<:::
0
1
n n
2 3
4
5
a)
6
7
n
II
8
·n
9 10 11 12 13 14 15 16
Poplar Clones
12
10
r:;"'
>
CJ
.....l
t:
8
·c:;
2
c..
6
CJ
"'
"'
-:5
t:
4
>.
(./)
c..
c..
(./)
2
I.J..l
0
1
b)
2
3
4
5
6
7 8
9 10 11 12 13 14 15 16
Poplar Clones
Figure 2. Influence of acute ozone e xposure (200 ppb for
4 h per da y for 1, 3, or 5 days) on the transcriptional a nd tra ns la tiona l level e xpression of the
aroA ge ne in trans genic hybrid poplars. a)
De nsitome te r scan of aroA RNA northern blot
from untreated and 0 3 treated untransformed
and transformed plants. Lanes 1, 5, 9, a nd 13
a re untransformed control pla nts treated with 0 3
for 0, 1, 3, or 5 days. La nes 2, 6, 10, a nd 14 a re
tra ns forme d clone 12182 tre ated with 0 3 for 0, 1,
3, or 5 days . La nes 3, 7, 11, and 15 are tra nsformed clone PT-1 treated with 0 3 for 0, 1, 3, and
5 da ys. Lanes 4, 8, 12, and 16 a re tra nsformed
clone PT-7 treate d with 0 3 for 0, 1, 3, a nd 5
days. b) De nsitometer scan of EPSP synthase
wes te rn blot from untreated a nd 0 3 treated
untrans forme d and tra nsformed plants . Lanes
are a s described fo r figure 2a.
72
mote r was not d etectab ly d ifferent from control conditions
a fte r 1, 3, or 5 d ays of 0 3 (figure 2a). Concentration of
modified EPSP synthase in transgenic popla rs containing
the 355 promoter was affected by 0 3 exposure w ith both
promoters (figure 2b). We do not know if this was du e to
redu ced EPSP sy nthase biosynthesis or a n increased
prote in d egra d ation ra te. H owever, if d egrada tion was
a ma jor fac tor, we woul d exp ec t to see an in crease in
deg rad a tion p rodu cts res ulting in n ew b a nds in fi gure
2b. Since this d id not occu r, we conclude that the main
in flu ence of 0 3 w as in decreas ing the trans la ti on of
m R A into protein.
Summary
The he rbicide glyphosate [N-(phosphonomethyl) glycine] ki lls pla n ts prima rily by inhibiting the enzyme EPSP
synthase (5-enolpyruvylshi k.i ma te-3-phosphate synthase).
A m u tan t nroA gene encod ing a modified EPSP syn thase
was used to convey increased g lyphosa te tolerance to hybrid poplar cl. 'NC5339' and othe r plants. While earlier
studies showed that tolerance of transgenic ' NC5339' to
glyp hosa te was increased, tra nsge nic 'NC5339' was not
imm une to recom mended commercia l app lication rates.
In this s tudy, we show ed tha t: 1) the 355 promoter was
more e fficient than the MAS promoter in eliciting both transcriptiona l and tra ns lational expression of the nroA gene;
2) the RU BISCO transit pep tide coding sequees successfully med iated trans location of the aroA gene p rodu ct to
chloroplas ts, and 3) acu te oxidative stress (200 ppb ozone
for 4 h pe r day for 1, 3, or 5 d ays) d ecreased the e ffectiveness of the MAS p romoter more s igni ficantl y than that of
the 355 p romoter in regula ting nroA gene expression. These
results sugges t that promote r selection p lays an important
role in transge ne ex p ression, and tha t we can design organelle-s pecific transge ne expression. Our res ul ts w ith
oxid c. ti ve s tress suggest tha t addi tiona l resea rch is need ed
to d ete rmine the e ffectiveness of transgene expression
unde r stress, a nd that stress tole rance of p romote rs should
be conside red in d eve loping long- te rm biogenic p roducts
in forest trees .
Acknowledgments
This research was partia lly s upported by the State of
Michigan Resea rch Exce llence Fund and by the Departmen t of Energy und er Inte ragency Agree me nt DE-A105.
800R20763.
USDA Forest Service Gen. Tech. Re p. RM-GTR-297. 1997.
-
Differential Expression of aroA Gene in Transgenic Poplar: Influence of Promoter and Ozone Stress
Literature Cited
Bradford, M.M. 1976. A rapid and sensitive method for
the quantification of microgram quantities of protein
utilizing the principle of protein-dye binding. Anal.
Biochem. 72: 248-254.
Bugos, R.C.; Chiang, V.L.; Zhang, X.-H.; Campbell, E.R.;
Podila, G.K.; Campbell, W.H. 1995. RNA isolation from
plant tissues recalcitrant to extraction in guanidine.
BioTechniques. 19: 734-737.
Donahue, R.A.; Davis, T.D.; Michler, C.H.;
Riemenschneider, D.E.; Carter, D.R.; Marquardt, P.E.;
Sankhla, D.; Haissig, B.E.; Isebrands, J.G. 1994. Growth,
photosynthesis, and herbicide tolerance of genetically
modified hybrid poplar. Can. J. For. Res. 24:2377-2383.
Fillatti,J.J.; McCown, B.H.; Sellmer, J.; Haissig, B. 1986. The
introduction and expression of a gene conferring tolerance to the herbicide glyphosate in Populus NC-5339. Proc.
TAPPI research and development conference. Raleigh,
NC, U.S.A. Atlanta, GA, U.S.A.: TAPPI Press: 83-85.
Fillatti, J.J.; Sellmer, J.; McCown, B.; Haissig, B.; Comai, L.
1987a. Agrobacterium-mediated transformation andregeneration of Populus. Mol. Gen. Genet. 206: 192-199.
Fillatti, J.J.; Kiser, J.; Rose, R.; Comai, L. 1987b. Efficient
transfer of a glyphosate tolerance gene into tomato using a binary Agrobacterium tumefaciens vector. Bio/Technology. 5: 726-730.
USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.
Karnosky, D.F.; Gagnon, Z.E.; Dickson, R.E.; Coleman,
M.D.; Lee, E.H.; Isebrands, J.G. 1996. Changes in
growth, leaf abscission, and biomass associated with
seasonal tropospheric ozone exposures of Populus
tremuloides clones and seedlings. Can. J. For. Res. 26:
23-37.
Mousdale, D.M.; Coggins, J.R. 1985. Subcellular localization of the common shikimate-pathway enzymes in
Pisum sativum L. Planta. 163: 241-249.
Riemenschneider, D.E.; Haissig, B.E.; Sellmer, J.; Fillatti,
J.J. 1988. Expression of a herbicide tolerance gene in
young plants of a transgenic hybrid poplar clone. In:
Ahuja, M.R., ed. Somatic cell genetics of woody plants.
Dordrecht, The Netherlands: Kluwer Academic Publishers: 73-80.
Riemenschneider, D.E.; Haissig, B.E. 1991. Producing
herbicide-tolerant Populus using genetic transformation mediated by Agrobacterium tumefaciens C58: A
summary of recent research. In: Ahuja, M.R., ed. Biotechnology of woody plants. New York: Plenum
Press: 247-263.
Shah, D.M.; Horsch, R.B.; Klee, H.J.; Kishore, G.M.; Winter, J.A.; Turner, N.E.; Hironaka, C.M.; Sanders, P.R.; Gasser, C.S.; Aykent, S.; Siegel, N.R.; Rogers, S.G.; Fraley,
R.T. 1986. Engineering herbicide tolerance in,transgenic
plants. Science. 233: 478-481.
Shin, D.-I.; Podila, G.K.; Huang, Y.; Kamosky, D.F. 1994.
Transgenic larch expressing genes for herbicide and insect resistance. Can. J. For. Res. 24: 2059-2067.
73